Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...

\ ?^p 6r.1 

CELL CYCLE CONTROL OF HUMAN H4 HISTONE GENE 

TRANSCRIPTION; CONTRIBUTIONS OF C'S-ACTING 

ELEMENTS AND COGNATE HISTONE 

NUCLEAR FACTORS 

BY 

FAMH AZZ 

\o 

->,' 

,9_ 

A Th63i3 submittgd to !h6 unlvoGity of Puhiab, Lahore 

ln Fulllllment ot the Requiromont fo. the Degr€e of 

/1" " /?ds- 

Doclor ot Philosophy in Zoologv (c.lland Molgcurar Biorosv) 

@ 

The oeDartment of Zootogy 

Univeclty ot lho ilnjab, Auaid-e'azam Campus, 

Lahore, Paklstan 

ln Conjunctlon wiih 

Tho D€parlm.nt ot c€ll Eiology 

Univorsiiy of ttla$achus.lts ttlsdlcal Centsr 

wo.c$tor, ltl'3.achG€1t., usa 

'32-a

CELL CYCLE CONTROL OF HUIIAN HISTONE GENE 

TRANSCRImON; CONTRIBUTIONS OF CrS-ACT|NG 

ELEMENTS Al'lD COGNATE HTSTONE 

NUCLEAR FACTORS 

By 

Farah Azlz 

t) Dr. A F. Snakoort, tuora$or.nd Cirtnnrn 

O.por|menl ot Zoology, Uniwrdty ot the punilb : : 

bhor€. P.lirbn. 

2) Dr. Janst L St ln 

o.t€dtn€ot of C.tt Btology 

Univendly ot tlr!..cfirerb U.rttcat C.d!.. 

Worct3ter, UA, USA

In tho narn€ of Alah, olt G6cloua, t o.gt Merciful 

Th. Ch.rlahf, and &lblmr of iha Worlds: 

Io.t Gr.dour, [o.t x..dtul; 

x..Lr ot !|. D.y of Judt.|n nL 

You (do.|.) w rorrilp, 

.nd You (.bn ) r. -( br lrlrr 

Shor u. rh. .tdgm I|y, 

r'|. r.y ol lhoaa on rh.xn 

You h!v. b.dou.d Yo.r G.rc., 

not ol rhoaa rl|o a.m Your an!.r, 

no. o{ thoaa rtro eo aalray. 

Anan 

(Ou/r|| &r.h l)

This disserlation is dedlcated 

to my Parents.

Acknowl6dg6msnts 

Contents 

Abstract .....,.., ............,,.,,..............,., ,. iv 

LisrolFigu.€E.......,.. .........................,.,,............vi 

Iniroducllon......-........-...-... ...,,,.,.,.._.........._... l 

Central concept ...-......-....,,.,, .,,.,,,..............................,..._.................... 1 

Cell division ..............-.................................._.._..._.............._................ 3 

Hislonesynlhesisduringth€c€tlcycte......._.._..........,..,,..,...............,..3 

Biological and blochomicat param€iors thar regutate th€ histono 

g€nee4fession...._........._................................................................ 12 

G€neral principles of kansdiption ............_..._.._..._......,,..................., 13 

DB novo synhesis of human hisron€ mANA fianscriprs ...........,,..._.. 15 

Hislone gon€ vanscription conuot by crs{egutalory 6tem€.ts ,,.,...._ 20 

Tfansc.ipriona modets for htstone gens regutation ,...... .....,...,.,,,.... 22

Muiagenesis olsrte tt bymorecutarctonrng............... 

lsolsi on ol DNAfragments..... 

Ligaonreactjons,.......-.,,..,.,-''-.'..''..'-..-................,,...................36 

DNA kansformalion into E. coti baclsria......... 37 

Prep.ralon of compere cers 

Bad€nal kanslormation 

Colony Hybrid'zarion .._.._.....-.---. -''-.''_.'-.. '.- -..._...._..,......._...__..... 39 

Small scale isolation ot ptasmid ONA ...........,....._......,,,.,..._............... 41 

ldentilicaion of c|ones.,........ 

Beslr dlon e.donucteass drg6ston 

DNA Sequence-anatysis .........,.,.'..._._''._.'...'...,..... .................._.... 45 

Prepara{on oi qlycerot stock bact€rial strains tor storage .......... . ... s5 

Largo scare preparalion ot ptasmid DNA by atkatine tysis ,........_.....,. 55 

ONAisolationusngOuragenion.€xchageresns ... 

Elechophofeticmobitityshifiassays...._..........._....'.'-._...,..'.,._._.58 

T4 Polynucleoride kinas€ .,... _..-.-.-- _- _- ---. ---.............._........... 62 

Transient €xpressio. anatysis .'...'..'.--''-'.'.''''.-_-,......_..........._..._.. 6s 

Calclum phospharo m€rhod 

DEAE-Dexlran mgthod .. .._,-,._..'...,.. ..,_,.. ......, ....... ......, 6,9 

Chloranphe"icol acetytrra st6,sse (CAI) assav . ........ . .... . 71 

Con9rucnon ot stabte @,t tines 

68 

73 

34 

36 

37 

38

Selection ol cell linss contalning chimeric H4 promoter CAT repoder 

geneconstrucls.,..,,,.._...........,........._..._..._..,...,..,,,...,...,..,,...,..........73 

Prepdarion oi glycerot stocks ior stabl€ c€ll lin6s ...._..._..............,..,, 76 

Soufhsrn analysis .....,.,.....-... ......-..-...--..-...-.......-.....,...,....,_,,............ Z6 

lsolalionofgeriomicDNA...,....,,..,...,..._.....,,,.,,.,,,.,,.,..................,...,.76 

Transf€r ot ONA to a msmbrarous sotid suppoft .,...,................._..... g1 

ttepar€tion ol €diolabeiled prcb€ ror Sourhem hybridiza on ......... 83 

Slafdardprocodurelorhybridizarion.............................................g4 

synchronizatonolsrabteHeLacelttin€s..........................................85 

suspension cullure of H€La s3 cell lines with integrat€d reponer 

genos ...................._............................_........................................... 85 

Doublelhymidn€blocksynchronizarionproced!rc.........................91 

Monitonng cell syncnrcny ..................-..,...,,...,.... .... .................,...._ 92 

O€terminarion ol ONA synthesis ,..,..,..,,..,....,............_...._..._..,..,,....,.. 97 

Nucl€arrun on lranscription: tsotation ofNuct€i ,.,,,..,,.............,..,. 97 

rsoralion oinlcei ................_......,..,,...97 

TEnscription reaction ......._....._.,,,....... 99 

DNASlot-8|oi.............................................................................. 102 

Pr€p8ration oi hybrldization membranas using ihe manlotd I sot 

lsolation ol totatcettutar RNA ton mamalian c6lts ........._.....,,.... .... i05 

Fibonuclease protection assay ............................................... ...... 109 

/, vr't o t anscripljon reaction ........,..,........_..._..._......._...... .....,.._..... 1 12 

Hyb.idiairon ol RNA transcripis and sampte RNA ,,,._.........._...,.,,,, j j 2

AnalysG ol the H4-Site ll cell cycle domain which inl€racrs wirh 

mullipletaciors..............,..,.....,,.,.,,..,..,......,...,...,..,..,....,,..,........... 115 

The dislal segmenl ol Site ll is €ss€nrial for high level H4 gen€ 

tanscriplion ...... __.._..............._...... 117 

rhe HINF-P/Ha-TF2 binding morit in lh6 H4 gene is disp€nsabl€ tor 

H4genapromoleraclivi9,t1v,vo.,,,,,,.,..,...........-......................... 123 

Specif c variations n ths natural s6quenc€s of analogous hlman fi4 

9€nesat€rlranscriptionalactivily...............-............................,.. 124 

Similariliss in mubrio aleil€cts on H4 gene transcription in dislinct 

prolilerating c€ll rypes displaying dilGrent cell growth and tissue- 

spociricphenotypicpropeniss..........-......-................................,..,,. 125 

Abolishmsnl ot specilic H4-Sit€ ll rccognition motits is dominant 

over eflecrs on rhe putaiive sparlal alignmenls ol H4 lranscrpuon 

laclors., ,,.,..... .. .. ...........,.,,..,.,..,,.,,.,,.,,.,.,,.,...,..................-... .. ... .126 

Targotsd mubrion oi rhe HiNF-[4/|RF-2 binding sil€ r€duc€s H4 

promor€r acriviy ............_.,..,...._.,.,,.,,.,,.,..,,..,.....__.....__.,__................ 130 

conr.ibutions ol lhe H4-sir6 ll binding prot6ins ro human histone H4 

96n€transciplionduringlh€c€llcycl€...............-......................... 133 

utrrrarcn ot RNase p.oreclon assays for gudying H4 g€ne 

ranscripliondungthecellcycl€,.,,.,,,.,,.,,..,..,,..,..,..,,..,..,,..,.,,.,.. 140 

Mutalion ol H4-Site ll lransc ption factor binding sites has subtle 

€fiec|sonc€llcycle@nl.olotH4 g€n€promote.acliviri .............. 146 

164

ACKNOWLEDGEMENTS

acRn@tedgMMts 

Acknowled ements 

I wolld liks to €xpf€ss my sinc€fe grarirude to my supervisor Dr. A.R. 

Shakoori, Proiessor and Chsiman, O€parLndl ot Zoology, University or ths 

Punjab, Lahorc ior hls guidanc€ and 3uppod durlng my work, and who has 

prcvided mo the oppodunlty ro work in a highly productivs laboratory. 

I am v€ry lttankful to firy co€upervisors Ors. Janet and Gary Stein as 

wellas Dr. Jane Lian lor allof ther guidance and sJppoit du ng my ysars ln 

lheir laboratory. Th€ nic€ Thanks giving dinnerc and Chd$mas pades (wilh 

kosher lurk€y) were very mirch apprcdat€d. 

I also wish to €xtond my sin€r8 gratitude ro or. andr€ van wrjnen lor 

his constanr suppon, €ncouragsn€nl and guidaice during my work. His 

contribltions to my work hav€ been padicularly €xlsnsive and have proven lo 

be invaluable throughout th€ courso oi my studies,

I would also lkg ro rhank Dr. Paficia vaughan, and Dr. I',,lark Eimbalm 

lortheir lriendshlp, support, va uabl6 sugges ons and dlscussions. 

My special lhanks 90 to Dr. Baruch Fr€nkel and Chaitali Bane.jee lor 

lh6i. help dlring my r€s6arch work. lam alsothanklulto Or. Lau.a Mcoabe, 

Dr. Heid Hotlman, Or. Elish€vasmilh, Or. Hyun-Mo Ryoo, Dr. Cong-lveiZsng 

and Dr. Bo Guo lor their help and ldendship during my stay in the lab. 

My special lhanks also 9o ro many tom€r labo€tory coll€agu€s 

includng Or. Vicloria Shalhoub for hor support and lriendship and Dr, Elen 

8rcen for he. helpd-nng my research. 

ol cours€ | cannot iorget lo exprcss my deep fee ings lor Bosa 

Nlaslrotolaro who has taksn ca.e of ms like a mother dling my 6nlre stay n 

lhe lab. My specialthanks 9o 

ro Jack Green for his gleat help during my cell 

cycl€ dpe.imsnls sndro Liz Bofiona for herfrie.dship and help. also, rhanks 

tobothJack and Lizforrhstissu€ cuttuf€ t€chnlquos tlsarn€d lrom thenr.I am 

gralelu io myti€nd, ShiMin Pockwinse for her help throughod my stay in me 

iab. I also wanl lo thank Cas€y Capparella, Oanielle Undenmuth and 

Jacqusline Marie Burrs rortheir hetp and triendship. 

lam gralelul Io Cathy Getuasl. Wnhout her h€lp I would not have been 

abl€ to complete this disserlarion. Also, I am grar€tu| to Jeannefte Landrie tor 

pholography. l arn also lhanKul to Judy Fask and Elizab€rh Bronst€in lorthelr

My special thanks go to my trl€nd, Falzia aslam, for her suppod and 

ior rh€ good tm63 we shar€d dunng my sEy in ihe us,a, lt was esp€€rally nrc6 

sbaring living accommodation, I am also |hanklul to Haroon Bhal, Fauzia's 

husband. tor the m8ny grsat limes r€ shared. 

I 6m rhankful to Fry othsr friands, sdEa nahman and Fa€h Mls€ila 

lor thet h€lp, trion(bhip and love dudng ny stay in lh8 USA I am lhanKul to 

Or Muhammad Ak&n ior his moral support during my studies. 

| 3m franktul to all my lamily mbmbers, 6spocially my brolhsrin.law 

Fiaz Tahi., ior ti6ir €ricolrag€rn€it durhg th6 €fnne p€dod of my studies and 

tor th6ir gred love and support, 

t

ABSTRACT

Abstract 

Historc proteins ar€ structural compomnls of chromatin and as such 

ars diucal packaging compon€nts of lhe DNA cloning and replicaiion. The 

expressi@ ol th6 five hisione gen€ classes is tunctionally linked to DNA 

rsplicalion. Athough conlrolol histons geno €xpfession occurs at muhipe 

r€gulaiory bv€ls, transcriptional r69ul6tion rsprssenis a prominent l€v€l of 

conrol. Cell cycle d€pends haslona genes r€pres€nt a paradigm lor the 

sludy ot proliieralion-specitic gen6.r6gularory mechanisms operatlve in a 

number of diveGe eukaryotes. Human histone H4 gen€ lransdipijon has 

beon nrost sxtensiwly examin€d wfth a highly €xpr€ss€d H4 g€ne designated 

as FO10A. This gene has be6n shown lo b€ trunscnbsd in a c€li cycle 

conlrorled manner and is regulat€d by a compbx aray or distaland proximal 

Marirnal hnsci ion of cell cycle conrroll€d histon€ H4 genes r€quires 

a mul panit€ and prollf€fation-specitic t v/vo genomic prol€in/oNA

i.leraclion elem€nt, sil6 ll, Thr€€ ssqu€nce-sP6cilic lr.n:icriplion tactors 

inr€r€ci wirh ovB.lapping recognition motib whhin sit€ ll: HiNF M, which is 

idenlicarto interieron .egulalory facior lRF.2, the H4 subtyp€_specilic protein 

HiNF,P/H4TF-2, as well as the muili-Protsin comPl6x HiNF_D whlch conialns 

fie cul/homeodomain protein coP-1, coc2, cyclin A and an BB ielated 

protein, To addr€ss the p.6cise contribulion ot th€ rocognilion €lements tor 

each ol these tacrors to $e level ot H4 gene lranscription, we pedormed a 

sysr€maric mutalional analyses ol sir6 ll transcri ional molifs. The resultinq 

site ll promot6r mutsn$ w€re l€sled tor ability ro bind each ol lh€ sn€ ll 

coqnate pror€ins. and subs€qu€ntly tundionsny 6v6lualed for ability to conrer 

H4 ranscriprional adivny using cttimeic H4 promot€/cAf fusion cooslrucls. 

Resulls show that the recognition sequdnce ol HiNF.lM/lFF-2 ls ths dominant 

componenr aid modulares H4 gene t ans.rpron bvsls bi 3lold. Howeve., 

lhe mllripre overapping rocognition sequences tor HiNFM, -P and 'o 

togethe. modulate H4 gene transcription levels by at least an order ol 

magnirude c€rr cycr€ analyses reveals rhd mutalims ol lhe IRF-2, HiNF-P and 

HiNF-D bndng sil€s, eilher separale o. in combinallon. have diflerent eflBcls 

on H4 gene transcription du nq lhe csll cycle. Ihus, it is proposed lhat the 

p.oper lemporal contol of H4 g€na transc.iption duing the c6llcycrs roquires 

lhe integrated activities ol mulrrpls trans€riPtion lactors at a composile c€ll 

cycle fegularory domain, H4'site ll. This composhe organizalion supports 

respo.slveness to muliiple c€llsiqnalling paihways moduLatinq lhe acliviuBs ol 

H4 qene ranscription lactoE du.ing the c6ll crcl6 to accommodale r€qure_ 

menis jor erpresslon in cells and llssuos with uniqus phenolyprc prcpedies

List of Fi 

1. Schemalic repr$enration of tunctional id€rr€la onships 

2. 

3, 

5, 

7. 

bolweenc6llcycl€{ogu|a|oryproieins.............................................. 

Reg-lsl,o_ o'hston€ H4 gen€ orpf€sslon ....... 

Regulation ol hislone gene lrans.riplion du.ing c€ll growth and 

d,l6ren aho_....,..,., 

Sdl8malic r€pr€s€nlalion of hanscyipton lactor inleractions wnh 

histons gene promorsr €bments du ng fte c€ll cycle and 

,b11ow1n9lheons6rotd,tferoneton................................................ 

Or$nizalio.rotth€H4genoproximalpromot€r.............................. 

Table of wildtype and mutanl H4 promotsr Sit€ llsequsnces -.......... 

Schematic diagmm showing the cloning procedur6 for H4 

mda.n promo er constucls. 

17 

25 

30 

32

8. Colony hybridizalion sc.eening ot transtomed baderia . . . .. . 42 

9, F6striclion enzyme analysis ol positivo clones . .,.... . .,.46 

ro. DNA sequence analysis ot H4 Promoter conslrucls I . -... ... -49 

11 ONA sequonce analysis ol H4 Promoler conslrucls ll . .,,... -. -. 51 

12. ONA sequence anatsis ol H4 promotsr construcls lll . ...... 53 

13. Schematic diagr€m illustating the prsparalion of plasmld probe 

15. 

17. 

14. 

20. 

21. 

22. 

23. 

24. 

25_ 

lo. use in gelshm assays -...............-..... -.. -. .. .. -. -. -. -.... ... - 60 

MoFhology ol human H6La s3 ceMcal carcjnoma cels (A), 

rat osteosarcoma (Ros) c€||s (B) and rcl ostsoblasl (RoB) 

cerls (c) . . 

ProdJdon ol H4 promo€ CAT slabl€ c€lllines 

Aralys s ol srabecell ines 

Souhern analys s ol staoly kansleded cell l nes . 

CAT acttrity ot stably tr&slsclsd H4 Promoler CAT c€nslructs - 

Sy'cnroniarol olsldble c6llli1es ...... - - 

Pholograph ol synchro.ized HeLa cels a hours alter release 

trom sec€nd rym'd ne blocl 

lsolato. ot n-c el 

Nucled run-on lranscription analysis ol synchronized slabl€ c€ll 

lsolarion o'toralc€ll-lar RNAlron synch'on zod ce ls 

Electrophor€ic mobilily shilt assay condi ons lor H N F P, M & O . 

Mutalional analysis ol H4 site ll protein-oNA inleractions . . . . 

Mutational afalysis ol H4 sit6 ll prolein oNA inleractions . . .,. 

79 

a6 

8a 

93 

95 

99 

'106 

110 

119 

121 

127

27 Mulalional analysis ol H4 sne ll protsin DNA inlsractions .. .. . - 131 

28. Transi€.t kansl€ctions ol various cell lyp€s wilh H4 promoter 

catconstructs.,,.,,..,.......... .- , 134 

29 Transi€nt translections ol various c€ll types wilh H4 p.omol€r 

CAT conskucls -........,...............,.............,...-................... .. . . 136 

30. T.ansent iranslections of various cell lyp€s wili\ H4 promoter 

cATconstruds-........,..........-.....-.......,....-...-...-..-....,...... - 138 

31. RNas6 p.otecilion asssay oi cAT mRNA in synchronized ells .. 142 

32. RNas€ proteciion assay of H4 mBNA in synchroniz€4 cells .... ... ., 144 

33. RNas6 protedion assay ot GAPOH mFNAin synchronized cells 147 

34. Analysis ol gsn€ exp.sssion in syn€hronized c6lllinss during S 

and lM.Phase ,,..,.,,,.,,..,....................,,,,..,,........... , ., ., .. 149 

35. ouanlra on ol T3/rg dala .....-......,,.,,,.,....-..........--....,.... -. -. - 151 

36. Graphic r€pr€senlaiion ol T3l'T9 data ,.,,...-...-......,.... ... -. . . 153 

37. Cellcycle analysis olth€ wid lype H4 Promoier CAT slable cel 

tine .....-.'..,...-......-._._..... ................ 156 

38 Cerrcycre anarysis olrhe suB-11 (hiNF-D Mulani) tl4 promol€r 

CATceline ....... .,........-......... .. 158 

39. Cell cycle anarysis ar the MsPl6 (HiNF-M Mubnl) H4 promoter 

cATce11|ine........................-..........,,.,,..-.............,.,,....................... 160 

40 Cel cyce anayss ol MPM-17 (HIN-D,M and P Mulanl) H4 

promoter CAT ell n€ ............................ .. -. - ,.... 162 

viii

INTRODUCTION

tzh F: C.n CYd. C@nd d ,U C@. t@ttttol 

Canlrll conc€pt 

General Introduction 

Hblon6s are small ba3ic prot€ins which havo a high content (10 io 

20%) ot ths basic amino acids arginino 6nd lysin€. a€ing basic, histones bind 

glnly !o oNA. Tho lour clr€ hislonos (tl2A, H2B, H3 and H4) are very similar 

in ditf€r€nt sp€cGs and ar€ pr€s€nt in oquivalsnt anE{,/nts. H24 H2B, H3 and 

H4 a.e sran$d in ocramets containing teo copiss ol e3ch protein p€r lwo 

hundr€d bas€ oairs of oNA. Th6 orolein ol lh€ hrslone octarner 6re an closs 

clniacl and th6 oNA is wrappgd 6round th6 prolain co.a io lom 5 

nucl€osoms.Hislons Ht is not cln$Iv€d b€twoen so€.$s and has a lissug 

sp€cific krm, on6 hislono Hl is pr6sent p6r 200 bas€ p6irs of DNA, is rather 

loosely associalod with chromalin and ls not a componed ol he nucl6osom6 

but is involv€d in maintsnsn s ol a hbher o.der lolding ot chromalin.

t t F: con cda c@tot d H. G@ r@Mbn 

Hislons oroteins 616 structural comoonsnls ol chromalin 6nd as such 

ar€ crnic6l c9rnponenF ol rho pacl(aging ot oNA cloning 6nd rcplication. 

sudying th6 mochanism by which hislone gsns €xp€ssion is r6gulal6d is 

important for our und66t6nding ol c6ll cycl6 conlrol ol eukaryotc gene 

€xprossiod, €6 w€u as control ol c€ll growti during tumorigenesis. Tha 

€tpr8ssion ot lhe live histon€ garc da$€s is tunclionally linked to DNA 

replicalion. Each class reprosents 6 mulllgsne lamily whose members encode 

essentialt lh€ same histon6 protsin, Allhough conlrol of histons g€ne 

expression occurs at mu[iple rsgubrory lev6lE, transcriplional r€gulalion 

rcprcsonts a prominsnt level ol conrrol T.anscridion oI histono gsnes is 

requned duing lho S phaso ol lh€ cell cycla, 6nd hi$on€ g€ne ltanscription is 

ssl€clively downr€gulalsd duing difl€r€ntarion. Because the tive hislone geno 

subtypss p€riom 3 joint tunctlon, lEnscdp on ol th€ss livs g€ne class€s is 

coordinatet rcgurat€d du ng €ntry into and €xit frorn s-phas€, as w6ll as 

wh6n c6lls c6as6 prolileration, Transariplional mod€ls ol hislone gene 

6rpt6ssion iNolve s€q€nc€-sp3cifc p,otein/oNA inls€ct'ons by hislon€ 

gsne pfomor€| factols. Th8s€ tactors m8y m6di6t6 (i) sp€cific recognilion ot 

the mFNA slart sir€, (ii) 6lficbncy ol iniuauon, (iii) s€bcri!€ discdmina on ol 

histone gene promolers, as wsll as (iv) $e tming ol t anscriplion relali!€ lo 

specmc siagos of lha cdlcycl€, Ths main locls ol rhis study is to und€rsland 

the cont bution ot sequsnce.sp€ciic promot€r taclors lo cell cycl6 regulated 

ranscription ol hsman hisbne H4 96n6s.

a2i2 F: cdt awte c.tuot d * effi I@tioti@ 

Progression irom a 9€m cell to an adult o.ganism is mediated by the 

complex interplay ol many gen€ €gulatory progiams. These programs contror 

prollleraton tom a singlo c€l lo a multicelular organism wilh dislincl 

sp€clalized tissues composed of d ifefonl cell types. The col s n each lissue 

6xprsss uniqus ssts ot genos, whichaf€ charact€rislic oi either prolirerating of 

dlriersntialod cslls.cell prolilgrarlon is a mul!.st€p procsss (revjewed in 

Baserqa, 1945; cross er a/., 1949; Muray and Kirschner, 1989i Pardee,1989; 

weinberg, 1989; Lovine, 1990). Ihe plocess ot c6ll diuisio^ is chaiacterized by 

a sequental series of biochemical evenls thar is tir$ aimed at the replication or 

the sukaryotjc genome during S-phase (Se€ Fig- 1)- Subsequenlly, mulliple 

ch.omosomes a€ segregated in an ofdsrly manner, a.i passed on to two 

daughter ells as a resulr ol miross. Th€ malntenance oi chfomatin slrlc1lre 

during the various phas€s ol th6 c6ll cycl€ is vefy lmponant ror the preciss 

sepaGton of the tuo sets oi chromosomes. The inkctness of the 

chromosomes ls reqlired lor completion of the norrnal celL grovih process, 

The strucruraL organizarlon ol ONA wirhin lhs nucleus ls medialed by histone 

Hisione synthesis d uring rhe cell cycle 

Histone genes musl b€ express€d in a highly efficie.r manner to 

accomodare a very rapid rat€ hislon€ p.otein synhssis. lt can b6 calcular€d 

thal, ior each subtype, severallholsand copies arc needed for each round ot 

ceLldivision. Thus,lhe celular p.orein synlhssls machinery is ded cal€d lo the

Azh F: C6n Ca/cro C@t.t ot H. G.n. t.ansdtpti@ 

: 

Fig.1. Schemattc rcpresentaiton ot tunctonat Inlerreta onships 

berween celt cycl€-r€gutato.y prot6'ns. Th€ ligure snows rn€ 

cascade ot regulatory me€hanisms mnt oling activation of cdk,s 

by rh6 c€ll cycle.dependsnt r€gutation of specitjc cyctins and by 

a series ol cdk inhibitors. Ths contriburiois ot ubiquirin and 

phosphatas€s (€.9., cdc25) to aclivation and downrogutalion ot 

cerl cycle-€gutatory compt€x€s ar€ indicated. atso o€sqnated 

are inreractions of cyctin_rstaed proteins wilh groMh ,actors, 

cytokines, tumor supprsssoG (plo7, pFB), TGF-p, and 

medialors ot c€tt-c€l conract, Competency tor cel-cycte 

progrsssion js rsfloct€d by conv€rgenca ot growth faoo. Lrumor 

promoers and suppressoE) siqnalting psthways with cyctin- 

r€lated prolelns. Ce|lcyct€ progrsssion is suppodeo oy 

ranscriptjonal coDtrot (activarion and suppression) oi genes a a 

series oi checkpoints (ag., c1ls snd c2l,\4) by rh€ cyctn- 

dep€nd€nt phosphorytarion o,tescriprion factors.

Frffi 

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Aziz f: Celi Ctcle Cotutol ol H4 Aene na&iblon 

produclion of hisrone prot6ins duing S-phase and it has b8en knownlormore 

|han rwo decad€s thal hislone symhesis ia coudod wirh the replicative phase 

o' the csllcycl€ (Bobblns and Borun, 1967). Lal€r slldl6s hav6 also shown 

that histone p.orein synrhesis is directly coupled wilh ONA synlhesis in lhe S- 

phase ot rhe celr cycre wirh advgnc€d r€chniqu€s ol mol6cular bioloqy. These 

srldies have r€vealed a complicated m€cha.ism ol cell cycr6 regulalon in 

which rh€ le@l ol histone mRNAs is r69ul6ted by both iranscnpl'onal aod 

posl-transcriptional contfo (see Fig. ?). Two vpes ot svalegi€s hav€ been 

used ro srudy hisron€ mRNA meraborism during th€ cell cycle. The firsl 

stralegy nvolved synchronlzed c€ll cuitlr€s whi6h wer€ used lo obsotu€ lhe 

acclnualon ol hlstone mRNA in the cellcycle withod any interrudion The 

second appoach involved rhe interrudion ol lh€ cell cyc.ls wilh inhibirors oi 

DNA chan elongalion, lo observe if oNA synthosls is af impodant event for 

conlinuols production ot hlstone mFNA. Ths majorresulls s::cwthallhe DNA 

replication is important lor rhe synthesis ol hisrone mRNA In mo$ ot the 

eukaryoric cerrs, hgh lever accLrmulaio. ol hi$on6 mFNA G obsotued dun.g 

rhe s phase. ThB level ot histon6 oFNA elevabs 15 - 30 ,old as cells pass 

rhrough rhe G1 ro s-phase and p€ak l€v€rs are obseryed i. th€ middle oi s- 

phase (3 ' 6) ho!.s at1e. rhe ell released lrom block (Ehinger €t al., 1990; 

Marzrull and Grave, 198.1 Srein et aL, 1984r Heintz e! al., 1983i Baumbach et 

ar., 1987iPlumb era/., 19S3; Mor s era/., 1991; Hads era/., 1991i Anishevsky 

er a/., 19a4). When DNA sydhesis is disiurbed, histong mRNAs disappear 

lrom the cyroplasm rapid y (Wu and Bonn€r, 1981j H6i.lz €tal., 1983i oeL sie 

er a/.. 1983i Snhan er a/., 1983). The higher eukaryoles

at F: c.n cyd. contol ol Ha a&. f@crlN@ 

n9.2. Fagulallon ot hldone H4 g€ne expt$3lon. Oop PansD 

Sch€matic reprosentation ol lhe c€ll cycle Gr, S, G?, Mitosis, 

lndicaling lh6 pahway 6ssociat6d wirh lh€ poBrp.olif€rariv€ 

ons€t ol difer€nliatoo initiel€d bllowing comple on of mihcsis. 

(Low€r Pan€D Repre3€ntation ol data dslining rh€ pdncipal 

biocbmical parafi€lors ot histons gen€ oersssioni hislon€ 

p|ol6in synih€sis and th€ pr€s€nc€ ol histon€ mRNA ar8 

r$ticl6d to S phas€ c€lls (DM synlh€sis). Conslilulive 

transcriAion ol hision€ 96nas occu6 lhroughout th6 c8ll cycl€ 

wilh an enhanc€d transcdploml l€vel dudng 33 y S phase. 

Thes€ . r€sults ssiablish th€ cornttn€d contribution of 

transcription and mBNA stability !o the s phas€-specilic 

r8gulslion ol histone lrc3ynlhasis in prolileraling cells, with 

histone mnl,|A l9v6b as th6 ralo limiung step.

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rsgulare hislone 'iRNA levels ihrough hr€€ palhwavs, Th€ lirst pathwav ls at 

lh€ G1lS boundary wh6n cells slat to sntar s-Phas€ ol the cell cvcl€ and 

nas@nt histong mRNA &ansdrp on elsvat€s 3 - 5 fotd owr th6 basal l€vel ol 

exoression whch is a characl€ristic ot c€lls when they ar€ ln G] phase The 

oih€r two palhways which control histon6 mRNA synth6sis are al the post_ 

r.anscriptional l6vel, These r€maining pathwEs a.s imPona ior elevating th€ 

hstone mFNA synthess level 5 - 6 iold in th6 s'phas€ ot lhe cell cvc€ and 

arso ror rhe degradation ol hislon6 mRNA lrom lh€ catoplasm. wh€n 

repli@lion is stopped by rr€ating silh r€a96nl, lhe$ bner lwo pathways ol 

Doslrdsdlprional regulallon aci in ditler€nt wavs on th€ lsvel ol hislo'e 

mRNA One olihe Nto pathways hdan imponant tol€ lo degrade the hislo'e 

mBNA in cyloplasm when DNA symhosis is dec'eas8d (Baumbach er a/ 

1987: Delisl€ er €1., 1983r Sive er 31., 1980; Moiiis 6i a/, 1991) Va ols 

d itle.enl techniq! es havs been used ro eslimate the hali lile ol hislone mRNA 

lr is lound thal the haf [r€ ot hislone mFNA is 30 ' 60 minules l/h€n lh€ DNA 

sv hesis is bLocked by us ng inhibrlo.s ol DNA chain elongation th€ halt life ol 

histone mFNA raoidiY decr€ased 10 ' 15 minules. Ssveral srudies have 

questoned whether this pathway might also w€rk during lh€ normal ce|| c-vcle 

when cerrs snrer inlo th6 s-phase wher6 histone nFNA is hlghlv elwared this 

rime rhe uliriation ol this pathw6y is th€ stabiianion ol hislonB mRNAlor lhe 

acclmulalon ol these t .nsc.ipls. A driving oprnim is th6t rhe ch4ges rn the 

srabilily ol hislo.e mRNA can ocdr du.ing lhe S'phase ol lhe cell cvd€ ?om 

rhe obs€tuation rhat h€li llls ol histonB nFNA can be r69ulaled eliicientlv a.d 

relersiblv in S-ohde. when cells ar€ t€leas€d lrom block, lhe histone mFNA

En F. Ce Cycl. Cantd d h. Gft ra.scdpnon 

d€stabilizalion is abolish€d and histone mFNAs rapidly incr€as€d in lhe 

cyloplasm as a r€sult ot a lengthon€d halt llf€ (Heintz 6r d/ , r983i sittman er 

a/, 1933i Sive er a/., 1984). Those concluslons strength€nEd lh6 idea that 

during the undislufbed corl cycle, ths synlhesis ot histons mRNA dLring lhe 

rare Gl and earry s-phsse ae siabiri4d whEn c€lls €nrer in s.ph6se. 

'rhis id€a has bsen support€d by anelysis o, H3 mRNA srability dudnq 

rhe HeLa cel cycl6. when rhe transcrip onal and posrlfansd pt onal 

regu arion were not linked (Moiiis d ai., 19ol) th6 H3 mRNA was 

approximaloly rhr€€ rlmes mo.e sr6bb in rh€ Early s-phase of 06ll crcle than in 

rhe G2-M-G1 phase ol the cells. Hoe€vsr, dilte.ent resuhs wers shown shen 

lhe hisrone mFNA hall lile w6 studied in syrchroniz€d CHO c€lls (Hairis el 

a/., 1991). During Gl €nd S-phas€ rhE hislona mRNAs had a 40 - 45 minut6 

hall lile which m€€ns rhar rhe srabillty of histone mFNA may nol vary 

signilicanrly in undisrurbed Gl and s-phas6 calls. on the other ha.d, ar naural 

completion ot replicstjon lhe degrada on pathway do€s work. al this slage lhe 

rare ol DNA synrhesis is drasricalt Gduc€d (Pbmb er a/., 1944) and hisrone 

mRNA hall liie has been observed ro be only 10 - m mi.utes (iiaiiis ea a/., 

199liModsda/,,1991), 

The second pathway ol posl.lranscfiptroflal modulalim works inside 

lhe nucreus to procoss rhe histone prs-mRNAs- For rhe production ol mature 

cyloprasmic mRNAi lhe hlstone pr€-mFNAs are processed by the 

endonuc eoryric cr€avage ar rh6ir 3 t€rmini (Pllmb et a/., 10841 Moss et a/., 

r994i Pandey eral., 1994i Eond 6r a/., 1991i Nagata er al, 1991).lhs socond 

io

A2it F: Cen Crcb Ctuot ol H4 GeN f,lMidk,l 

pathway is assumed ro be acli6 already at th€ G1-S phass bolndary. This is 

rhe same phass whero ONA synth€sis is adivaled. This was lirst shown by 

i.ealing mouse temperalure sensitiv€ mastoqdoma mutanl cells wiih a specilic 

remperarure, which blocks rh€ cells G1 phase, In thos€ lsmpe.atu.e treated 

cerrs, rh€ hisrone gen€ exp.ession conlinues ril irs highssr lsvsl bd the 

nascent histone mRNAs wer€ not proc€ss6d activsly and some or the mature 

histonB transcriprs re.6 in the cfoplsm (Lusche. 6l a/., 1985i Ha.is el 

'ound 

a/., 1991 ). A len lold inc.ease ot mature h stone mRNA was obs€N€d torlow ng 

entry inlo s-Phase when h6so cells w6r€ rer€as.d tom ihe G1 block. The 

rransiro.y rera onship beMeen th€ accumulation of hisrone transcriprs in rhe 

cybprasn and lhe aclive .ucl€ar proc€ssing ProPoses thal th€ p.oc€ssing 

pathway may have strong conrribution in the increas€ ol histons mFNA levels 

when the cels 6nterlntolhe S-Phas€ ol tho cell cycl€. Ths vi€$s lrom Harfls el 

a/. (1991) and Stauber and Schump€ni (1988) are compatibl€ in fiai lhe 

hislone mFNA rhat coud be accu.ar€ly processod but nol degraded 

conrinues lo accumurale in $€ s-phas.. lr may be trus rhar lh€ conrol ol 

hisbn€ pre mFNA processing €vent ls th8 malor eve.t of posrlranscriptiona 

pahway which is utilized in high€f eukaryotic c€lls wh€n c8lls ent€r into lhe s 

Phase. on lhe olher hand, lhs control ol histone transcripts stabilization may 

be an important event €ith6r ro 6n inFrmit€nt S.Phas€ or at h€ ond ol lhe 

The pathways (descibed sbov€) gdsrning the level ot histone mRNA 

in vertebrale coll cycle does nol necessa ly apply to all €ukaryotic cell cacl€s. 

tt

,r1z F Cett cy.te connot ol H4 Gene ftansc.ipt.n 

For example, oliat€s and lunqi do not show rhe post'transcriplional Pathway 

which regulares lhs processing ot pr€ mRNAI bul lho olhel rwo pathways are 

operaiv6 and all €ut6rycli€9 ll is known lhai lhe 

hanscriptional rBgulalion is mo.e Prominenl lhsn the Posi_lransfiiptional 

regulalion ol rho level ol hlstong mFNAs ln som€ lowsr eukaryol€s The posl 

rranscrip onal coolrol of hislon€ mFNA sccl,mulalion in the cetl cycle ot lowe. 

eukaryotes has been syslematically studlad only in budding yeast For 

example, ifl yeast th6 hislone gene fianscn ion is hardly se€n in ihe eany G1 

Phase, blt tacl ilaled 10 . 20 lold in lal6 Gl Phase (Hsrcford d ar , 1982) This 

me4s hal lhis incre6e i. vansc ption is the sam€ as 

accumllationol mFNAduring lh€ S'Phds6. (Hareford €l a/., l9SliHeretordel 

3/., 1982) s.d,u her wien yeast colls 6ro block€d during s'phase ihe major 

effect ls to tum ofl lhe transfiiprion machin€ry ol hisbn€ genes ralher rhan to 

deslabii2e th6 hislon€ mANAs (Lycan €l 3/., 1987) lt is shown thal wh€n 

some oher yeasl hisloDe geaes were axprcssod conslilutivaly signilicanl 

fuctuations we.e obs€rved in the lev6l ol hisionB mFNA du ng lhs cell cycle 

so rhat these mANAs clnunuously accumulaled in s-Phase (Lvcan er a/. 1987; 

Biorogical and blochemicat Paramel6.s ihat regulal€the hrstone 9en€ 

The several reouialory mschanisms conirolth€ timing and coordrnalion 

ol hinone gene expression. For examPle O non'cen cycle .eguhed histone 

gene expression can occur during allstag€sol d€vsloPm6nlln manv dllferent 

lisslest (ii) Hi$one gene expression can be resPonsive to c6ll signallng

a2tz F: cellclde ca.notal H4 G e fanscdp an 

mechanisms rhar derermine rhe initiation ol csll division tom quiescence or 

d€termine ths c€ssation ol prolilsralion during dilf€r€ntjation; (iii) sil€ncing ot 

histone gen6 expression is r€versible in difierentialed cells during 

tumorigenesis; (iv) l-lstone gen€ €xpr€ss on is coupl€d wth DNA synth€sis 

during S Phas€ ol cellcycle. 

Ihe biochemical @nrelcs lo. histone g€ne r€gulalion include (i) an 

ofdered serles otg€ne regulalory l€v€ls that permit conlinuous, stoichlomet 6 

changes in th€ synlhesis ol histone proteinsi (ii) irnctional redundancy (,1e., 

'laiils3fe" mechanisms) in gen€ r€gllatory levels to preve unconvolled 

expression ot hisbne genesj (iii) padi€iparion ot lhs elsments which control 

requlatory leve in coordinating hisiot gsne erpr€ssion wnh expression ot 

other S'phaso r€lat€d genesiand (iv) €tficlsnt m€ans by which to slence and 

aclivate mlliple potential gen6 regulalors, ard deinitive histone gen6 

o(pression as c6ll €nte. inrc rhe cell clcl6 or when €xit lrom th€ cell cycle . 

General prlnclplo3 ol transcripilon 

Genes arodlvided irto lhre€ cLasses according 10 the types olpromol€f 

lhey have and €ach class ot gens is transcibed with a diferent RNA 

polymerase. Ribosomal FNA is lrans.ribed by Pol I locat6d in lhe nucleoplasm 

and its relativ€ acl vity is 50 - 70%. Messenggr RNA (mBNA) is vanscribed by 

Poi ll located in nuc ooplasm, with a rslative activily ot 20 - 407. and lransler 

FNA (IFNA) is transcribed by RNA Pol ltt, slso localed in nucleoplasm, wilh a 

relatve activity ol 10%. Inilialion by RNA Pol ll can bs sponsored in 'nany 

d tterent ways. To nitiaie transcriplon, nNA poymerases n€ed som€

A2i2F: Cettcdt cotubtot e@IrNiotid 

accessory lacrors. In eukaryotes it may be th6 accessory faclors rather uian 

lhs polym6rase enzymes themsolves that are princlpaly reqlired for 

fecognlzing th€ oNA sequenc€ component of rh6 promoter iniiialon site. The 

common iearirre is that transcription tacrors bind ro the sequence elemenls 

concenlrated upslream ot rhe ransciprion start poinr. Facrors binding ro DNA 

Iom a complex in which protsin-prot€in interactions aro imponad RNA 

polymeras€ binds as a part oi rhis cohplex and ihen inilialLon can slad at 

p.omor€r sgquencss which are fequ r€d for initiation. anorhor rype of site is 

.eqlired which enhances th€ inleraclion of such sequence caled 

enhancer.The c€mponent ol enhancer resemble those of lhe promoler which 

@nsist ol a variery ot orher elements. FNA Pol ll r .tlires some other 

aclessory iactors lo nitiale lranscrlpiion. The TATA box re atsd iactor which 

bi.ds to TATA bo( s r€qunsd to bind poym€.ase, TFllo, TFIIA TF lB and 

TFllE. Since TFIIO binds dnectly to rhe TATA box and other lactors blndtolhe 

Gsemblies complex and rhese laclors may be pan ol rhe rranscrip[on 

apparatus which is imporlant lor assembling a complex ln which RNA 

polym€raso is able ro i.itiare lhe lranscription, 

The pfincipl€s operal ng rranscriptional regulallon ol prokaryolc (Jacob 

and Monod, 1961; Glben and tMulter-Hill, 1966: Emm€r er a/., 1970) and 

eukaryoric viral gsnes oooz€, 1981) hav€ bsen frmly esrablished. The inst 

promoler ONA elements ol eukaryotic g€nes were idenlified by sequence- 

alignmenG (Brealhnach and Chambon, 1981) and mulallonal analyses 

(Grossch€dlsnd Sinstiel, 1980; McKnighl6nd Kingsbury, 19S2) oilhB regon

,rn F: cat o/.t. co.not ot H4 cnre f@nq@ 

mmediately upsveam ol the mFNA stan-sito Tj an and coLlaborato6 identitied 

lhe lirst ceilu lfansciplional aciivaiors with seqlence_speciic oNA binding 

actuny incllding Sp1 (Dynan and liian, 1943i Kadonaga a.d Tjian, 1986i 

Br€gs er a/., 1986r Kaddasa sl a/., l9a7r Pugh and ]lian 1990) and 

cTFlNF-1 (Fossnlsld a.d Kelly, 1946i Jon€s €r a/ 19a7)- subs€quently' 

smiar trans.adivating lactors w6r6 lsolsled jn many oiher rabo.ator6s 

(.eviewed in cufian a.d Ffanza, 1988i Johnson and McKnighl' 1989i 

Kadonagaand Tiian, 1986iStruhl, 1987i Mitchsland Tiian' 1989i Llschef and 

Eisennan, 1990 a and 19sob; wing€nd€r, 1990; zitt, 199o) 

D€ rovo synlh€6ls ol human hlslone mFNA ttanscripts 

T6nsc plion olriistone genss l€diiionatly has b€en studied duing lho 

cel cycle wth severaldisti.cl synchronization proc€dures thal €n.ich l0r cells 

ar deined stages dling each round oi prolireralion bv lsing chemicalv 

indlced blockadss, separalion prolocols based on phvslcal difiersnc€s 

between cells, or grofih laclor and nuried deprivalion techniques These 

merhods may penurb physiological coolrol mechanism ro vtious degrees 

and sludying codrol ol hisbn€ g€n6s duting s phase wft any or lh€se 

rechniques ls slbjecl 10 mulrlplo l€chnical consideralions lhat qualit lhe 

i.rerpr€ralon ot resuts. For examPl€, call cyco studies on hslono gene 

reguldon mayb6inlluenced bythe ext€.ito which th€ cellol6xpe mentat'on 

displays propenies ol the trans,omed PhgnotyPe (lto ela/. 1989i Horlhu6 et 

a/., 1990). In this study, we used lh6 Hela s3 c€ll line; this cell tvpe is 

polyploid and has been de.ived lrom c€tuicat carsnoma cells Although rhe 

p 

15

Ntz F: Ce Cwte Contd d 114 Gm tanscbnon 

ability to srop cell proliferation is compromis€d in HsLa s3 cells, thes€ cels 

maintaina reqlifementlof cellcycl€ conirolled expression of histone genes in 

conjunction wnh ong.ing ONA synlhasB during S ph46 progr€ssion (s€€ 

Fig. 2). Thus, HeLa 53 cells .epresent a slilabb model lor sludying conlrol ol 

hisrone g€n6 rranscriprion at lhe G1/s pha$ boundary. 

Transcription ol human histon€ g6nes in HeLa s3 ceruical ca.c noma 

c€lls occurs constitltively during lh€ collcycl€, and a iransient and coordinato 

increaso (approximably 2 to 3-fold) in tanscription rales occurs at ths Gl/s 

phase boundary (Baumbach er a/., 1947)- Similady, ranscription is inc.eased 

sev€rauold towards s'pha* when c€lls enter lh€ cell clde lrom a qu€scenl 

slare (Go) (wighr ei a/., 1992). Histong g8ne t an$npton is qu4tilatively and 

coordinar€ly downregulared at lh€ induc€d c€ssalion ot proliferalion and onsel 

o, diflereniaion in human HL60 iromy€loctnic bukBmia cels, as 

demonslrated by rh€ shltdown of rotal H4, H2B and H1 hislonB g6n€ 

transc.iprion and undolectable le!€ls ot individual H4 (Fo10a) snd Hl h stone 

(FNc16)genes (colrart el a/., 19aa; Shalhoub etal, tgagr stein €! a/., 1989). 

Similar obsetoations have been nad€ dldng induced differenriation ot mouse 

3T3-L1 adipocttes (BorleLl er a/., 1992) and rat myoblasls (Bird 6a a/., 19851 

Larso. 3t a/., 1989), as well as dudng lh8 developmentar sequence ot 

difierentiatng ral primary calva.ialosl€oblasts in cellcultlro syst€ms (Owen 6t 

a/., 1990a,b) (see Fig. 3). Apa.t lrom lhis, lluctuations in hislon€ gene 

lransc ption have been obseru€d when human CF-3 loreskin libroblasG 

swirch lrom an actively prolilerating mod€ lo a quiescenl state and vic6 ve.sa 

16

aziz E c.n aydo co"tu d Ha a@. nscddbn 

Fig.3. F€gularlon ol hlalon€ gene lrlnrcrlpuon during c€tt growth 

and dnef€nlb{on. organization ol the humsn hislone H4 gen6 

promor€rrcgulBbry €l6m6nb (silss l-rv) is ilusfared. the 

lransa.iplion taclors that exhiblt s€qu€ncaspgcitio inreractions 

wnh thoso do.nalG aa€ Indical€d during the S-phase of rhe catl 

clcle when th€ g€n€ t9 acuvgty traNcrib€d, 

'17

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NizF: CelC6t Cdtol ol H1G.N f@dDtle 

(wrighr eral., 1992). Thus, regularory pror€in/DNA inte€clions may existlhat 

aredependenr otrh€ cel groMh$ar€, and aro ralelimitingln lh6 prolitera on- 

speci'ic tansc.ipion ol he$ g6n€s, 

I€nsci ion €.tes have also b€€n measured pr€viously i4 several 

srrains ot rransggnic m'ce conraining vsrious rePorter genes lused lo the 

human H4-FO108 histone gene prohot€r (van W,jn€n et a/., 1991a). In 

general hislone gen6transc ption (as rBll€clsd by reponer geng Bxpr€sson) 

paralle s histon€ gen€ expression at lh€ mFNA level in several tissles ol lhg 

adull mous€, i. paniclla liver, kidn€y, sploen and thymus tanking 

in ordef of 

increasing reralive leveis ol e4rcssion). Because ol the tighl coupling 

beheen hisron€ gsne expression, oNA roPlicduon and cell prorile.ation, lhis 

indl€les Ihat histone gene transcription rafl€cts the level ol cell proilleration 

wfhn rh6so lissuos. Th]s suppons |h€ €xistence ol proiteraron-spBcllc cis- 

acting el€msnls nluencng h stone g€ne transcrpUon. 

High levsls ol reponer gene €xprsssion can be obsetusd in aduh braio 

desplte lhat histon€ mRNA lev€ls ars almosl b6low lh€ level ol deledio^. This 

tissue conla ns a small populalion ol cells with proliferativ€ potsntial (4,9. glial 

cslls), bul consists pimaily or non.prolil€tating neuronal cslls that by 

deiinrlon do noi require DNA r€pllcalion depsnd6nl histonos, olher than the 

lmiled quantity lhat rnay be n€cessary tor lormation of chroma n during ONA 

repair synthesis. These iindings cbany su996st that, frst, mulliple lwels ot 

hisbne gene .egllalion are op€ratve in lh6 iniad animal and, second, $al 

redundancy n conro mechan sms is lunctionally relaled lo lhe prevenron ol 

19

AzzF cett Crct. ContolatH4Gen ldn*tipttan 

abe(ant histone syd$esis. Therelore, is not slrictly necsssary to i.voks 

negative regulalory transcription lactors io ordsr to strlng€ntly conl.ol histono 

oornrogualion ol histone gen6 tanscriPtion as measu.€d bv reporter 

genes has been obserued du.ing muine hepalic development .etlecting lhs 

onset ol in v,!o qui€scen@ and ditlercn0ation (van wijnen el a/., 1991). These 

modilications in lranscrlption colr€spond lo similar d€cr€ases ln histon€ 

mBNA levels, This ind cates lhattfafscrlplonalregulation is a primary mod€ ot 

gene control dLring developmeni, Becaus€ transcriplion rales may or€ciy 

corelate wilh the presence ot ralo-limiing kansacting lactors, lhese nndings 

provide a i rst ind cal on for cell grofih relaled, developrnenla reguallon of 

hisrone gerj6 transcr ptron ractors 

Hisrone g€ne tanscription control by cls-.egulatory eremenrs 

This laboratory has been involved lor a long iime in the study ol rhe 

regLrlation oi hlstone gene expression th.ougholt th€ cell cycle oi cullLrred 

human cels.The h stone gene rspfes€.lan €xc€ll€nt modsl ior the study ol 

rumorogenesis or the genes which do€s not express inaLly h dlfferentaled 

state, bln rather lurned on and otl in a t'm6 dependent lashion. The frst 20o 

basepans upslream of fte histon€ Protein codi.g sequence has been 

desgnaled as lho proxlmal promoter, and sequences beyond that as he 

dista promotor ALthough lhsr€ is svidenc6 suggesllng lhat dislal y ocaled 

promoter elements are capable oi inllu€ncing rhe levol ol hisbne g€oe 

exprcssion (Krc€ge! er a/., 19a4, hese pulative distal regulatory eEmenls ar€ 

20

atz F: cdt c/.t. c.Dtd ot H4 G@ TansctiDtl@ 

dispeosible lor c€ll cycle conlrol of histone H4 gene tansciption (Ramsey- 

Ewing et a/., 1994), Theretore, in this study rhe tocus was on the histone 

proxma promol€r rsgion otthe H4-FOICA hsbne gen€, which spans several 

seqlence morrs consetued a_rnong hisron€ genes (van wrjn€n el a/., 19asi 

wells and McBrid9: 1989) 

rt has boen pr€vrousry shown in lhis laboratory rhat lhe TATA-box was 

the lksr lranscriptionar erehe.r which ls tlntionalty involved in human histone 

gene lranscr ption, becalselhis soqu€nc€ is sulficlent tor mediarng synrhesis 

or coiiect y nital€d ranscripts,n /,to ol th€ human ft4.FO1CE hislonB gene 

(sie(a er a/., 1983)- sludies using lho anatogous H4 histon€ gene H!4a 

conlimed thes€ tindings and provlded indicaions lor auxiliary sequences 

capable ol s mllating l, v/lro transcrlption (Hanly el a/., 1985), and lurrher t 

has been shown rhar rhe H4-Fo108 hlstong gene has simit additio.al 

stimuratory seqlences (Wriqhr er ar,, 1992). Thes6 and ohef cis,acting 

elemenF identitied by ,n v,lro t anscipiion assays are discussed in he nexr 

chapters. and felared to lhe pdetn/ONA .teractions occuring at these stes 

/n v/vo ana ysis ot a series ot d6te on mtiranls spanning ih€ 5 ttanktng 

reqionolthe H4,Fol0ahistone gene has rovealed that the region inmedrarely 

upsream or the TATA.box is requir€d lor erpression of this gene (Kroegef el 

a/., 1944. ln rhese and parallel studi€s (Helms 6l a/., 1987). ndicalons were 

aso lound for dislaly locared e ements capable oi influencng Bxpression. 

Flnhenore, p.olen/DNA interactons in vivo have been established in the 

21

a2i2 F cdt c$t. connd d H4 Gm Tdnscionon 

proximar promorer r€gions ol rhe human H4-Fo108 (Paurier at,, 1987) and H3- 

sT519 (Paul er a/., 19ag) histone gsnes wi|h genomic oNasel tootprinung 

assays (using solatBd nlcei) and g€nomc oMs tingeFdnling assays (lsins 

i.ract celrs). ao|h 9en6s appear to hav€ two protein/DNA interacrion domains, 

desiqnared H4-sire I, H4-sire rr, Hlsir6 | .nd H3-sn6 rr, r6spediv€1y. These 

domaids rep.ese c.ucial cis acling cortrol elements, and are lo.ated wilhin 

the lnst 0.2 kb lpslfeam of lhe coding region. The number ol pfotein/ONA 

co.iacts at glanlne.esdles detecled wilhin these stes is consisrent with 

each doman infer.cling wth more than one ONA binding prc\en in vivo. A 

major local poinr ol rhe studies prese €d here is related to lhe lunctionar 

cha.acterization of rhe tacrors binding ro H4-sit€ lr. 

Transcriplional modsls ror hlston€ gan€ 169ulatlon 

ar rh6 ons6r or rhis srudy (JUy 1992), rhe initiar concgpts for 

lnderstanding huhan histone H4 gene l.ansc.iprion had bo€n eslabllshed 

(sreinera/, 1994iHdntz, r99liOstey 6ta/., 1991). The study d€scib€d 'n 

this thesis rep.es€nts an elpansion ol previous studies on histone gene 

regllarion (A.J. van wijnen, 1991, Ph.D. rh€sis, Departmenr ot c6ll Bioogy, 

unNersiry ol lvassachuserts, 19s1: A.L. Famsey-Ewing, Ph.o. thesis, 

Oepadmed ol hmunoloqy and M€dic€l Microbiology, Universily ol Flo.ida, 

1991). h rhese srudies, seral H4.sir6 lr rranscndio. taclors we.e 

characterized, and s€veral nucleotides implicatsd in cell cycle contro! were 

ldentiied (s6€ F gs. 3, 4 a.d 5). How6v6r, limitod intormallon was avallable on 

the specitic conrriburions of H4-sr€ ll bnding protsins to H4 gene 

22

A* F: Ca|| Cycle Cbtd at H4 cere rran*iotian 

Flg.4. schemallc repr€senlatlon ot transcription tactor 

htoracrions wilh hlslode gsrle promoter etements durtng 

ths cell cycle and to otvhg ihe onsel ot dlferentiaiion. The 

figr,fe shows rhe iour p nclpal regLrlatofy €lem€nis of a ceu 

cycrs-regulated histon€ g9n6 (t, , t, and tv)_ Modilicarions in 

the interaclions oia seriss of ubiquirous (a9., Ap-1, Ap-2, NFl, 

ATF, SP-1, TF2) and c€ll cycl€regutatory (€.9., tRF, cdc2, o/clin 

A, FB) raclors ar6 indicar€d duing c6llcycl6 p.ogr€ssion and 

posrproltemtvely in difierenriated ce[s. The upregutalon of 

hisron€ gene ranscdprion at lhe G1ls phase ransirion poinr tn 

lhe cel c)cle is mediat6d by phosphoryaiion dependent 

interaclions of IFF and H|NF-D (cdc2, cyctin A, RB) at the si!6 n 

cell cycle.regularory etom6nr Downregutation ol hjsrone gene 

tansc.iption at lh€ prolif€ration/diflgrentia on transirion poinr is 

runclionally related loloss ot lranscriplion tactor bindtng at sites 

ll. Occupancy of siro lby HiNF-4, SPl, afd ATF is constitutive tn 

proliferaiiru, quiescent, and differontialedcelts. 

23

o 

rl$orrE GEIE FEEI{IXU 

=; 

FrN -!r 

9l@"tr1'*r 

@f (ffi) 

iif- 

\

lLF dq.!:M! dtu?!r*dp6- c*"ntkryy,* 

Flg.5. 

Organtzallon ot th€ H4 gene proxtmat promotor: H4-site I 

inieracts wih ai teast |l|re€ ctassos ol trEnscriflion tacrors hst 

are dll'€.e.!a[y r€gujatsd €nd bas€d on th€,rjdBnr,hss Eppear ro 

operate in difter€nt c€tt signsJlng pahways. Facior HiNF_M 

wl|ich rec€nty has b€€n id€nrifsd as fle oncsgeDs-gncoded 

protejn lRF.? and $6 rslatod turnor-suppressor prorern rBF-1 

recognizE th6 sam9 elomsnt (M,box) tocated in lhe mo$ d6ral 

segment ot H4,Siro j HiNF-P/H4-TF2 is a publiv€ H4 gene 

sp€cilic tactor which binds prcxjmatto HiNF-M/|FF.? but its sire 

(P'box) ovortaps in part wilh th6 M.box; HiNF-D r6pr€sen6 a 

muftjcornpon€nt p.otsln cod€ining th€ clclin dependent kinase 

CDC2, cyctin A snd an BB rclarod proiein, and HiNF.D inte€cts 

with th6 enlif€ Site |, inctuding the reglon ctoss to the IATA bo( 

locarod in lh6 most pronmal segrnent ot Sit6 l,

!e 

3lhllrllon c.I cycl. control 

aa a at- 

."q,"."",gtff ocf ocPgg'clggarac'cn cltrclc,cccAAf 'a 

nu l lnor ll cnox llP-Dor I lrarabor | -t 

--r 

ICTGGICdIAYAoiCNCTATAIC^

A2t2 F Cer Ctrte cannd d tlr Gde r@apt'a 

ransq pllonal conirol during lhe @ll cycl€- lh6 liEt obleciiv6 ol this study was 

lo assBss rhe i.dividla contributions ol Hlstone Nuclear Factors (HiNF's) 

interacling wilh H4-site ll ro the level ol transc ion. S€condlv we sludLed 

involvem€nr ot these HiNFs in $e l€mporal .egulation ol histone gene 

rranscriplion d! ng th€ cellcycle. 

Whil€ lhis work was in operalion a.umber ol oth6r labo€lories hav6 

also ini at€d and/or codlnued sludios on histon€ g€m otganizaton and 

expresson, For examPle, s€v€ral hlsion6 g6n€s wars recenlly clon€d and 

charact€rizod in many ditlerBnl sp€cies (Albig d a/ , 1991 ; Oobn€r er a/.' 1991 i 

Drab€nt et a/, 1991, 1993r Nakayama €.d S€roguchi,, 1991i Oonq er €/- 

1994). l. addton, lho €rPrBssion pattsrns ol disrinct hislone geres has been 

analyzod d! ng diflgr€.lalion (Koppel €l a/., 1994i lvanova er 3/ l994ai 

Budeind s? at., rs4i albig el a/., 1993i Col'ad er a/., 1992) or i6 ths c€ll cycle 

(Famsey el a, ., 19o5; Brocard el a/., 1994r H€ints. 1991i wolle and GriFes, 

1993; choi and chas., 1993i Kremer and Kis er, 1992i Eilers ea a/' 1994i 

sreinel€/. 1994ivanden E el a/., 1994; Fams€y el a/ , 1994; Boute.ia d a/., 

1993j gonner er a/., 1993; Lim and cha€., 19924 Gom€z-coadrado €t a/ 

1992). Fudhefinor€, a nLrmb€r ol cis-acling 6lemer't hsve been described io' 

many hisrone qenes (Hatch and aonn6r., 1995; lvanova er 3/ 1994b1 

Khochbin and t?wrence, 1994; Ohshig€ el a/, 1993r Khochbin and wolre, 

1993t Hlnkley and Perry, 1992i G mes€la/,19S2: ChOi and chae 1991; 

Wolto and Grlmes, 1991; van winon €13/, 199la b,c) and m€ cognate vans' 

acti.g iactors have also been lde.lili€d (Madinelli and H€inlz 1994i van 

w',nenerar 1904. Sh€@ood el a,., 1993, van W',qnen el a/ 1992abc 1991i 

27

A.E F: Cet CEl. Cotuot ot H4 t3.M f@btbn 

Huh er at, 1991 I Palb €l a/,, 1S). D$pil6 lhb $densivo s€l of dala oblai.ed 

by difierent hboralodes sp6daliznE on hlslorE genl3, hduding lh€ stein 

labo.atory, the work pr€s6nt6d ln lhis th€sb rspresants th6 frrst 6vld6nc6 tof 

rhe imponanco ot rhs co.nbin€d adn ni€s ol rruhbl€ lactoG in the rcgulation ot 

hisrone gene 6xprosion d!.ing rhe c€! q/d., 

a

MATERIALSAND METHODS

Crct C@nd d H. GNf,'/MAi@ 

'2izE:Ce 

Materials and Methods 

MUTAGENESIS OF SITE II BYi'OLECULAR CLONING 

lh€ hisron€ H4 gens promorg (F19. 5) us€d in this sludy is detivsd 

lrom plasmad FOIOa which was cloned in this laborarory by Si6na er at 

(1983). The 0.2 kb prcmoter ol d|e H4-Fo108 gene was llsed lo lhe caT 

g€ne by or. Anna Aams€y-Ewing (Fam36y'Ewng €t a/., 1994) Th€ res!fting 

4.55 kb iusion construct was desiEnal€d pFOICaCAT; this plasmid repf6s€nts 

rh6 witd type conEttuct, which was used hroughod ltE mutag€nees studi€s' 

Muranr p.omolsr c!.rsLucls co.tanhg th6 c T rcpo.ler 9en6 w€.€ Pr6pded 

by an origonucl€oridE cassetle apprcachs (Frgs, 5,6 a.d 7) . 

Plasmd us€d in lhs studY contalns the H4 proximaL prornol€f ol lh€ 

pFOlOa gen€ and spans nuclsolidos (nt) '240 to _38 (rslativs ro $e 

a

tzr2 F c.a ctdQ cnanol ot tk GM fntsctl.dd, 

Flg.6. w dryp€ and mutant 84 promotor ste I sequ€nc$. 

Subsrftrrkn, ins€nion and d€t€titon muratioos rv€re inroduc€d 

into Sire ! within the cooiaxt of ih6 H4 proxm& promor€r lo 

eslablish the r€qrlrement tc,r bindhg cd HINF s io site L capibt 

|enors and dash€s show th€ witd type s€qu6n@, sma tenels 

show substitutions or Ins€dlons and da.k lni.angtss show the 

deletions,The €ft€ct ol sach rhutalion on the binding ot HiNF-p, 

HiNF-M and HiNF-O ts indicst€d In the cotumns to the rioht, 

30

9C| 

-= 

= 

, 

o:li?eiP 

:< 

:::? 

il il 

7E2H9FFFgE6

a2t2 F: call cyds contol d u4 G@ f,wiPl/ol 

Fig,7. sch6hatic dlagnm showlng the ctonlng procedore tor H4 

fiutant promoler conslrucis. (A) Ths top $rsnd oi lhe t€st 

oiigo and bottorn strand ol rhe adaPtor oligo w€€ each 

phosphorylatsd and ann€alBd ,rte trayod ends w8re frll€d in 

wth Krenow and digested wirh Psl L (a) pFo10€ sas d6av€d 

wirh Pst | / Nae I and lh6 oligonud€otide casselt€ trom (A) was 

rigated into ih€ Pst | / Na€ | sires ot H4 veclor puc19 which 

r6sults in rhe inlermedialr dorc A. (C) pFOl0€ CAT was c{€av€d 

with Pst I ro isolar€ lhe caT tragment and ligaled into linearlz€d 

inbem€diate clono A, rssulting in site ll H4 mutant promoEr CAI 

conslrucr (o). 

32

s.tudi. dhcrm or do"rn! 

-l 

/4 

I 

I 

I 

_-r--# 

\ 

\ 

*// 

/',r 'q 

I

ND F: C.ll C\cte Cdtuot ol l1a GM I@Eaoti@ 

translalional stan codon; lh€ mFNA cap sit€ is localed at nt _3o) (van Wjnen et 

a/,, 1991). This conslrlci lvas frst dig€sted with th€ r€striction enzym€ Nae I 

ioltowsd by ethanol prgopnaiion. Tho dig€sled DNA was then cut wilh Pst I to 

relsas€ rhe wild lype H4 site ll seqlonc6 which spans nt_93 lo .3a 

lsolatlon ol DNA lragmEnts 

fte large Nael/Psll lragment (containing th€ remainder oi lhs H4 

prcmor€r fused to puclg) was isolaled Lom 1% agaros€ gel a.d eluled in 

dialysis rubng (moecular weighr cul-oil of 12,000 - 14,000). fte lLrbing was 

prcparcd as descibed prcviously (rlranialis el ar, 1982)- The tubing was cd 

inro piec€s of con!€nienr lengrh (10 - 20 cm) and boiled for 10 minutes in 2% 

(w/v) sodium b calbonale and 1 mM EDTA (ethylons diamine tetra acetic acid) 

solulon. The tlbes were wash6d ssveraltim€s wilh dslilled H20 and lhen 

boilad agBin ior t0 minures in 1 mM EoTA (pH 8.0). The tubss wsre kept 

storedar4'cin a large volum€ ot TE bufe. (10 mM Tis.HCl, 1 ml"{ EOTA, pH 

8.0). Thrclghout ths procedLr€, lh€ dialysis tublng was hand ed wrh 

Th€ srltsd oNA was purilied by passing the eluate through ElutiprM 

min-collmns (Schlecher and Schuell) according to the ma.ufacturers 

suggestions- In bri€i, Elulips were cut at the botton and wash€d with 2 ml high 

salt bufer 0.0 M Nacl, 20 mM Tris-Hct, pH 7-3-7.5, 1.0 mM EDIA) using a 

syfng€, subs€qlenrly the syinge was washed wilh 3-5 rnlof low sall blfier 

(0.2M NaCl20 mM Tris-HCl, pH 7.3-7.5, 1.0 mM EO'A). A iiher was lixed lo 

lhe Elutp ro r€move gel padicles and lhe DNA sampls was passed hrough 

34

aziz F: cel crd. cotuot d 81 cd. fnBdidlon 

the tip wrh rhe l ltor. The oNA (2.9 kb) was rccovgrcd by ethanolprocipltalion 

and used jn subsequenr cloning experimen$ as lhe v€clor lragnenl- 

synrherio oNA l.agd€nrs conraining speciric muraio.s in the binding 

sires ol disrincr H4-Site ll binding prot€ins (HiNF's) were design€d. Ihese 

oligonucleoridas span ihe sam6 Nael/Pstl sequenc€s as removsd irom lhe 

orgina pFPr conslruct (see above) and wer€ rcins€ded in rhe Nael/Pstsit6s 

ot the 2.9 kb vector r.agme by unidjrediodal cloning. si.gle-straded 

oligonucl€olid€s w€rs ftst traclionared by slectrophoresis in adonaluring 12% 

polyacrylamid€ ger(S€q!ag€[M), ellred by dirlusion 6nd recoverad by ethanol 

The murant promoter was designed in such a way thal lhe test 

oligonucleotides (lop strandi nt -931o -53 ol the sense slrand) hyb.idizes wilh 

rh6 u.ive.sal adaptor AOP.3 (bonom sl.andi nl $7 to '3€ oi lhe anli sense 

strand plus 12 nucreotlde oi linker sequ€nco) (Fig.7). Bolh rhe bp and bonom 

oligonucleotdes were each phosphorylat€d with T4 kinas€ and annealed. The 

sing e-ska.ded ov€rhangs oflh6 hybridiz6d oligonucl€otides wer€ I lled in wilh 

Kenow polymerase (New England Biolabs) and th6n cd wnh Psi L Th€ 

ponion span.inE nt -93 1o -38 which has a 5 blunl 6nd 3 P$ I €nd was 

separated lrom the shon rnk6r sequenc6 by ethanol precipital on, and clo.sd 

inro rhe Na€l/Pstl srr€s oI lh6 pFP-l v€ctor fragmont. Th€.€sul ng s€i ol 

prasmids in which €ach oi rh€ cloned mutant H4 sita ll lragmenls is iused lo 

lhe remainder ol rhe H4 pfomorer, will b€ rslored to as intermediare hltanl 

H4 promoler constructs. Ttu lhird st€p in the proc€dure was to prepare lh€ 

35

aziz F: call cr'cl. cqttnt at H4 Gqe 7ldns.detian 

mulant H4 promoter/CAT tusion construcl- To engineer fte cAT repoler 

gene in ifo ol rhe mulant promot€r sequsnces, lhe inlemedialo conslruds 

were dig€st€d wilh Psl I, dephosphorylaied as d€scribed below and puriied 

Th€ llneariz€d inrermediate plasmids w€r€ lr€aled with calf lnlestnal 

phosphataso lo remove lhe 5 teminal phosphale group. This step reduces 

self ligation dlring the lgauon reaction and w€s Pedormed as described 

prevjously (Maniatis el a/., 1982). In biet, lhe working butfer lor the snzym€ 

conbins 500 mM Tis Hcl (pH 8.5), i mM EDTA and 1 mM spenidlne, and 

's 

prcparcd irom a 10 x bufter obtained lrom lhe supplisr (Boehringer 

Mannheim). Ihe desied ONA fraqmenl to be d€PhosPhorylated was mrxed 

wilh 0.01 unil per pmol 5'ends at 37'C tor 30 minules. After the li.st 

inclbaton, a second allqlol of th€ 6nzyme was added a.d lhe sample 

rncubaled tor an additional30 minutes at 50'c. The reaction was stopped by 

adding 0.1 [4 EGTA (elhyleneglyclfbis(p-aminoethyletre.) N,N,N',N:telra 

acetic acid) and lhe enzyme was h€at- nactivaled tor 30 mlnlies at 65'c. Ths 

DNA was €nraded twics wnh an equal volume of phenol/chlorctom/isoamyl 

atcoholi'25:24:1vlv/v)(saru€led wirh 10 mM Trls/Hcl, pH 8.0 and 1.0 mM 

EDTA) afd recove.qd by ethanolprecalat on. 

The nsert lragm€nt was prepafed by digssling pFOloEcAT wlh Psl I 

ro rerease lhe 1 7 kb Pstl/Psrr kagmenl spanning lhe chlo.amphenicol 

36

a2lz F: celt cyd. c.ntol d H4 G@. fansqbtbn 

acevlt.ansferas€ (CAT) g€n6. Thls CAT lragm6 was inserted inlo the 

irnermediate constructs to yield th6linal mutant H4 promoler/cAT conslructs- 

Ligation reacrions we.o set up by adding lhe vector and Lnsert f.agmenls d a 

rnolar ralio oi 1r5 (vectoiinsed) tor cofstruction ol ihe inlerrnsdiate plasm ds 

and 1:3 tor ligalions lnvolving rh€ cAT fragment and the intermediale mliant 

clnslrucl- Feaction were iniliated by addition ol 1 unil of T4 ONA ligase in a 20 

rl reacrion vollme which includes 1 X ligase butfer (50 mM HCIpH 7.6, 10 mM 

Mgc2, 10 mM DTT (dthlothretol), 1 mlM ATP,50 pglml BsA, bovlne serlm 

alblmin). Each Ligation mixtu.e conrained a totalof r00 n9 oNA. To check ior 

lhe p.esence ot recombinant DNA Lagmenr, as weli as lhe efecliveness of the 

ligalion reaclion, 5 rlol ligalion mixtur€ was loaded on a 1% agarose gel. 

oNA rransformallon inro E col/ baclerla 

Frepa.arbn ol compelenl callr 

A l0 ml culrlre of e co, DHSd was grow. wirhout ampicillin by 

inclbaron overnighl ln a shaking inclbator. A sampls oi the culture (2 

ml) wasinoculaied nro 1@m oiLB medium in a side-arm flask wilhout 

anpicillin. To monito. bacleial Eowth, tr€ optical densily ol the culture 

was determined d 30 minur€ ideruals until lh€ c1rllure reached a 

densry of 0.400 A@. Bact€ria cultufes we.e vansl€fied into lwo 50 rnl 

conicsllub€s, and chill€d on lc€ lor 10 minules. Bacrerial pellers woro 

collected by c€nritugalion ar 2!0o rpm for 5 minutes at 4'c in an lEc 

rotor. Each p€llel was rssusp€odsd in 10 ml ol CaCl2 bufier (60 mM 

CaCl,, 1s% glyc€rc|, 10 mM Pipes) and again pe leled by

tniz F: cen c\tto contat ot H4 G@ I@.ttott@ 

cenlrilugation. The bacteria wsrs resuspended in 10 ml ol CaCl2 bulfer 

per rub€ and chilled on ic€ tor 30 minLtes. compot€nt c€lls were 

co eded by cenr lugation as dessbod above and rosLrspendsd n 2 

mloicacl, bulier pertub€. Aliquors (10o,,1) ol the compstent bacteia 

wer€ transleiied into approximatsly 40 Eppendort tubes (1.5 ml) and 

f.ozen down in liquid nit ogen al -70'C. 

Baclerial translormatlon 

Ligalon rnixtures wer€ lransiormed into DHsd by slendard merhods 

(Manlatis el a/. 1942).In bri€t, approximately 100 rl ol competent cells 

were lhawed on ice and 25 ng of plasmid DNA c 40.80 ng ol igated 

oNA mixt!rc was mixed g€nlly and incubaled on ice ior 30 minutes. 

The tubes were then transteiied ro a wat€r bath s€l at 42 0'c for 

exactly 90 second lo heat-shock lhe cells. Subsequently, 1 ml LB 

medilmwirhoLt ampicillin was added and lhe tubes were incubaled al 

37'c tor 30 minlrtes whte shaking. For bacteria translormed with 

igation mxrures, tlbes wer€ cenkfuged lo. 10 second 10 colect lha 

pellgls These pe leG were roslspended in 1 / 

10 ol ihe oflg nal volume, 

and ih€ resultlng bacterial suspensions were sprsad on ampicilln 

(anp) conhini.g LB agar plares. For the cornrol tanstornations w'th 

unrr6at6o c.rc-ldr plasm,d oNA (whEh translorns fud' morP 

elilcienrly) 50- i 0o p oi rhe or ginal ranslormallon rnixur€ was spread 

overamp/LB ag plares. Plates were incubated in an invened position 

ar 37'c lor 16 ro 20 hr, lesulring in bacterial colonies wilh a diamete. ol 

38

A2iz F Cett cycta contat al Hl Gene I'ansciPtiah 

0.5 to 1.0 mnr. Clones $€f€ eith€t Picked and dirsctly Prccessed ior 

min|pbsrnd prcparadons, or itanslgr€d b a solid suppo'l 

(nilrocellulose membran€ discs) and lysed in situ as a pr€Ude lo 

colony hybridizaton rcp.€ss s a laPid and otlectiv€ technique lo 

rsorato and id€nfry rccomblnant plasmids grown on LBlanljbiotic plal6 

by lransf€r of bacierialcolonieg lo nilrocsllulos€ inembranes (Hanahan 

ard M6s€lson, 1943). Alt matsials 4d solutions were aLnoclaved 

beto.e uss. Nitrocellulose m€mbranes were wrapp€d in duminum foil 

and auloclaved ior 15 cyclos. Ligstion mixturas wsrs lransformed inlo 

DHsd and spread on LB/anlibiolic agar plal€s. Th€ pLates w6rs 

incubated overnig al37'C i.rntilthe colonies were grown 0.5 mm in 

diameter and removed from rhe incubabr' 

Nitrocellulose membran€ discs were wotred using lorceps by placing it 

over LB/anllboric agff plates wLthoul colonles Ths discs wer€ lilt6d 

and gently placed over a second aqar pLat. containing baclsriar 

colonies. fte membanes wers slighlly pressed to albw rhe colonies to 

adhsrc lo th€ d scs. The postof ol lh€ membrane drscs were ma {eo 

by pu.clut lng aneedlohrough bothlhe membranaand the agar' Ths 

is idporlant tor orientng lhe membrane and lutu.e idertification ol lhe 

positavB colonles. The memb.ane which now conlains a replica ol lhe 

original bacleria colonleswas removed and temporarily slored onto the 

39

,n|z F: celt C@to Conn , ot H1 Gene f@qtdi@ 

agar plate used lor the initial wening ot the mgmbran€i the m€mbrane is 

positioned race-up' (re, trrs bacl€rial sids is on lop) Ihe oiginal 

plales w€re saved and incubated ove.n ght at room temPerature Io 

regain bact€rial colonies. 

Th€ membranes containing bacterial colonies were lysed dnedly on lhg 

membrane disc by placidg il onlo whahan 3MM paper lor 5 minules, 

whch was pre.soaked in 0.5 N4 NaOH solution. The m€mbrane disc 

was n€utralizsd for 3 to 5 ninltes by placing it onlo another 3MM 

Whahan sheet, which was saturated wilh a solution ol 1 M Tris/Hcl 

{pH 7.5), The membrans discs were ina$ated tor an additonal 5 

minures on anoth€r set ol3M[4 whahan sheets, saiuraled with 0.5 M 

Tris-HCl pH 7 5 and 1.25 i,,l Nscl, and th6 discs wore air drled. Th€ 

DNA from th€ Lysed bacl€.ia was lired tothe membrane by Placlng the 

disk under the UV,ighl tor 1 minule, and stored al 4'C in a sealed 

plaslic bag. DNA {ragments spanning the inse.l was radiolabeled wilh 

rhe PrimentrM Gndom prim6d oligolabeling kit (Stratagene) as 

dessbsd by ihe manulaclLrr€r. lvlemblane discs wers pfe-hJ7bddized ln 

25 ml of pre-hyb.idization solution containinq 5 X ssc (5 x ssc was 

obhinsd from a 20 x ssc stock which contains 0.3 M sodium cltrale 

and3 M sodium chloride, pH 7.25),5 x Denhaidl s soluion (lrom a 100 

x srock conla.i.g 2% polyvinylpyrol done, 2% licol, 27o bovine serLrm 

arblmin), O.O5 i.,l sodium phosphate bufier [pH 67i a 1 rr,4 slock' 

solution ol phosphale buftff pH 6.7 is oblained by mixing 6€5 ml ol 1rM 

40

E2 F. cen crct. Connat al H4 c*e Tansctipnon 

NaH2PO.6nd 315 ml ol 1M NatlPo.l' !o !g/ml denaturcd slmon 

sperm oNA and 500,6 lormald6hy@. Th8 m6mbran6s wers incubated 

wirholr radiolabeLed DNA in pr€.hybridizalion sollllon tor o.e rrour 3l 

65'c. This solullon was renov€d lrom lho cylind€r and .eplac€d by 3_4 

ml hybridi2alion solulion [5 x sSC, 5 x Denhardi's soiulion,002 M 

phosphare bufer. pH 6.71, gl)% to,maldehyd€ Badio'lab€led DNA 

Prcbe (2x1@ cpm/ml) was boiled inth6 P€sence oi 100,rglmlsahon 

spefm oNAlor l0 minltes and add6d tothe hybridyzal on mlxtlre 'rhe 

membranes w€re hybridized lor 20. 24 hours at 42'C Subsequentlv, 

lhe memb.anes were rinsed twice with a wash_solution (6 x scc and 

0.1% sodium dodecfl sulphat€ lsDsl) at 65'c Each rinse was 

periormsd in a shaking wale. bath foi 30 minutes. The membrane was 

washecl o.ce wllh 2 x SSC 0.1% SDS al 65 C tor 15 minules. 

Membranes w6r€ died on a lilt€r Pap€r and subjed to €utoradiographv 

by ovemight exposure at -70'C using xiay f|m (Kod.k X_OMAI aR, 

Easrman Kodak). The onginal aga. plates on trt'ich lho coronres were 

qrown. wefe alig.ed lo lhe X-rsy li m for idenlilication and processing ol 

positve clones {see Fig.8) 

Small sc.le isolation orplasmid DNA 

Prasmid oNA lrom 2 ml cultures was prepared bv th€ boiling melhod as 

described previously (Hohes and Auigl€y, 1941). Singl€ colonies were 

lnoculared in 2 ml LB medum and incubated at 37'C ovornight, Alqlols ol 

each cullure (15 ml) were ransle(€d into 1.5 ml EpPendod Ubes and

A2t2 F: Con Cat t C@nntottla6wlruEdEd@ 

NNhLadnentds 

Fig. & colony hybddarton sq€€olng ol lranstorm€d bacteria. 

Et6ck spots indicaiB the prE€nco ol po€itve CAT clon6 shich 

w€r€ lal€r isolaied by aligning tho nilrocellulose m€mbran6lo lh€ 

agar plaies conlainlng lho orlgnal colonies 63 dsscdbed in 

"fiitstsdal and Melhods". (A) repres€nts th6 wlldtvp€ TM-3 snd 

(8, c, snd D) rcprssenls lho niltanI oT-9, lN91o and suB-lj 

42

Colony hybridization 

4a 

aa 

a 

a

AzE F: Cel Cvcle Cotuat at H4 eqe ftanscttoltan 

c€nrriluged lor 30 second at 14,000 lpm (Eppendori microiLrge) to colect the 

bacterial pellet. The supernalant was discsrdod and the ce|l p€llet was lysed in 

150 /r or STEI bufer (50 mM Tis HCr pH 8-0, 50 mM EDTA, a% sucrose, 5% 

Tdton). The conrenb ol the rub€ w6r6 boited exactly tor @ seco.ds a.d the 

rub€s wer€ canrrituged for 15 minutss al room temperarure st 14,000 rpm io 

collect tlra pellet. The pellgt which r6pr€sents bacterial debris co.taining 

chrornosomal DNA, was removed with ast€r16tooth pick. Ths plasmd ONA ln 

lho romaining lysate was prscipitatsd by addlng 200 /rl ol isopfopano and 

micro-cenl.ilugaton ol the mixtule tof 10 to 30 minutes at 4'C. The pe l€l was 

washed with 70% elhanol (1e, 10O &l was added to the pellet, and the lube 

was spun 5 ro 10 minutes, Ihe pellst was at dried and the oNA dissolved 

'or 

in 40 rlolTE buffer. 

ldentltlcatlon ot clones 

Restriction endonuclsase dl9ssllon 

To identify co.structs containing oligonucleotide inserls, plasmid 

clones we.e digested wirh specilic restrictjon enzymes. Presenc€ ol lhe 

oliqonucleoides cassetles would recreate a site lor Ava ll (lor all 

mlranrs sxcepr GT.g) and increasa the size ot one ol rhe Taql 

l.agments. Pst was used to conlirm thatrhe oligonucleotides contai.ed 

lhe proper proxmal l€rmlnal soquences. Thereiore, lhe mini 

preparations of pDNAs wefo individually digesied wilh lhe restricron 

endonucleases Ava ll, Taql, Psl I and E@Rl. Typical .esviclion 

endonuclease reaclions morained lhe desired plasmid DNA (4 /l) and

,dd f . c.t cfi|e cotud d H4 cd'e f@daton 

2 sr 10 X readron bunor (suPPli6d by N.w England Blorabs; 10 X Bulrer 

3 or 10 x butfer 4 was us€d; 1 x 8ufisr 3 contains 50 mM Tris_HCl, pH 

79, lOmMMgCl, IOO mM NaCl, 1mMOTI| l XBufi€r4 contains 20 

mM Tr s'ac6tar€, pH 7,9,10mM megngsium acetal€,50 mM polassium 

acetate, 1 mM DTT), Th€ fsacton volumewas b.ought up to 20rlwllh 

distiled wal6r. Feactions w6rs InitlaGd by addilion ol 10 units ol 

enzyrne (added in 2 ,rl a iquots lrom diluled €nzyme stocks) Fsaclions 

were rncubated al 37 c lor 2 . 4 hours, The samples we.e lradionaled 

by 1% agarose eleclropho.Esas. lollow€d by erhidium b.omide $aining, 

and eEmination using long-wave uhr6viol6l light p10 nm 6lter) (see 

Fig.9) 

DNA €eq!enc*anarFl6 

Each mulanl co.strlcls was sublsct€d to s€quence analvsis oi lhe 

reglon m lhe H4 promorer suiiollnd ng Fl4 Sil€ llin bolh lhe sense and 

antisense stands. Ths p.ocedur€ was pedom€d to connrm pfesence 

ol the deslg.ated mLrlatlon, and abs€nca of possible mutations 

genera@d during lhe Kl€now medialed cloning prod€dlre. sequence- 

analyses wer€ pedomed by uslng a commsrcially avaibbb oNA 

sequencmg kn (sequenass v€rsion 20i usB carabgue number 

70770). As described by rh€ manufactursr, app.oximalelv 4_5 pg ol 

clean DNA (lreated wth FNas€ or CsCl preparation) was h€aled with 

one primer (ehhe. the foNard primsr or rcv€rso primor) at 95'C lor 5 

minutes. Sequenase 5 x reaction bufi3r was added and tubes were 

45

Mz F: C.lt Cttl. C@tol ol H1 C@e tuffictlp|@ 

M.bntls and Mohods 

Flg.9. Ae4rb on en:yms.n!ly3ts ot posluve clon*. Each plasmid 

prcpsrutbn sas dig6E!6d with the charsclsristrc reslriclion 

6ndonucteases to decft lh€ pr€s€nc€ of sp€cific mutauons or 

th€ presencs ot t\s cAT g€ne an t€ corEtruct. uc, E, ET and 

Ava r€presents uncut oNA or sampl€s digest€d wilh EcoR l. 

EcoF l/raq I and Ava ll restrltlon €nzvmos, r€sp6ctiv6lv. The 

mol€cllar weight markff (m) was I DNA digssted with Hind llr 

and EcoF l. Molecirtar R€rghts of the marker bands are as 

iotkMs: 21.23. 5.1{8, 4,97, 4.26, 3.50, 2.027, 1.904, 1.544, 

1.375,0.947, 0.831, 0.564, 0.125 (Kilobas€ pairs) 

46

Restriction Analysis Of Mutants 

e'b:e'b: e'bi 

f--f-Fl' 

9,ni 

I lel I | | 

g.r j s.a i e,5i 9'rs 

47

a2t2 F: cdr cw. ctuc ot H4 6.n. f@d4@ 

raostered io ih6 37.c wat6r bath to. 15-20 minul€s tor annEati.g, 

Dunng |his lncubation, ths did€oxyructsotird€ l€mina$m mtes (2.5 / 

each ol ihi lour dille.enr basss, i6., dA! dT, dc and dc) werc added in 

small 5OO pl €ppendo.l tubss a.d tstt at rcom tgmp€€turc. |.;beting 

mir (provid6d in the kD was dlutod 1:5 wilh di3iitt€d tt, and 2 Et of this 

dilurion kas added in e8ch rnn€ating r.action 0o a totat votume ot 10 

pl). Each annoa,ing r€adion was turther 3uppi6h€ntisd with 1 / ot 0,1 

M OTT, r rlol lssl dATP {10 /ci/,lti sp€cilic acttity 1OOO Cilmmot) 

aod wilh 2 /l of a 1 t7 mixur€ of th6 s€qu6nas6 €nrym€ and saquenass 

dilution bufi€. (linal votum€: 14 /t), Aflar indbation room 

temperauro ror 5 minutssi 3,5 ,, ol €acn ,6action was add€d to !h€ 

.ucr€olida spocilic tsrminarion mix€s (,,€,, cra, cIT, dc and dc mixrures) 

akeady aliquored. Tubas wer6 irct,bated tor an additionat 5 minutes 

and slopp6d by adding 4 /t ot gsl to8ding buf6r. AI samptes werc 

heared lor 4-5 minutes 6t 8o'c b€iorc toading on a 6% 

polyacrylamid6/0.7 M urca d6naturi.E gst. Th€ gat was c6st€d with a 

shark s toorh comb. ons hou bsfoG toading, lhe get was p€run lo. 

15-30 minul€s, and was run lor 2.2.5 hours a,r6. toadtng tha sampres 

(gr€crrophor€sB !m6 dep€nds on rh6 si26 ol DNA t agm€nts and the 

@ii6spondn9 s€qu6nc€s ro b€ Fad). IhE g€l was dried und€r 

vacuum and e4osed to Kod6l( i-oMAT AF film overnight and 

deveroped |he n6lt mo.ning (s€€ Fi$. 10, 11 €nd 12). 

48

,d, F: ccl crc!. cqfrol ot H10d fdtsld@ 

Flg.10. olla aaqsoflcr lnalyab ol H4 promoiai conalucb I H4 Sit6 

n wildtpo TM3 srd tt€ rdsrt GT€, INS-10 6nd sUB11 cAT 

constucts wBr€ $q!€nced udno the puc19 iorward pdmef on 

ih6 H4 pfomobr s€ns€ stand. Each 3€q$nc€ r6ads tom ghi 

b bn ss A T G C.Ih€ loca{on ol Sn6 | 6nd Sft€ ll s6qu6nc€€ ars 

indc€lsd. P and M indicat6 P-bo( and M-box, resp€clivet 

GATC r€pr€senE a linkgr s€quencr dld Psl I ard Mun I 

rsslridio; ondonucleas€ sitos arE shown x,ih a.rows. 

49

DNA sequencing of 

wt IM-3 m GT-g 

I 

! 

q 

a 

I 

h I I 

---h 

-rg 

PUC 19 

t 

t 

3 

H4 promoter constructs 

m INS-10 m SUB-11 

l1 

l- 

* 

.l 

torward primer/H4 oromoler sense-slrand 

50 

;

AtIzF: Cd CJ.L Cdtuotol BaGq:€lat'!€ipns N.lddsad Mdhdh 

Flg. 11. DllA s€quenc€ snllyl|3 or H4 promotet consltucts ll. H4 sne 

ll ftu.iant caT construcE Mc-7, TCNn2, FAM-14 and FAM-15 

wer€ sequencsd uslng lhe puc19 iorward primBr on the H4 

s€ns€ strand (s€3 Fig. l0). 

51

DNA sequencing of H4 promoler conslrucls 

m MC-7 m TCN-12 m FAM-14 m FAM-'15 

-rt : 

PUC 19 lorward primer/H4 promoter sense-staand 

52 

t 

:

,.8 F: 6t W @tlol o, rk 0.,t rn',dfi,F., 

fr.12, 

oru r€$.noa rdFb ol rn P.oior.t co.}!{r!d. lll. $dd 

typ€ R)IOS H4 Eb ll p|tn Lr Cr'T corEt|.td 3in HI'{FLl 

bhd|E ste rruaii cal cql3llJob M8P.l6, MPiF17, lnd IGM' 

18 w€re sequ.nc€d u.tng lh. puc19 torward prlmv on t4€ H4 

!€.86 srd (3€o Flg; 10). 

53

DNA sequencing of H4 promoter construcls 

wt FO1O8 m MSP-16 m MPM-17 m lcM-18 

a 

:l= 

" 16 

""1 

PUC 19 lorward primer/H4 Dromoter sense-stGnd 

]'

ldD F: Cett CrL Cotuot at t14 G* Iwion@ 

Preparation ol glycerol stock badlorlal strairu lor slorage 

sacterial cullurcs 0O mD Mr€ prepared by inodla on tom a singl6 

colony or trom a previous glycerol stock (25 _ 50 pl) and ovsrnighl incubalion 

at 37'C wlh shaking. Ihe nexl morning 500 rlol g.own bacteria were mxed 

{fth 5oo pl ol sterile 8o% glyc€rol or 1oo % glycerol in cryotubes. The 

suspefslons wer€ mxed by inv€rting th€ tubes mulliplgllmes and stored al _ 

70,c. 

To ensure the clrr€cl idsntily ot plasmld DNAS grown Jrom thess 

bacrerial stocks, construcls wer€ analyzed by both lestricron enzyme 

analyses and seque.ce.analysis ol large scale Plasmid preparalions derived 

lromlheslocks 

large scare prepa.ation ol Plasmid oNA by alkallne lvsis 

Large scale prepration of plasrnid DNA was initial€d by prepanng a 

loml ove.night cultwe which was grown io late log phase to an optical density 

or O.O al A@. The culll]re was lransloned to 5OO ml TB medium Oeriiic Brcth: 

Ausubeler a/., 1987); the lquid culure was s€t !p in such a way that the arto 

liquid ratio was appronmately 3:1 in each culture llask (reqardless ol size) 

Cuhu.es were lncubated at 37'C ov€rnght in a shaking incubator with 10 

mg/nlampicilin. Bacteria we.€ hatuesled by spinning samples in a sorvall 

GS3 roror at 4'C ior 10 minutes at 5000 rpm. 

Bacler alpellets wers dissolved in ao d ol ST€ solution (0.1 M NaCl, 10 

mM T s HCl, pH aO, 1 mM EDIA PH 8.0). Tubes we€ centrillrged lo corlect 

55

Alir F: Cel cycta conxot ot t14 G@. t@cdpuan M.tlnate and Mahads 

% 

lhe cellpellets as abov6, and the p€Iets w€.€ r€suspsued n I rnl o, soturion I 

(5O mM gru@se,25 mM Ttis-cr pH 8.0,10mM EDT pH 8'O), 1 mt of ,reshty 

prepared ryso2ym6 sotution (10 mg/m|n 10 mM ris_ct, pH 8.0) and 20 hr ot 

soruliof ll (0.2 N N€oH and i% sDs)_ Tub€s wor6 invert€d manytimos lo mir 

r.6 conrenls genly and incubared at room temp€ratu€ tor 5 minur6s. 

slbsequendy. 15 mt ot ice.cotd sotdion t (sotlrion t is compossd oi 60 mt5 

M potassium acetat6, 11.5 mtgtaciatacatic acid, and 2B_5 mt dis ||ed H2O) 

was added and tLtbes were inv€ded sev€rat ltmes to mix rh€ slsFension. 

sampres were incubaled on ice tor m minut€s and centnfuged tor30 minules 

al 15000 rpm d 4rC in a J2-21 Beckman c€mriftrge using rh€ J-20 rotor. 

Supernah.ts w€re sbved rhrough the ch€es6 ctoth and rranstetred to another 

srerile tube. DNA was p.ecipitated with 0.6votum€ oi isopropa.otand washed 

once wrh 70% erhanot. 

oNA peltels wer€ djssotved in I mt ol fE-bufier, and exac y 9 gm o, 

CsCl p€r 6 mr or ONA soturion was addod to tha samptes, The cssium slt was 

ossorved at room remporarura and 1 mt ot €lhidium bromide sotltio. (10 

mg/m ) was added lhe conrenls of rh6 tubss w€r€ lhen tan$t6fied nio qurck 

ssalllbes Tlbes wer€ spun in a ]lTO ro!o! tor a minimum-period ot06hours 

ar 48,000 rpm d 20,C, Tubes were removod whjt€ taking cars not to disturb 

rhe cesum chlorid€ gradie.r The ptasmid oNA band was id€ntilied wilh short, 

wave UV lght and putt€d our ot the lube using a syinge, Th6 DNA was 

gxiracred s€vefal tim€s w h wateFsaturat€d blianot to r€mov€ €rhidium 

56

A2E F. cdt crcto Cantat at Ha Gqa ronsctiDtion 

cscl was removed by dialysis (dialysis rubing wi|h a molecular weiqht 

cul-otl or 12000-14,000and a diamete.of 10 mm). Ssmples wef€ dialysed in a 

Large beakorco.tanlng 1 llerTE butler wh ch was subject to constanl stirring. 

The TE bufie, was replac€d two times and ONA was r€covered by €thanol 

precipilaton uslng 2.5 vollmes ol95% e|hanol, ONA pellsls were wash€d wilh 

70% etha.ol, and dissorv€d in 1 ml ol TE-bufior. DNA qua.rirared by 

determining iho optical densilies al Ad and Aa 

DNA isolallon using Olagen lon-exchang€ r€slns 

DNA solalion was p€dormgd using lhe Oia96n-klt (Prcmega). The 

protocol is based upon a modili6d alkalin€ lysis procedu.B tollw€d by bi.ding 

ol plasmid ONA 10 Qiagen anion-exchange .esins under approp.iale 

conditions (low sart a.d pH). rmpuiries presenr in bEcteid lysat6s afe 

removed by washing rhe resin with medium sah w4h, and.ec@ry ol DNA by 

ellt on wrh h th sarr buffer lolow6d by isopropanol procipiratio.. 

Aliquots (l ml) oI treshly p.epared 10 ml miniclltur€s were ino€ulated 

n 100 ml ol LB medium wrh 10 mg/ml ampicjllin, and incubaGd at37'C n a 

shakifg i.cubaror. cells w6r6 ha.vesred n a 250 rnltlask lor 10 mi.ules at 

5,000 rpm ln a J2-21 Beckman cenriluge al 4'C. c.l pellels were 

rcsuspended n 10 mi ice cold buffer Pl (50 mM Trs.HCl pH 8.0,10 mM 

EOTA pH 8.0 RNase A loorg/mD- ThB conl€nts wero Ira.sr6(€d lo 50 ml 

centrLruge llbes and 10 ml ol lysis bufe. P2 (20O mM NaOH, 1% SOS) was 

added, tubes were i.vened several limes and incubabd tor 5 minvles eraclly 

al room rernperalure. N€utraization bufler P3 (3M potassum acelate pH 5.5) 

57

azn F cett qcl€ c@@l al h4 Gane r@sc4ptt@ 

{10 mr) was rhen added mixed and [ibgs were incubared on ice tor 20 

mrnules Tubes we.e ce.kiluged at 15,0@ rpm tor 30 mindes at 4.c. 

Olagen columns were eqLrilibrat€d with 10 mlot equllbration bufter QC 

(70 mM NaC, 50 nt\4 MOPS (3-[N-Moehotino]9rcpane-sotphonic acid), 15% 

erhanol,0.15% lriron, pH 7.0). ard rhe butler was dGined t:. n the columnby 

g.aviiy llow The plasmid soruton (r'.e., lhe cleared supernatant obtained afte. 

cenl.itigarion ot rh€ bacrerar rysare and si€ung lhrough che6s€ cloth) was 

added to lhe column, iorowed by addi on ol30 mt of washng buiter 0000 

mM NaCl, 50 mM MOPS, 15% ethanol, pH 7.0)- The rinse,slep was repeated 

wrh a second 30 mr ariquor ol @shing bufier, and oNA was €luted wilh 15 ml 

or erution butrer QF (1.25 M Nacr, 50 mM Tds-Hct, 15% erhanot, pH 8.0). DNA 

was.ecovered by precpbtion usng 0,7 voum€ ol isopropano The pelet 

was washed onc€ wlh 70% ethanol, dried by elposure to at and dissotved in 

TE bu(e. The DNA concenkation was carculargd by dsr€rmininQ rhe 

absorbance ar wavsr€nEh 260 nm and 2€o nm {A@ and Ae). 

Elsctrophoreitc mobtltty shift ass.ys 

The ooclrophor€tic mobilty shilt assay (aso reienod to as gel shift 

assay) represenls al€ch.iqu6 that is us€d to analyze lhe binding ot tacto.s to 

gene p.omorers /, eillo (Ausubel er r/., 194D. Th6 assay is bas€d on rhe 

panciple thal rho orocrrophore c mobility of DNA fragmgnrs is rel6rd6d upon 

nleradon with ONA b.dng pd6i.s. As a resllt, mixing of pfomoter 

iragments with nlclear pfoteins will r€su I in a characte stic panern oi shifted 

prolein/DNA complexes ( gel shilts'). Sequence-specilicity ol prorein bindlnq 

58

Aziz F: Cdl Ced. Cd*ol ol H4 Gse ran&donan 

lo radio-lab€ll€d ONA iraqments ls €ssess6d by comp€ttion assays n which 

10 - 100lold €xc€ss ol unlabell6d oligonuceotides is added 10 lhe binding 

reaction. As posilive carnrols, oligonuclaotjdos wilh lhe same or slmilar 

sequ€nce ar€ added as sp€cfic comp€lrtor DNA, whereas unrelaled or mutanl 

DNA lragmenls arc vsed as negativ€ conlrors. To reduce non.specitic binding 

of prot€ins to probe DNA, bindifg feaciions also cornain a relatvely la€€ 

amount ot non-sp€ciiic DNA. S6veral lyp€s ol non'specllic DNA subslrates 

can be us€d, inclldnq nalive oNA lrom salmon sperm ore cor, or synlhelic 

co polymeE such as poly (drdc)'poly (dl-dC)l'poly l/C oNA'l of poly(dc' 

dc)'poly(dG.dc)t'poi G/C ONA'I This lochniqu€ rs also uselullor idenlirying 

rhe actual prorein ihat binds to ths DNA; for example, anlibodies raised 

again$ proleins whch are belleved to bind to the DNA tragment, can be 

inc uded irilh€ r€action and lh6 6ft6cl oftofmarlon of prolein/DNA complexes 

In lhis sludy, gel shift assays w€ro used lo compare binding ol HiNF D, 

HiNF-M and H|NF-P in nuclear prcrein p.eparations lrom HeLa 53 cells to ONA 

tragm€nts spannlng wildtype and mL'lant B4-Site ll sequences (see Fig. 13)- 

Thelirsl sl€p in these qe shifr assays was to mak€ lhe probe whch spa.slhe 

Hind lrllMun Itragment ofth6 wildrype pFP-1 construct or analogous mutanr 

promoter consrrucrs. Plasmid oNAs wsre cut wirh Hind lll by incubalion in 

digestionbufi€l (50 /lot plasmid DNA(1&g/1rl),10 plo{ 10x NEB Buli€r+2, 

5 ,!l {20 units/rl) ot Hind rrr and 35 /rl oI Hro) ior several hou.s ar 37'c- 

Enzymes wer€ heat.inadivated at 65.C for 20 minut€s followed by elhanol 

59

tzh F: c.t (r.J. cstuot ot H. e* fd@td@ 

Fr9.l3. schemallc dLgram llluslrstng lho Pr€9arallon ol Plasmld 

probe lot use h get shllt aacrF. ol.lA s6mde5 w€re linearized 

with Hind lll, phosphorylalod wllh 32P tIATP and ihen digested 

wih a s€.ond resrictlm gnzyms (Mun l) whicn cds a! the 

boudary or sit6 l- a fiagin€nt of aPprodmat€lv 90 bp isolabd on 

a 4% 3Or1 polyacrylamid6 gel Dark line shows th€ polylink€r or 

l0 bp, 

60

o 

E 

o 

!!tH 

I rtog 

5l 

€s 

c_g 

- t- 

PLEE 

i5== 

a 

I 

ll 

t a

Arn F: CeI Cvcte Ctuot ot H1 6ene ttesidid 

p.eciptailo.. The dephosporylation r€action on the 5 ov€.hang ot rhe Hndlll 

dlgesled ONA was perlorngd by dissolving fta pellot in 80 !l of TE bltl€r, 

10 rlot 10 x Ca[ Inr€stinal Ph6phata€€ (ClP) bdler (Bo€hinger Mannheim), 

a.d 10 /lot (1 unit/pl) alkalin€ phosphatas€. The DNA solulion *as incubatsd 

lor one hou at 37'C, and lhs rgadion was slopped by th€ addilion ot 0.1 M 

EGTA and tlnher incubaton of lhe r6sullin9 mixturB al 6a'C br t h.. Th6 

soldion was extracred wih phenol-chlorolo,m to inadivate arry residual 

e.zyrnalc adivity, and lhe DNA $€s pr€cipibted with €lhanol. The DNA was 

dissolved 'n 50 !lol TE bu'i€r and 9orcd ar -20'C 

T4 Polynucleolid€ kinase 

Labeling of iho dephosphorylaled S ends was pedomed lsing T4 

polynlceorde kinase in 1 X kinase bufier {10 x kinas€ bltfer is 70 mM 

Tris/HCl, pH 7.6, 10 mM MgCr2, and 5 mM OTD, A rypi€l 25 pl kinase 

reacrion conrains 10 /g (1!9/1/,1) dephosphoryrat€d DNA,2.5 pl ol r0 x 

kinase bliler, 100 l]Ci (usua ly 10 ltoilr ) ol 1 ATP sp, and 1 p (10 U) or T4 

poynucleoride kinase. The T4 k nase catalyzBs the tansl€r ol lhe phosphar€ 

Lom lhe ATP to rhe 5 hydroryl terminus ol rhe DNA. The readion w6s 

incubatedlo.30 minures at 37'c. subsequ€ntly,80 !lol TE burler was added 

and rh€ reacr on was hear.nactivared at 68'c lor 10 ro 20 minures. The oNA 

was precipllaled wilh ethanol |o r@der th€ DNA, and lh€ p€llel was dissolved 

in 90 al ol TE bufier and 10 d 10 X Mun lbufier. Th6 .adio-rabelled DNA 

lragmonts were ihen digesred with Mun I (25 unils) which cuts lhe plasmid 

once (and eractLy atrhe boundaryol sir€ l)to r€laas€ lh8 radio.labened sii6ll 

62

kir F: cettctttd contal at H4 cen. fnrcutptt, 

Mateiats adt Mott'ods 

lrom vector sequences Folbwng ethanol preciplr:lo' the p€nels 

'ragm€.t 

were dissolved in 40 r, TE bufigr, and 10 ,rl ot 5 x dy€ was add€d pnor ro 

Samples were loaded omo a 4% (3Ol) polyac.vlamide gel contanrng 1 

x TBE butle.. Gels were prepa€d by mi{ng 10 ml ol 10 x TBE, 10 ml 40% 

(30:1) acryiam d€, and 80 ml ot HrO) Tho solution was subjecl€d to a vaclum 

( de.gassed') lo remove oxvgen which inhiblE rhe polvmeizaio' reaclion 

Tho gel was polymgrized by adding 50 rrl ol TEMED (NN'N'Nr 

lelramelhy'erhylenediamine) and 5OO d ol 10% ammonium persulphale Th€ 

soluiion was gently swided (while avoiding fo.mation ol an-bubbles) and he 

solution was pour€d in a ca$ considing ol 24 cJn X 12 cn precision_cut g|ass_ 

prares (Hoele4 and allowed to polvrneizs lor ons hour' Ihe gelwas p'e'r!n 

for al ea$ 30 mlnules. The gels containlng tM probe sampleswere run lor 2 5 

hours rhe Localon ol ihe corect DNA band was idenli'ied by 

aubradiography ot th6 wel gel, and th€ €orr€sponding gel slic€ wes isoralBd 

using a scalpel. The ONA in rho gel slice was recovered bv el€clrophoresis 

inro dialysis bags and ihe DNA was recowr€d wilh ethanol ptecipita on 

The second step involvod pr€psra on of nuclea' prot€ins Nlclear 

exvacls were prepa.ed accordi.g 1o lhe Dignam pr@€dure (Dignam d a'' 

19s3) lslng HoLa 53 cels (d€.sily 7-9x10! cels/ml) as d€scribed pr€viouslv 

(vanwiine. elal., 1991b,c), bd magn6sium_salt was.€placed i' allbur'€rs by 

1 mM EGTA,0.2 mM EDTA, O 75 mM sp€.hidine add015 mM spermlne and 

a broad sped.um proiease inhibitor cocktarl was used {PMSF' leupeptn' 

63

Azi' F. Cet Crcl. C.nttat ot H4 G.n6 fans.4ttoh 

pepstarrn,4psin inhibiror, TPoK, EoTA, and €GTA Bo€hringer, Indianapotis, 

rN) Also, desaring was nor penormed by diatysis btjl by ditdion wi|h srorage 

buffer wthout KCI (2 gtycerot, 0.2 mM EDTA, O.O1% Np4O, 25 mM H€pes, 

pH7.5 and 1 mM DTT; kNO b!ff€4, Att€.nativ€ty, samplEs w6rs desalted by 

gel tihration lsing Po-10 cotumns (p-! Biochemicats), or uttrafillraion using 

c€nr.iconro unirs (amicon, Lenngton, MA)_ Finat protein conc€ntraions were 

6dlusred wilh storage bufier {KNo bufi3r wiih 1clo mM Kct = KNlm bufie4 

Prorein.DNA bindlng reactions ior deleciing BiNF.D were caiiBd our by 

combining 10 rl ol a prot€in mixturo (in KNICTO storage boltet 20% gtyoerc, 

100 mM KC1,0.2 mM EOTA, 0.01% Nonidst (N) p-40, 1 mM dithiothreitot, 25 

mM HEPES lN,2-rlyd.otyerhytpiperaine-N:2-ehanesu onic acidl NaOH, pH 

7.5)wrh 10 rror a DNA mirllre contaihing 1 plot probs oNA 0o tmotin TE 

bun€4, 2 rr poly(dc.dc)i(dc-dc) (2!g in TE bufier), and 1 pt of pory(dt- 

dclt{dl-dc) {0 2 !g in TE bufie4. Atternartvety, binding reacrions io. delectiog 

HiNF-P/HiNF-M wer6 prepared by mixing 10 ,rl ot a p,orei. sotulion (in 

KN100/MZ b!tle., whlchis ike KNICrO bur inctudes O.ZmM MgCt2andO.2mM 

ZnCl2)with 10 plof a DNA mxrure containing 1 /l ot prob6 DNA (10 fmotjnTE 

bule4, 1 ! ol salmon sperm DNA (Sigma) (2 /rg in TE bufis4, and 1 !t ot a 

divalg cation soluiion (conraining 2 mM MgCt, and 2 mM ZnCt,). 

competirion analyses wer€ pertorm€d by mixing 10 |lt ot DNA mtxture 

conlanng 10 imol ol probe and 1,0oo tnot (,1e., 1OO iotd motff 6xcess) or 

lnrabe|ed specific compeiiror doubte-srr€nd€d DNA with 10 !t ot protein 

mi{ue conlaining approximately 1 ro 10 pg ot Heta nuct€ar prorein, tolowed 

64

arE F: CeI Crct Cotuatat 114 G40 fa@ ptian 

by incubation lor io to 30 minutes at 24'C and elect ophoresis wh€reas 

H NF.M and HiNF-P prctein/DNA complexes were rosolved ln 4% (20r1) nal ve 

polyacrylamids gels using 1 t TGE Ois_base/Hcl Glvcine' EoTA PH 8.5) 

b!ffer (Ausub€l el a/., 1987). Bindlig readion mi{lres lor HiNF_D were 

elactrophoresed in 4% (8Ol acrylarnide/N,Nh€thvlenebisacrylamide weight 

relio) poyacrylamide gels in 0.5 x TBE bulf€r' Oel supershift assavs were 

carried out by p.einclbalion ol nuclear proteins (10 pl; 2_5 ,rg) wilh antibodv 

0 pD lor 15 min on ice, followed by additon of 10 r,l oi ONA mixtur€ and 

lncubation ior 15 min at room temperalLrrs pior 10 eleclrophores s 

Translenr ery.ession analys's 

wd rype H4 promoler cAT constrlrcls (ie, Fo108_cAI) and ths 

analogous muianl promolsr cal construcls w€rs tansrecled in human H€La 

q cetuical carcinoma cells, .onaldiploid rat calvarial oslsoblasls (FoB)and 

ratosl€osarcoma 1712.8 (FOS) cells (S€€ Fiq.la) Helacels were cultured in 

Dulbeccos minimal €ss€ntial medium (DMEM) usinq high glucose 

concentrations and a combination ol5% feta calt s€rlfr and selo donor hors€ 

serum. Ihe medium was supplemented wilh antibiotics ad amino acids at lhe 

lndicated linal concentralonsr respeclively, 100 lnil/ml penicilln l00 rglrnl 

slreptomycin and 2 mM of L-gldamin€ Monolayer cuhlres were se€ded at a 

density of 1x1@ per 1oo mm lissue cullur€ p'ate in 10 ml of olvEM and were 

incubated at 37'C lnder atmospheric condi ons ot 5% co, The nert day 

ce|ls were translected lvilh plasmid DNAS or inle.esr'

Aziz F: Celt Cyd. Cantd d H1 6ffi f@ctlpion Mat lak aal Mttha

c 

A 

B 

e'l' t i 

_ f '', \J..$.'

azn F: cetl cde cotuat 6 H1 646 htndiottoa 

Two di(erenr mehods were used lo |ransfecl c€lls dep€nding on the 

cell rype. HeLa cells were transl€cted wiih rh€ calcium phosphate mehod, 

whereas Ros 1712.4 and RoB cells w€r6 ranslscled wilh rh6 DEA€-dextran 

calcium pho6phal6 merhod 

The calcium phosphate m€thod (Ausub€I, 6l ar,, 1987) involved hlgh 

efiiciency translection ol HeLa cells using lh€ 8Es bufie.ed system, 

which gensraLly allo$s lh€ DNA prscip{ale lo torm n rhe m€dra 

lollowed by incorporstion into tho 6lls, Th€ 2x BES b!fier conlains (5O 

mM N,N-bis (2 hydroxy€ihyl)-2€mino€thane sullonic acid [BEs 

carBrocheml, 230 mM NaCl, 1.5 mM Na2HPO., pH 635 800 ml H2O' 

pH 6,95). Because sl€rilily and th€ pH ol lh€ solution ar€ very cntical 

paranalers in this melhod, aliquols w6r€ k3Pl lroz€n in 50 ml at -20'c 

a.d rhe pH ol rhe sollrion was checkBd a.d re-adiusted lo pH 6 95 at 

rhe day ol tansfecrion wilh 1 N NaoH. Th6 solulion was st€dized at 

room t€mperature by nltra on rhrough a 0.22,m nitroc€llllose liter 

(Na gene). Calcum chloride was fr6shly prePared lo a mncenral o' ot 

o.25 M by dilutng a lo x cacl2 slock_$lutlon (2 5 M: slored at _,O'c) 

in sr€rie H2o, The DNAmlxlure us€d lor translsclion wis prepared Ln a 

1.5 ml llbe by combining 20 llg ol Plasmid ONA (no cari€r ONA was 

added) wjth 50O rll ol0.25 M CaCl,, and 50O,rlol 2 x BBS bufier. The 

sorurions w€r€ wel.mix€d lsing a vorid 6nd lh€ tub€s were ]€n 3t 

room lemperalure lor ar leasl2ll minrnes until a tanslucB lant_blue 

6A

Nz F: Cel Ctd. Ctuol at h4 Cq. faBdiDtidl 

9r6y preclp tale devoloped, subs€quBnlly, ihe DNA mixlures we.e 

added b the mediud in th6 plalos in a drotrwis€ mann€r. Tho plates 

were gonrly swnbd and placed back in th€ incubalo. at 37'c lo allow 

the DNA to€rnerhe cells, Ths nsxt day, lhs c€lls w€re fel€d with 10 ml 

ol lresh OMEM medium. Folowhg 48 hours, the plates were washed 

twice with PBS phosphal€ bufier saine (PgS, 1 lil€r Pas was prepafed 

by mixing 0.2 g Kcl, 0.2 q KH,Po.,8 q NaCl. 1.14 g NaHrPO4' the pH 

was adjust€d to 7 4 and the sol! on was st6riliz€d by using 0 22 ,rm 

i lre4. The csns were hatuosted by aspnaling the medium, adding 1 m 

ot PBS and scraping lhe plates with a rubb6. polic€man. cell 

suspensions we.e lranslerr€d to 1 ,5 ml c€nlriluge tub€s The cells wsre 

subjected to cenrilugalion lot l minqtg at 14o0o rpm and the 

sup€rnatant was aspnai€d. Th€ p€lle|s were sav€d a.d slored al - 

m'C, o. processed lhe same dav lor chloramphenicol 

acevrransr€rase (caT) assays. 

DEAE-O6xlran M€lnod 

ROS 1712.8 and secondary ROB calls wsrs translgct€d with the DEAE 

dexrran m€thod (as desdibed by Ausubel, €4a1 1987) One dav prior 

to rhe rransfection, Ros 1712.4 cells w6re se€d€d d a densily or 

o.4r1G c€lls/1()o mm tlssue culture plal€ in HAMS F12 media 

supplemenled wnh 5%i€lalcafi serun, 100 un't/ml penicillin 1o0 !g/ml 

st€promycin, and 2 mM L-glutamins (kom GlBco)i nde het Ros and 

FOB cells r€quke a gap ot at l€ast 12 _ 14 hours alt€r plaung ror proper 

69

Aziz F: Cen ddo Contot ol H4 Gere l@sqiol@l 

adhesion to the plales andto Prevern the Peeling ol lhe cells from lhe 

plai€. Similany, nOB cells wero plated at a densily ol0 4xlCF celb per 

1oo mm plate in minimal ess€nlial m€dium (MEM) containing Ea e s 

salt and supplemented wiih 10% l6tal call serum 100 units ol 

penicillin/mJ, 1Oo ,rglml stfeptomvcin and 2 mM L-glllamine Th€ cen3 

were microscopcally inspeded belore transfection ro ensure thal cols 

are actLvely prolilerating (ie.,40 _ 50% confluent) and devoid or 

microbial conlamination For esch 1oo millimeter plste lranslection 

mdures were prepared in 2 o ml cenlrillge tubes serum'tre media 

(1.5 ml)was combinedwith 1.5ploi chloroquine (slgmaistock soluton 

oisO mg/mland slored al -20'C n a daA containe4 afd 6 ! oroEAE 

Dsxlran (Pharmaciarstock soution ol50 rng/mland stored al4'C) to 

nnal conc€nlrations ol oo5 mg /ml chloroquln i'd 0 2 mg/mr DFAE 

Dextran. The mlxtlres w€r€ complemenled wilh 20 rg of plasm d DNA 

in a minimal voLume ol TE bufler (ie up ro 10 ,rl) 

Plales were wasneo 

twice wilh PBS, and theeLlswere covered with the 1 5 mloNA mi*ure 

lolLowed by i.cubar on fof 2 5 lo 3 holrs al 37'C After lhis pe' od' the 

DNA was aspraled from lhe pates and the cels were subjected io 

glycerolshock byth€ addilion oi 10% glyc€rolin serumJree med a and 

incubatio. for exaclly 90 sec.nds The glycerol 'nixu'e was removed 

by aspkation, the plates rete vvashed tlvice with Pgs and elB were 

reled with complele nedia- The plales were incubat€d ior 4a lo 72 

hours at 37'C, and cells were hatuested as describ€d above tor Hela 

70

Aziz F: Celt Cyct. Contd d H4 6@e lanscttOtbn 

chloramphenicol acelynransteras€ (cAT) assay 

The CAT assay was perlormed as des.ribed bv Go'man er a/ 0982)' 

C€lls were washed wilh 1 x PBS twic€ and wet6 s'raPed with a rubbe' 

policeman in I ml ot TEs bufier (40 mM Tris l nM EOTA, 150 mM NaCl pH 

7.5). cells ca. be easit peeled ofi nom rhe plate, €sp€cially FOS and ROB 

cells by using TES bufior and tansl€ded in 1 5 ml c€ntrdug€ tub€ k€pt on rce 

Th6 tubes sere cenlitlg€d lof 5 minul€s at 4 C th€ super'atant was 

dis€fded and lh6 pellel was saved Subs€quentlv lOO rlol 0 25 M Tris' pfl 

7.8 was added, ihe pelletwas resusp€nded w6ll and the suspension paced n 

an ethanol drynce barh lo. 5 minules Th€ samPles wer6 ih6n tansre(od rc a 

37 c watedalh ior 5 minul€s' Thls p'oc€du'6 was ropsaled iwo times (i € ' a 

total oi lhree lre€z6thaw c-ycles) Th€ sampl€s w€re th€n centriflged lor 10 

minltes al iul speed a.d the supernalani sav€d lor the acetvlalion reaction A 

smallalqlol ot 6ach samplg (2 }ll) was sav6d lorlhs dotBrrninallon of prol€ln- 

cofce.lratio. 

Prolein-concentrations w€re dBterminod as lollows A s|andard'cldo 

was obiained bv d€lermi.ing th6 absorbanco r€adings ol a saries ol saFpr6s 

wirh known protei. co.ce.l'ations betwe€n 1 T g to 7 rr9 ot bovine serun 

albumin (BsA, $andard slock 1 mg/ml) Aliquots (2 pl) ot each sample were 

mded wilh I ml ol 1x coommassie prol€in assav teagont (Pierce) (obtained 

by dillring lhe slock soldon2lold wnh dd H2O) and the opii':3ldensilv ol the 

mi{ure at 595 nm was detemined The absorbancs values ol lhe iesl 

samptes was compared wllh ihose ol ih€ BSA srandatd and corlespondlng 

pro€in concedratonswerecalculaled 

71

Azh F: C.tt cycla cdt@t al H4 c.@ ftansipn@ 

Itatetuts dnd Mer4ads 

The CAT reaciions were pedorm€d in a Inal volume of 150 pl 

coniaining lcro - 250 !g tola prolein in97.5 ploi €xtract, io llloll lv T'is/HCl 

(pH 7.8),2.s !lolrrc ch oramphonlcol0 25 roi(AmeGham) anda0'rlol39 

mg/ml acelylcoenzyme A (Sigma)(final conceniration: 4 mM) B€adions wers 

incubated lof 1 to 3 houG depending on the level ol cAT activlty in the 

samples, The approximate levsl oi CAT sctivily was determined bv pre- 

evaluating a subset ol ihe samples bv slandard reaclio's io establish that lhe 

CAT assay conditions remain in lhe linear r6nge (e q'' 510 10 rl oi HeLa cell 

tsate to. t hr, 30 ril ol FOS and ROB ceLl lysaie lor 3 hr) The rcact ons were 

stopped by adding 1 ml oi cold €lhylaceiate ssmples were vortexed ior 30 

seconds to mix lh6 organic phas6 wit'h the aqu€ous phase The phasss w6rs 

separaied by briel centrilugation and th6 organic phase conraning lhe 

chLorornphenlcaL prodLrds was iranslercd io enolher lube The orqanic 

solvenl was allowBd rc evaporats over'ight und€r lh6 hood or dried using a 

Speed vac apparatls The samples wer€ rcsuspended in 20 _ 25 pl ol 

ethylacetate and loaded onlo a silica gel thin laye' chromatographic FLc) 

plale. Plates wers placed in a mixlur€ ot equilibrated chloroiorrn:methanol 

(9s:5 V/v) in an ascending develoPing tank and lhe samples were run lor 45 

minlrtes. Ihen plates were air d ed ior 30 soconds und" |he hood and were 

exposed lo XOMAT AR Fih ovehighl at room tenperature wilhout 

intsnsiting screen. caT acriviiv on lh6 TLC plates was determined bv placing 

rhe plalss inthe Belascope 603 analyzgl (Bstagen) ior30minutes The levelot 

erpressiof lor each mutant cAT construcl was evauated as percent 

convers on r€laliv€ to thewildlvp€ construcl' 

72

azE F. celt cy.b contol al H4 Gene rtanscnptian 

CONSTRUCTION OF STABLE CELL LINES 

selectlon ol csll lines conlaining chlfierlc 

H4 promot€r caT rePortsr gens consltucls 

Mutanl H4 promoier/CAT r€porter gene lusion construc'ts wers 

cotransleded wilh the psv2 Neo plasmid at a 2o:1 w/w talio in Hela cells The 

plasmid SV2 N€o (5.5 kb) (southem and Be.g, 1942) caiiies lhe bacrerial 

neomycn phosphor.bosvl transtorase lll (N6o) gen6 whrch was rsoralod lrom 

Tns transposon gene 19 (Beck el al 1982) The Neo gene coniers resislance 

to ihe aminoqLycosde antibiotic G418 in mammalian ceils HeLa cels wer€ 

translected wth the calcilm phosphate method (lsing lhe BES bulf€r 

condtion) b€calrse we observed a hlgh efficisncy ol selection with lhis 

melhod. Aller 48 hours ol transledlon lhs monolayer cell cuLtu'es were reo 

wjth G418 (neof,ycin) containing media al a working or io 

'oncenraion 'Joo 

4CrO ,,g/ml of adite antibiotic. h platss containing suvivng colls lhe cerls 

were reled with 2 day intedals wth lrcshly prepared medE contamrng 

neomycin. Aile( b€tvveen 2 to 2l weeks slable coLonies started to emerge as 

feiLecl€d by sing e conglomerates of ce ls at difiersnl spots on the dish Each 

otthesecolones is derivedlrom a si.gl6celand represents a monoclonalcorl 

llne. Polyconalslable celLllnes were €slablished by Poolng e lthe colon es on 

the plate. These polyclonalcells f€lled aheterogenous Population ol cells wlth 

dilfefe.t random chromosomal inlegrarion events (see Fig 15) 

73

t2izF cen cuft c@d o! H4 6@. f@ttpti6 

flg.15. 

Mtteats'dtrtttdt 

Producllon ol H4 prornot€r cAT stable c€ll ltnes' (A) Control 

H€L3 c6lls t ansl€ct€d witln wild type constluct FO108 CAT and 

without neomvcin resislant plasmld (psv2 Neo) All csLrs w€r€ 

d6ad withln two w66ks oltr€atment wnh G418 (B) Hela c€nsco' 

t€nsfec{ed with FoloScaT snd psv2 Neo plasmids The ce er 

oi a G418 resistanl cllorry is shom (C and O) P€ripheral view ol

A 

Geneticin (G4'18) rssistanl HeLa cell coloni€s

azn F cett cwh codtot ot H. a@ lan*tiproh 

Pfepararion oi glycerol stocks ior stable csl, lines 

The monolayer ol stable polyclonal cell Linas were grown unll 

conlluency, and lhe cells wsr€ lrypsiolz6d wilh t/psin/EDTA sol!{on (0.25% 

trypsin and 1 mM EOTA). Each dish was sPlit i o 10 dishes at a densily or 

1xlo6 per 1OO mM plate, and lh€ cells wero allowod to grow ior 2 to 3 days 

until conlllency. The plal€s wer6 irypsinized again and wash€d with PBS lo 

r€move the media. Trypsin solLllion (2 ml) was added lo the cells ior an 

incubation Period ol30 seconds Plal€s were examined under|he mrcroscope 

lo establish that the cells were t€leased lrom the culture vsssel snd the 

kypsin was inaclivaled by ihs addiuon ol complete m€dia C€lls were counteo 

and co lecred by cenlrilugation at 15OO rpm lor 5 'ninules using an lEc 

ce.triluge. Cellpellets were resuspend€d in storage media (70% Dt{E['| 20% 

letalcall serum, 10% glycerol), and storcd al a densitv ol l oxlot cells per ml 

in 4.5 ml cryotubes (4.5x10? c€lls per viaL) or 1 a rnl cryotubes (1 8x107 cells 

p6r v al), at - 1 aO c. For each establishsd cel llne several pLates w€re lsed ro 

assay th€ evel ol cAT aclivlty to check ior expression ol the reponer gene rn 

addllion, two Plates weie used lo. the lsola on of |he genomic DNA ro check 

ior chrcmosomal integ@ton and copy number oi ihe repone' gene conslrucr 

- lsolation olgenomlc DNA 

High molecular we tht 

g€riom c 

isoLated lo delermine lho copy 

suspensions of HeLa cerls wer€ 

76 

oNA trom HeLa stable cell lines was 

number bY squthern blot analys s 

cenvjluged in 50 ml coni€l lubes al

NizF: Cen qcb cttuat al tu G@ ft|tlMipl@ 

tua@ats aftJ tttdi'ns 

15oO rpm io collect ths cells. Media Ms asPiratsd and penet was 

washed with 10 ml of PBs (PbosPhaie buifered saline) cells wer€ 

resuspended in 1 volume of digesion butler (1!-r mM NaCl' 10 mM 

Tis/Hcl, pH B.o, 25 mM EDTA o 5% sos 200 rqlmL 

proteinase K; 

noler SDS and prot€ifase K wer€ added treshlv lo a stock-soldon 

conlainng the rcmaining componenis) A lix€d volume ot digeston 

blrller (3OO rli lhis volume will accomodats up lo 3x107 cells) was used 

and fie suspension was incubated overnight ai 37'C The aqueous 

solution conbining |he genomic DNA was eiracted with an equal 

voLume ol phenoL chlorotom ad dvact€d onco wilh chLorotorm 

isoamyl alcohol The DNA was precipjded by the addition ol 3tl 

sodilm acetat€ (NaOAc) and 2 5 volumes oi ethanol Al lhis stage' the 

qenomic DNA was kspl in elhanol and slored at -20'c lof long lerm 

storage. For subsequenl use, |he precipnate would be collected bv 

spinning lhe samples at 14,oOO rpm lor 15 minules at 4'c in a micro' 

centituqe The pellst was washed with 70% elhanol an dried' and 

.esusPended i. TE-builer. 

App.oximately 20 /g ol genomic DNA lrom individual cell lines was 

dillted lo 3oo ul in TE butler' The genomic DNAS wefe digested with 

Hind lll (NEB bufi€r 3) and the sampl€s were incubatsd at 37'C 

ov€rnght with o.e unit per l,g ot DNA. Aller th€ rgskiction dlqestion' 

rhe samples were trealed with DNas€ t r.ee RNase 00 mg/mD lor 30 

minules. subsequen{v, the samples were subjected Io elnanol

,nE F: c.tl Cwle C@tol ol H1 Gfle lznxnptlan 

Motqiats a'd M'thods 

oreciDitalion and lh€ DNA was dissolv6d in I0 lo 20 pl 01 TE_butter' 

In psrallellolhe abovo procedue involving the genomlc DNA samples' 

2 rg ol plasmid oNA (19, the wildtvpe FOIO8 H4 promorcr CAT 

rcporter gene clnstruct) was also dig€sbd as a control ln a 200 xl ot 

rcaclron, rhe plasnid DNA (mncentraiion 0 01 pg//l) was incubated 

with Hind lll enzyme overnighl follo$ed by heat inacilvalion ior 10 

rninltes al65'c. Subsequenlly diLuiions were mad€ to Prepar€ gel 

erectrophoresis samples containing exactly 10 pg 20 p9 50 pg and 

1cO pg ol digested pDNA Thls slandard curue ol digested plasrnid 

oNA, as well as molecular w€ight markers (Hr:jln/EcoRl digesl ol 

lambda phage DNA) was Io€ded along side 10 rrg or each ol ths 

senomic oNA samPles on a 1% TAE (Tis/Acetate/EDTA pH 8 0) 

All genomic DNAS wera qoantiiated by speclrophoromslrv lercre 

eecifophoresls 10 ensurs thd 6xacl amounts ot DNA were loaded on 

the gel. The dgesled DNA safipes w€re r6solv6d by electrophoresis 

whichwas performed at 50 v tor 1a hr' G€ls were stained with ethldium 

b.omide in disli{ed wale. and deslained lor 30 minrites in dislilled H2o' 

DNA was visuaLized by lonq wave (312 nm) UV iuorescenc€ and 

phorographed w.rh Polaroid Tvp3 57 h m (se8 Fg 16) 

7A

M F: Cd W W.l h. 4.. 7'.t'll'Pn'd 

Ftg.1l, 

Anslyals ol attDL call s{!s. Agero€€ gFl of gt,nomb DNA 

lgoht€d lrcm tteb srabla crl if6 afid digBtod rih tho 

r6s rlctlon onzyn6 Hind ltl (ls{! bn") and Sr'a I (doht hn€3j' 

Th6 mldclg lanss clhdn pl$inld conrol (pFo108 CAl) 

dlg€ii6d $tlh Hlnd ltt (oad.d in pg emo(nts nol vl$trle) l Hiid 

llt/EcoB I ws3 run along tE stnplelt s e me €r (o('€me 

dsn).

Matur I Hlrd lll/EcoR I 

Gsnomic DNA 

r2laaarae|0nu|lr1!l!|7|lrr2o 

80

'dit F: ce qcle cotuot ol Ha Gm ra@dp&m 

i.ansfer oi DNA ro a membranous solld supporl 

To rransle. DNA tom agaross gels lo a soid supporl, agarose g€s 

were placed in larce glass dishes and soaked in a denaturing solulion 

(0.5 N NaOH, 1 M NaCi). O! ng rhis proc€duro, il is imporlanl thal lhe 

geltloats lr€€ly ln the dish ro lacilitare rh€ denaluration process, O shes 

were plac€d on a shaking pladorm tor 30 minutes ai room iemp€ralure, 

8nd gels we.e neutralized by replacing the lirsl soluion witb neutralizing 

solulion (1.5 rM Tris HCl, pH 7.6 and 3 M NaCl). Each g€lwas subjecled 

ro genrle shakng loi an addlonal 30 minutes at room temperatur€. 

vfnile the g€ls wer€ incubating, 6 sheets ol lille. psper (whahan 3MM) 

as well as one sh€st ol zera-probs msmbran€ w€re cul exaclly the size 

oi the gel. The rnembrano was soaked ln distled r?ater for ive rn nutes 

before placing it on lhe gel. The g€ltray (in which the gelwas iun) was 

placed in an invened positionin a largeglsssdish, and then enough 10 

x SSC bufigl was added so that lhe tay was tiled up lo 50% wirh 

transler solllon (equires abour 2 L). Then a ong pfecur sheet ol flter 

paper (with a s ze exactly lhe width of lhe gel lray), was soaked in 10 X 

SSC and placed on lop ol lhe gol tray. Ai bubbles were etlruded by 

rolLing a glass pipetl€ over the sLr ace. Three sh€els of fl ler paper were 

soaked in 10 x SSC and piac€d on top ol lhe long sheet on the gel lray. 

The gel was praced crelully on rop o, lhe lilrer sheels wirh rhe wells 

lacing rrp. Again, a qlass pipon€ was rolled over lhe suriace to extrude 

any lrapped ar blbbles, and fou pieces of plastic (e.9. lsed X-Ray 

rim) were praced undemearh (and surounding) rhe borders ol lhe ger. 

81

azi, E. c$ q,tte CMtat ot H1 Gffi Iran*dpt.n 

The plastic stips s€rue as a bafiier 10 pr€vs liqud i.om llowlng 

direcrly t om rhe r€servoi. ro lhe pap6r rowets, which witt be ptac€d on 

Presoakod zera prob€ membrane was ctn at one corner to mark the 

orlentation ofthe gsl, and was placed ovortheg€t, Slbsequen y, thr€€ 

presoaked sheets w€re placed on top ol zeta probe rnombrane, and 

each lmo an bubbl€s wer6 removad afi€r €v6ry addiljon of sheet. A 

slack ot soft papsr tssues (one to rwo boxes or faciar rissues, 

KleenexrM) was precisely pLac€d on top oi the iihor sheols (A straight 

stack of lissues is imporlant as it pr46nts papBr lrom hanging ove/ the 

edges of lhe gei and conract rhe transter blftsr. This could calse the 

inefiioien! lranster of ONA to tho membrane). On6 pack oI paper row€ts 

were placed an the paperltssles and th€n agtass ptate a.d a medium 

weight object (e.9., lead viat, gtass bor e) was ptaced on top or the 

entre slack lo leep pressurg on rhe blffsr absorbing pap€r lissugs 

The exposed edges ol the larg€ glass dish were covered with ptast'c toil 

(saran Wap) to avoid evaporation of ths 10 X SSC burier, and ONA 

transfer was conrinued lor m lo 24 hours. Th€ zsra proo€ memorane 

wascareulry remov€d lrom th6 gel and p ac,.d i. n UV chamber ior one 

mint e to ix th6 rranslerred DNA on the membrane. Then the 

membran€ was placed in a s€al€d ptaslic baq (sea-a-mea )to ke€p th€ 

membrano hoist during srorag€ at 4.C.

M. F: Ce|| Crd. Cqnrot ot Ha Gerc TEneDiM 

Prepararion ol radlo-labelled probe tor soulhe.n hyb.idization 

Radiolabelled DNA probes lor South€tn hybddizallon werc prepared 

by radom labelling. h b.iel, the 1.7 kb CAT insen iragm,eni derived 

lrom pFO1o8CAT was radio]abded wllh the PrmeltrM randomly 

prlmed ollqolabeing kil (Stalagene) as descrited by lh6 nafulacllrer. 

h bri6i, 25 ng oi CAT ONA in 1 plwas mired rrith 10 !l ol random 

oligonlcleotide primer, and ths reacrion mixturg was adjusred wirh 23 / 

high qual'ty deionized and disl/tred H2O ro a lo|at votume ot 34 rt. The 

tube was hear treaied lor 5 minules d 95. to 10O' in a beaker. The 

rlbe was cenl.iluged brislly at room tempe.aiure 10 co tect ths 

cond€nsed liquid on the cap ollhe tube The lolowing reag€nis were 

added i.lho readon rube: 10 !tot 5 X dctp prtmer buti€r (conlatning 

dATP, dGTP and dITP), 5 pl ot labell€d nucrsotids [4J,P] dcrp (10 

/rcilpl - 3000 Cilmmot) and 1 /it or ExoC) ktenow enzy'ne (5 unir/ltt). 

The conterns ot the lubes were mixed by pipene mixing. Afie. bdef 

centritugarion, the samptes were incubat€d at 37 . C fo. i 0 minures. The 

read'on was sropp€d by adding 2 pl oi stop mir (recip€ according to 

uninco.porated nucleotides wero removed by psssing th€ p.obe 

through Quick SpinrM columns. Eelore usir'g rrre spin cotLrm., the 

unopened corumn was ihverted severall mes to mlx the G.25 s€phadex 

beads in the blfter, The lop ld was removed tirst lrom lho c$ umn and 

rhen th€ bonom iip, which is impo.lanr to maintain a smooth flow oi rhe 

83

kn f: c.n cyd. cdnot ot H4 6@0 rtanx'igion 

bufier trom lhe column, While keeping lhs column in a straight position, 

the bufier was allowed ro rrav€rse rhrough tho column by gravity ltow. 

Th€ ljp ol the column was placed inside a coLtection rlbe and 

centriluged a! 2500 rpm lor 2 mi.ut€s. The lir$ lube was discarded and 

lhe collmn was paced of top of €nother cot€cUon tub€. Th€ probe 

sorurion was added in th6 center of the s€phadex beads, and thg 

sampre spun down lor 5 minltes at 2:@ rpm. The cotumn which 

conlains the majority ol rhe free tab€twas discarded in rhe radioacrive 

wasle bin (!h€ top cover and bortom tip clver were pur back on the 

corumn ro prevent the spilling ot iadioacrive beads). The approximate 

speciiic aclivity of rhe pfobe was delermined by anatyzing rhe yietd ot 

radio-acrlvityr 1 rl was added in 4 mt of scintiltarion oprifluor and 

counl€d in scinila on counter, 

slandard procedure tor hybrtdtzalton 

Hybn:dization was perlormed tn glass hybridization cytinders. prioi ro 

use, lhe cylinders wer6 washed and siticonized wirh 5% siticonizing 

solurion in chtoroiorm. rhe UV dosstinked memtanes (conlaining rhe 

genomrc ONA from the c€tt lines) we.e removed tom the seated bag 

roned up and placed ln lhe gass cytinder. To lhe cylinder 25 mt of 

pfehybridization sotution was added (1 mt\,i ot EoIA, 0.25 t\4 NarHpq 

pH 7.2 and 7% SOS) to coar rhe membrane, and the cytinder was 

incubated for oie holr ar 65.C with aqilarion. Then 15 mt ot 

prehybridizarion solution was removed tom the cylinder ano denatured 

84

azi. F: cetl cttcL ctu.t or Ha Gtu lftMtip.ion 

prob€ (1x106 cpmlml) was added. The m€mbrane was incubarodinthe 

presenc€ ol prob€ tor 24 hours with agiratjon_ Th€ membrane was 

removed lrom fte cylinder and washed iwice ar 65.C in solltion i (1 

mM EOTA, 40 mM NEhpoa pH 7.2:5% SOS) Ior 30 to 60 m nll€s 

whlle shaking, iotlowsd by two washes in sotllton lt {1 l.ntv EDTA, 40 

mM NaIHPO4 pH 7.2 and j % sos) tor 30 to 60 minltes at 6s,C. rrs 

moist membrane was wrapped in ptastic toit (Saran wGp) and oxposed 

to x,Ray oMAT-AF fih, and ptaced at _70.c with an inlensifying 

screen overoighl. 

The titm was devotoped and the membran€ was subjected ro rh€ Bela 

scope 603 anaiyzer (Betagen, MA) over.ighl. The intensily oi tie 

genomic DNA iragmed spanning the tnregrared hisrone H4 CAT 

reporter gene plasmid w6s used as a m€asure tor the copy nlmber of 

he ntegrared constructs (s6e Ftg. 17). Th€ copy nlmber vaiues 

obtain€d this way were us€d lo catcLrtal€ the tevet ot expression por 

inbgrated copy ot each H4 CAT consrtuci. (See Fig. 18 ) 

synchronizarbn ol srabte HsL, c€[ unes 

- Suspenslon cutiureot HeLa S3 C€[ lin€s wnh dablyintograred 

F/ozen siocks oi stable Heta Sg monotayer cetis were lhawed in a 37 . C 

water barh. cers wers grown sl a d€nsity ot 3.ox1o5 c€ts/m|n SMEM 

(Joklik-rnodltod minimLrm esse.tiat medtum) supptemen€d wrrh 5% 

carr serum, 2?q holse serum, l0O unirs/mt peniciJrin 1oo /rglml 

85

M E C.l *,b cctol ot x. q.L f ld.a'el 

Frg.17. sodnam lndyeL d .iatty ltaiaLctad caf lln-' Hlnd lll 

dgsd g..roitic Ol.|A fdn vatbiB lsb c€I lln* 6 

rarBl€flld io z€ta !ro6a msrnbnne along wlh lh. Indica!8cl 

rmoun o, Hhd lll dgE€t d pFOISCAT td tty!.*t d wih o' 

r+dCTP radlob€ll€d CAT DMlrr€mdr 6.7 kA).

kiz F: c.lt

o 

lu 

r60 

120 

80 

Con3lruct 

binding: 

Stabtg cell llnos ot H4 promotsr CAT conslructs 

HINF-tl blndlng slto mutants 

toa 

wl 

usP-16 ltPr-r7 tGM.18 

P+lrI-lO+ P-l)l-lDt P-/lU r/Dr 

69

ktz F. cen ctct6 ctuat at tu 6e rftN.tipti@ 

stepromycin and 2 mtM oi L-glllamine c€Lls wefe maintalned in 

suspension llasks, with 6 capacity ot twice lhs volume of lhe cultur6 As 

lhe c€ll lines were initjally gtown on plates, it was necessary ro adapt 

|he siable calllinssto growth ii srlspension by slowly adiusllng mediLlm 

conditions. Inil ally, c€lls wers Prov d€d with h gh concenlrations ol letal 

call serum a.d L-glutamin€ and lhen gradually shiffed to comdeb 

medium as lollows. The lirst two days, lhe cells were led win5 7% lelal 

cali serum, 2 r,rlv L-glllamin€ p€nicilin and sreflomvci. as desdb€d 

above, and a 1 x mixture ol non_essential amino acids. The cells wer€ 

checked daily under the microscoP and fed al regular dailv inlervals 

with pre-warmed lresh m€dia. After two davs, !]e celrs rere ted wilh 

s[,lE[4,5% l€ral ca]t serum,2o,6 calf serlrrn, 2 mM L-grulamne, 

peniclllin/strepromycin as abov€ wilhout non-€ssenlial amino acids 

Subseque.tly, the cells were shilted to SMEM supplemented with 2% 

fetal call serum and 5% call serlm. Alter two additional daily feedings 

with lhe latter medilm, lhe cells wsre led wilh SMEM supplemenl€d 

with 1% letal cai s€r!m, 5% cal' se.um, and 1% horse donor serum 

Finally, cells were sh:ned ro SMEM supplemenled with 5% cll serum, 

2% donor horse serlm, and 2 mM !-G[nam ne Pe.ici]lin/slfeptomyon 

The c€Lls were grown at leasl 4 to 5 days in comp eie med a GLycerol 

stocks were made by spinning the cells down at 1500 lpm in the lEc 

rotor and lhe cell pellet was resuspended in sbrage media (70% 

SMEM,20%l€lalcali s€rum lovo glvcerol) Ce ls were stored in 4 5 ml 

cryotubes al a d€nsity oi 1x107 cels/ml (4 5x107 cels per lube) and 

90

,zn F: ce ct.le c@not ol H4 Gqe ,anscdpttan 

slored at - 150 c freezer. 

Doublethymldine block synchronizallon Procedure 

Th6 synchronywas pedomed by blocking the stabls Helacelllines by 

the admlnistralion oI2 rnM thymidine Hela cels w€re maintained Ln 

suspension at the densrv 4-t1ot c€lls/ml in SMEM supplied wilh 5% 

call serun, 2% donor hors€ s€rum 100 u.its/ml penicillin, 10o pglml 

sveptomycin and 2 mM L-glltamine (JOKLIK modiiied minimln 

essenrlal medrurn). Prlor to the flrst block, cells were counled and 

diMed 10 3.75x105 ells/ml. (All media, glasswa,e and souions were 

pewamed to 37'c). All processes were cariad oul in a 37'c warm 

room usng sleri e lechniqrss, Thymidinewas add€d to the suspension 

ceLlculiure at a linalconceniration of 2 mlM. Cels were blocked io. the 

duralionol12 16 hours (th€ blocking procedur€ n€ver erceeoed mors 

Cels w6r6 r€leased from th€ tirst block by sPinnlng down in lh6 

cenl.iiugg 15oo rpm for 5 . 10 minutes. cells were washed lwce wllh 

pre wa.med serum+ee medla to ger id or resid!3rs lhymldine (S gma) 

The c€lls were rc-suspend€d in fresh compl€ne media wilh 2- 

deoxycytidino at a iinal concentfation of24rM The celLs @re reLeassd 

lor exacrly I hours, and ihs c€lls were again sublocted to a second 

thymidln€ block tor 12 - 16 hours. The cells wer€ released trom the 

second hymidine block by decanling lhe medium lolJowing 

cenrrilugaion oi the cllture al 1 500 rpm lor 5 - 10 mlnlies ce I p€lrots 

91

Azi2 F: Cen Cvcte Contot .t H4 Gere ftaEdieti@ 

were washed lwce wilh s€rum-free media and were re.slspended In 

lresh compele medls with 2-deoxycylldine. Nucle were isolaled at 

multiple time'polnts after release irom the blockade lor nlcleat run'on 

transcription analysis ro obrain a direcl measure of promo€r actvity at 

dilterent time poinrs during rhe cell cyde. RNA was also isolated from 

synchfonized cels at every singls lrou (110) and was used to pedom 

ribonucease prot€ction assays Ths ls a very sensitive assay lor lhe 

deteclonolhRNA nacompoxsampemixiureoitotalcellllarFNA. 

Monnoring cerr synchrony 

The enenr ol cell clcle synchrony was determined by assaying the rale 

ot ONA synlhesis by pulse-labelling c6lJs with 3H.lhymldine lor 30 

mlnlles at lhe G1lS boundary (0 hour; ta , the pr6re ease lime_point), 

as wel as lhrolgholt S'phas€ and th€ remancler ol lhe c6ll cycl€ at 1 

hr lime.inrervals (ie., posr.releas€ pornls wei€ taken €very hour 

between 0 - 10 hr) The rate ot oNA synthesis which is required lor the 

replicarion ol fte entke genoms du,ing s-phase is ma.y mes hiqher 

lhan lhe rale of DNA synlhesis, which is requned br repairol damaged 

DNA. Honce, delerminalion ol DNA synthesis ratos provides a di.ect 

ndicator oi cel s progressing lhfough S-phase (Fig.19). Ce ls w€rc slso 

examned lnder lh€ microscop€ 10 obserue ihs abundance of milotic 

cells (ar a, 9 and 10 hr afier .elease irno s phse), which /euecls 

progression pasl lhe G2lM boundary (see Fig.20). 

92

.:''.'|' 

eE@cyaci'dac,l,o.Df*Wt 

''i1..'. :.. 

FE.19. synchq zfo|r ot .dL oau fn-. F{ Tlrvntidtu 

hcdr.don rB tLs,cd e ritc frcca"o 6nt63 bbriE 

rds hm 6td rymdrF Uoalq d|d f-dta ae o9r€ss€d 

c courG par mLrt ot acb puFesd Frl lvffin€. as 

d€.dfl€d h marBtd mdlD&.' 

&l

I 

200000 

1mmo 

DNA Synth.3l3 In Synchronlzsd HeLa calls 

10 

hours Dost-release 

94 

12

E F.c|dcll,'r,@tfit dHlcerw 

Ftc.20. Plrtogrrgt d arialr!|tad lg.a c.lls I hol|lt 'llf 

ral{aa trom 6rcotrd itry| dha uoct. A Hgh mltollo l'dat 13 

r..n, hdlcdtifE a lleli rynchrcny (s€c next pag6) 

95

High mitotic index 

alter relsase trom 

in HeLa cells 8-10 hours 

doublo thymidine block 

96

aniz F: cen ctcl. cantat ot H4 Gene rtanscnpt@ 

Derermlnallon of DNA synthesrs 

3H-lhymidine (10 llci = 2 rrlol lh€ spci/ml slock solltion) was added 

ro 2 mi of suspension cels whch w6rs aliquoted in p.€'warmed qass 

tubes. Tub6s were ii\ed on a.olating whe6la.d incubatgd in lrie warm 

room (ambied temperaturc: 37'C) ror 30 minutes Cells were collected 

by cenrilugation at 15oo rpm lor 3 to 5 minutes, and medium was 

removed by car€lully aspnaling lhe media. Th€ cell pelLet was washed 

wlh 2 ml or serum.free m€diun (SMErv) and cells we€ co ected agam 

by cenlriiugalion ro. 5 mnules. The meduir was discarded bv 

aspira on and the pellel wss resuspended bv adding 4 mi ol 10% 

trichlo.oacetic acid ficA). Ths tubes were incubated on ice lor 5lo 10 

minules1o achieve oplimai precipitalion The samples wefe spun r' rhe 

centriiugo ro obtain a celpel€t, the slpernatanl was discarded by 

aspirar on, and lhe sbove slep was rep€at€d The linal pellet was 

dissolved in 5co d ol 10% sos and transtefed to a scinlillaton vial 

Liquid sclntillation counling cocktail (16 mD was added and the amounr 

olradoadlvity evelwas quanlilal€d n a liquid scntilaion speclromeler 

(see Fig. l9l 

Nuclear run on ttanscription 

Nucl€lwere lsolated lrom Hea celLs lhatwere haNesled atvariouslime 

pol.ts after release from lhs dolbL6thymld ne block procedure c! Ur€ 

samples w€re subjected to centrilLrgalion ior 5 minulos al 1ao0 lpm al 

97

ND F Cen atct6 cannot ot B4 6ene rrctctlptian 

37'c (wam foom). cell pellats w€re washed lwic€ with 10 ml cold 

'soronic bufi€r which is (125 mM KCl.30 mM T.is/HCl. pH 79 5 mM 

MqCl2, and 10 mM !-mercaprcethanol). From lhis point on cens we.e 

kepl cod througholt the romalnder ot lhs proc€dlre. Cells w€re 

centiluged at looo rpm and r€susP6nded in 10 ml cold hvpotonrc 

('swelling buller': 10 mM kcl,30 mM T.is HCI pH 7 9' 5 mM MqCl2 10 

mM B m€.capioelhanol) and allow€d to swelltor 10 ' 15 minubs Cells 

were dou.ce homogenized wilh a tight Pesrle lo. 10 - 15 sl'okes; a 

smallaliquot w€s stained with 2% trypan blue und€r |h6 mrcroscope lo 

checl ii cells w€re lysed). The lysato was transl€r€d lo th€ 15 ml lubes 

and cenr.ituged at 15oo lpm lor 5 ro 8 mind€s in lh€ lEc rotor' The 

slpernalanl was drainod carefully, and the nucleiwere resuspendeo n 

storage buter (50 mt\4, (NH.), SO1' 30 mM Tris/HCl pH 80 5 mM 

MgCl,,1 mM MnCl,,lOmM t-morcapro€|hanol 25% v/v qlvc€roD The 

nuclei were coumed and divided inlo aliq@ls ol approximalelv lxlot 

nucle/1oopl. Nlcei wer€ imm6dietely l.ozen i. Liquid nitogen and 

stored at -85 C {See 

TranscriPiion reaclion 

F g.21). 

Assessmenl ol transcription rates in isolared nuclei was accomplished 

accordng 1o 1he procedure by (Gr€enbeQ and zlt1, 1944) Fozen 

a iquob ot 1t1O? n!cl6i/1oo /rl wer€ lhawed our on lc€ a'd qlickry 

added to 15 ml cofex tubes conlaining 1oO ,,1 o, 2 t reaction buffer (10 

mM Tns/HCl, pH A.O, 5 mM M9Cl2 03 M KCI). which was 

9a

Nuclei isolat€d lrom synchronized HeLa cells 

100

NE F: Cell C6t cotuat .t H4 cffi fhNtiotlan 

complem€nled with unlab€lled and labetled nucleolds triphosphales [2 

mt!,t dATP, I mM dCTP, 1 mM dGTP and 100 pciol 9PUTP (10 rlol a 

10 llcilll stock-solullon with a sp€cilic aclivty ol 3000 cilmrnos)j. 

Nlcl€as€-lree glass tubes and solutons were ls€d thfoughout the 

prccadurc. The samplos (olal roadion volume ol 200 /]r) werg 

indbared al 3o'c for 30 minutes with intermit€nt shaklng Alier 

incubaring the sampies lor 30 minut€s at 30'c, 600 !l ol nNase free 

DNase | (Promega) in high s€ t bufie. (0.5 M NeCl, 0 05 M rr/gc 2 0 002 

M caot and O.o1 rM T.islHCl pH 74) was added. The samples were 

r4uspendod by.epetitiv€ mixing wlh a Pasleurpipelte 

which lacilitates 

lhe breakdovin oI nuclei and DNA, end lhe susp€nsons were 

incubated al30'C for 10- 15 mlnjJl€s. To each oilhe r€acion m xtures 

2oO /rlol protenase K (2omg/ml) n proteinase Kbufier (5%SDS 0 5 rv 

Tris pH 7.4, 0.125 M EOTA) was added, and tubes were incubared ar 

37'C ior 30 m:nules wnh gen e shaking at regular in€tuals. Reactions 

were stoPPed by addlng 300 !l(.e. 1/10 oI the odginalvollme) ol2 M 

NaoAc pN 50 lo a linal concentralon 02 1\,l The volume oi lhe 

samples was ilrther increased by addi.g 2 ml ol 10 mlr,4 T s/Hcl pH 

8.0 and 1 mM EOTA Reaclions were extlacled several iimes with an 

equalvolume ol phenol eqsilibraled with 0 2 M ol NsOAc (pH 5 0) and 

10 mM EOTA. The radiolabelled lranscriplion reaclions were 

ranslerr€d lo 30 ml cor€x nrbes and nlc eic ac ds were precipilal€d n 

ihe prosence or 75 /19 glycogen (Boehringer l'lannheh) and 25 

lolumes ot 95% mld elhanol. The sampies {€re kept at 20'c 

101

,2iz F: cell cttl. canlrct ot H4 Gene I€ns.iprt@ 

ovemighi or incubated for t hour at _85'C. RNA was recovered by 

centdlugario. (J-21 Beckman) al 10,000 rpm lor 30 minutes- The RNA 

perlets werc wash€d with cold 70% gthanoland at di€d. samples were 

resuspendod n 1 ml ol pfshybridlzation solution (125 mM sodium 

pyrophosphats,1 M Nacl, m mM Tris pH 7.4,2 nM EDTA,0.1%SOS 

10 x Oenhadrs solLtion). Small aliquots ol each sample (5 ,!l) we.€ 

procipitat6d with I rnl ol 10% TCA and 150 pg of yeast FNA (15 pl or a 

10 mg/ml slock) lo dst€rmin€ lhs spocific aclivity of RNA iransfipls 

The mixlures were incubal€d on ice ior 15 lo 30 minules and 

pr€opital$ were lillered lhrough 0 45 pm nilrocsllulos6 H_A tvpe liftels. 

Filters w€fe washed iwice with 10% TcA and diied by exposure ro ar 

ior 5 minutes. The i lters wer€ thsn transf€rr-'d to a scinti|]aron viai and 

dissolved n 1 ml cerlusolvern (polyethyleneglyco monoethyl €lhe4. 

Atter lhe lher dissolved, 10 ml ol scintillation lluid (OptiiuorrM) was 

added and were subiected to liquid scintillation spectrometry' 

Slot.blots w6re prepared on zeia_prob€rM nvlon membranes The zela 

prcb€ membran€s we€ load€d with inseit lragmenls ol interssl (2 rg of each 

oNA to be tssl€d), as we|| as plasmid DNA @nEining 6 qenomic inse.t 

encoding ribosomal RNA (LS6j 4 rg) DNA and various olher contro DNA 

fragments. Each sampe was propared n a l 5mlllbs by adding lhe requted 

amounllor €ach bLot (2 llq), and ths volume was brolght up to 500 !lwith 10 

mM rris, pH a and 1 mM EDTA. The DNAS were denalured by lheaddnion ol 

142

A22 F: Cett cytl. Connd d H4 64. rnnsoipnhn 

1/1olh volume (5opD of 3 M NaON -nd incubarion tor t holr at 65'c' 

Folrowing this slep, the tubes were placsd on ice and 55O /lof 2 M NHTOAC 

was added. samples were diluled wilh 1 M NH.OAC to obtain the desired 

vollme to be load€d in each slot. In th€ case oi rh6 ribosomal RNA encoding 

plasmd oNA (LS6), oi6 slot was kopt 6mply on all sid€s es the hgh sgnal 

expecled radio-lab€lled iRNA tansdiPts might inleri€ro wilh quanfta ve 

'or 

deteclio. ot signals lrom olher slols. 

Preparalion ol hybridization m€mbra.es usl.g lh€ manilold ll slot 

The Manifold lt slot-blol system {schleicher and schuell) w6 used br 

loading the sgmples onlo lh€ mambrano.lwo whalman 3MM a'd one 

zeta prob6 nylon fiombrane w€r€ clt exacllv the size of the lemplale 

provided in the box. The apparatus was wash€d throrolghly ov 

soaking il in 0.25 N HCllor hal( an hour and rhen nnsed several lmes 

wirh dislilled waler The two sheels ol fltor Paper 

(whatman) and one 

sheet of zela probe membran€ w€r€ soak€d in dislillod waler' soaked 

in 1 M NHroac a.d then placed on the apparalus The rwo sheeG ol 

I |rer paper were pbced on the surlace ot lhe apparalus on lop ol the 

/ed spacer gasket lhe red spacer in lurn was placed on the mylar 

(lransparent plast c sheeD At 6ach slep an bubbLes were removed 

wnh lhe help ol a g ass piPede, which was qenlly roll€d over lhe surlac6 

ol lhe pap€r. Th€ z€la prob€ nvlon m6mbran€ was placed on top ol lwo 

whalmansheeis, Agaln, an blbbles w€re.emoved wilh aid oilhe glass 

103

aziz F: ce dde c@no| ol H4 Gqe fanscdorbn 

pipe e. Tho Manifold il was plsc6d on top ol th€ m€mbrane and the 

davice was ciosed at both sid€s using plaslic closure pieces (provd€d 

wilh the apparatus io the boi)- The apPaatus $as hooked up lo th€ 

regular laboratory vacuum lines. and vacuumwas appled careillly Ths 

wells were nsed wilh 1 M NH.OAc and 400 rrl ot sample was loadsd ln 

eaci slot, slots wers washed with lM NH.oP€ again. Ths danitold w€s 

femoved carelully and lhe nylon m€mbran€ was air dri€d The DNA 

was nxed by placing the zeta probe membrane under the UV llght for 1 

minlre andthe membrane was stored in aseal€d dasric bagal4 C 

BIors were prehybidi2ed in cylind€ls using 25 ml hybridianion solulion 

(12,5 mM sodum pyrcphospfral€, 1 lv Nacl,20 mM Tris pH 7.6, 2 mM 

EDTA pH 7.5, O.l% SDS, 10 x Denhardt's solulion and 250 rglml 

lr4hly denatured Bdom oNA lrcm E colt or salmon spem)- 

Membranss were incubaled at 65'C lor 2 lo 4 hou.s Blots were 

hybrdzed n 3 mioi hybidizauon solullon as above wilh the addition oi 

2.0 to 2.5x1Od TCA precipilabls counts of radiolabel ed FNA transcripls 

Hyb.idization was performed by incubaton st 65'C lor 72 hours Elols 

were washed wilh shaking at 65'c lor 30 minlt€s ln a solunon 

cootaining 2 x ssc and 0.1% qDs. This slep was repealed two rLm€s 

The membran€ was washed lwics with another solution (containing 0.2 

x ssc and 0,1% SDS) with each wash pertormed tor 30 minltes at 

65'c. Flnally, ths blots wero wash€d once wilh 0 1 x SSC/o1% Sos 

lor 20 minul€s at 65'C. 

104

hnF cet cyttd cannot ot B4 6end ltunrc ptlan 

abG we.e airdried and expos€d lo prcllashed X'OMAT AR (Kodak) X_ 

Ray fih botw66n tlvo Cronex Lighling PlusiM i.rensitving scr@s al _ 

7o'c. Blols wore quantnaled by subjecling $€m lo a p_emEson 

analyzer (Beta scope 603, B€iagei) The valu€3 w€re expressed 

relativolo ribosomal RNA after co sclion io. non-sp€cilic binding by 

puc19. The r€sulls ol rhese transciptional run_on analyses {see Fig. 

22) were tound to be hiqhly va.iabre and wore nol us6iul lor anarvsis. 

The rbosomalFNAwas iound lo b€ Pa.ticubnv v4 abl€ du.ing the limg 

cou6e (dala not shown). Ihorsloro, lh€s6 erp€ m6nlsw€r€ penormed 

with RNAse protection assay, dsscnb€d in the lolloring section 

bo|arion oi tolalcellut3r FNA trom msmmalian cells 

Totalcellllar BNA was isolaled from synch.onizsd stabl€ Hela cels al 

various t me points after release lrom he synchroniza on piocedlre bv 

established procedures (choimcynskiAnd Sac.hi 1987). cell cultures 

(co.taining approxlmalery 3.7x1or cells/ml) we.e translered lo 50 mL 

plaslic rubes 6nd centrauged 4'c at 1500 rpm lor s mi'utes cell 

per els w€r€ wash€d lwico In phosphal€ b!n6r saline (l 50 mM NaCl l0 

mM sodium phosphare pH 6.8), and €rlpollets w€r6.€susp€nded in 4 

ml ol guanidinilm soluion (5 M guanidinium, 0-75 M Na cirate pH 7 0, 

0.36 M p mercaploelhanol). The slisponsion was hor'iogenlzed !si'g 

rhe Po yrron, wh ch 

nucl€i and genomic oNA subseqlently' 

'ragments 

1/loth volume ol 20% sarcosyl was added and 500 ,J ol lhe ninure 

was alquoled in 2 ml sl.aigh cent iluge tubes (Ph€nit) The samples 

105

lzizF:cdto'e!.@ototuao@fd@lttLtt 

.--_ 

Ftg.22, 

nttb'iarst'ditolttods 

Nuclear run-on lranscriprbn analysis oi synch'onit€d sttbE 

c€ll tines. wild Vpe FO1OE CAT atd IVSP_16 CAT (H|NF M 

mubrn) stabl€ c€ll .lin€s w€re svnchronizsd bv a double 

lhym'dho block (See Ma€dab 6td Merlods) Transciption €tes 

of dirersrt genss rere me6surcd al 1,3,5'7 and 10 hours by 

nuclear Run-on hanscription (See Matedals and lr"lethods) cAr 

and H4 repr€s€nt siably transfecled cAT lranscn s ano 

endogenols Histone H4 tarudipts r*p€cnv€ly Puc' was 

used as a conlrol ior.onspecillc binding 

r06

Nuclear Run-on TranscriPlion 

wr Fol08 cAT 

h rs 

l- 

-(D 

-- 

m l\.4sP- 16 CAT 

L 

.- 

1- 

hrs 0 1 

7 

t- .D 

10? 

I 

- t- 

- 

10 

10 

- 

- 

UA 

CAT 

RJC 

H4 

CAT 

PUC

Mz F: Cel Ctct. Contot at Ha Gffi fta@tlDn@ 

were t ozen down at -70.C or lsed direclly icr RNA sxkadion. Each 

cell lysale (sri! rri) was supp e.rrenled with 50 ,rl 2N,4 sodium scelate, pH 

4.0, extracted with 5o0 /ltoi phenot (equitibfated with 10 mtv Trs and 1 

mlt €OTA) a.d 100 /lo1 isoamyt6tcohot. Tubos were vortexed tor 10 

seconds aod incubatBd on ic€ tor 15 minutes. Tubes we.6 spln down 

in a micro.cenrituge ar 14,000 rpm tor l0 mindes at 4.c to separate 

the organc and aqueous phases. Th€ aqueous phase was ransrerred 

lo anoth€rtube and t ht ot isopropanotwas added ro precipirale aNA. 

The samplas were tetr in -20.c to. 1 to 18 hr and th€ FNA was 

corrected as a pellet by splnning rhe tLrbss in fte microcentrifuge tor 10 

mrnures at 4.c. The FNA pe ers wers washed with 75% ethanot and 

RNA was dissolved in 360 pt of nucieas€ tree H2O wtlh potyainyl 

suphale (smg/rnD. Then, 40 lrt ot 10 x DNase I b!ffer (T s ph 8.0, 40 

mrvl; NaCl 10 mMi Mgctz 6, mM) snd 20 d ot nuctease lrce ONase I 

(P'omega) 1000 units/mtwer€ added. Tho rubes wer6 incubated in a 

waler bath ar 37.C tor 30 m'nures Tubes we.e removed aftor the 

incubarion and eilfacred wirh 4@ ut at 2s:2at g/v/v) 

phenol/chlorolom/ soamyl atcohot mtxture. The tubes were spun 

down and rrre supernatant was trgnsferied lo anonef tube. The 

sampes w€re exrfacted onc€ with chloroior. Fotowjng ehanol 

pr€optauon, the RNA pottet was washed ons wilh 750,6 ethanot and rhe 

linal pellet was dissolved in 50 /t of ûclease iree warer wilh 5 mg/ml 

r08

NlzF Cett cr.t. canuat oI H4 Gene f6nsc ption 

polyvinyl sulphate. The optical density ol the nucleic acld preparation 

was del€rmined by spedrophotomelry, and 5 rg ol RNA was subjed to 

eieclrophoresis in an agarose gel ro check the inregdty aDd the puiily ot 

FNA (See Fig. 23). 

Flbonucrease protecilon a3say 

The ribonuclease prolection assays (FPAS) were perromed by usjng 

lhe commercially avaitabte RpA lt'M kit (ambion, Inc ). FpAs are apptied to 

deled and quan tate th€ nRNA trom a compiex of ioIat elutar RNA seples. 

To p€rtom /ibonuctease assays, tab€Ied .iboprob€s were prepared, which 

we.e complem€ntary b tho targer FNA, Th€ Mari ScriprrM kit was used to 

synhesizs rhe RNA rranseri s. To generar€ lhe Ribo p.obe ot interesr, ule 

linearized romptate pTFl cAT and pTAj-GApDH w6re !sed, which w€r6 

obtain€d tiom the manufacru.er (Ambion, hc,). The pTFj CAT anti sonse 

conrolremptate contans a 152 bp tragfient ot the chtofamphentcot acelyl 

rransbrase gene. Th6 pTRl-GApoH huma. anrisBnso contro temptare 

Lor'ams a 316 bp l agnenl o. t-e nund- gtyce-aoeh/o€ 3 p.osphate 

dehydrcgenase {cApOH) coding sequ€nce d€rtved lrom exons 5-8. Thes6 

ragments were doned inlo a generic transcrip on veclor (pTBtpLE scripirM 

veclors) which contain the Sp6, T7, and T3 promolers atranged tn landen 

(thrs conskuction alows tor ths use of any ol tho retaled phage RNA 

polymerase tor synlhesis ot tGnscipts). The H4 ribo,prooe was ranscnb€d 

trom a crnstruct which conlajns .ucteotjdes -2r5 b +279 (ie., spanning lns 

109

leE F: C.! W. @turl ol u o.n lld,.tt tun 

Ftg2& 

' 

faodo|| ol 6l oCdar RllA tqn ryr|o|||o||Ecd cr&, 

'FOIG C.Ai db H€l3 c.l tE vr.s sf.drortzcrt by dotlCc 

fiyrddho blod< !d l[{ sr!C.. wsr botid.d dfio tndtcd.d 

er|. 3nd run I m ! 196 a€rrode (696 t'$nebehyde) 96l. 

'110

RNA isolated f rom cell synchrony 

Hou rs 

0 1 234567891 0 

111 

jziz F: cell cycla cannat ot H4 Gen. f@sqFtq 

smal/Nml lragment ot pFo108x) ot the human hisione H4 g6ne, This 

canstuct was kindly provided by Ihomas J. Lasl. 

ln vtro transcripllon reaction 

Tho reagonts of lhs SP6 lM€xi ScripltM kii were lhawed a^d placed on 

ice, The reagenis were added in RNase f.66 Eppsndorl lubes (1,5 ml con cal 

size) in the tollowing ordsr: 2 !lol 10 x tansciplion bufler, 1 !l ot 200 ml',| 

OTT, 1 ,rl ot 10 mM ATP, I /l 10 nM CTP, 1 /l 10 mM GTP, 1 lll RNaso 

inhibitor, 1 /rlor linearized template DNA (1pg),3!1o10.05 mrvl UTP,5 rrlor Ic- 

kpl UTP 800C /mmol a.d i rl ot SP6 BNA po ymeras !r The contenls of the 

lubes were mixsd and lhe tubss wer6 incubaled at 37.C tor 30 minutes. Atr6r 

lhe incubalion, 1 pl oi RNase{ree DNaset was added to €ach reaction and lhe 

conlenls were mlxecr by ticking the tlbes. The samptes were briefly 

centriluged and incubared at 37'c tor 15 minutes. Tlbes were rranstered to 

r@ and the transcription reaction was passed lhrolgh nuctease-tree ouick 

Spinrt collmns to pu.iry rhe radiotabetted p.obe tom unincorporated 

radLoactive ibonucleolideriphosphales. The spgciic acuvity was derermined 

by counting 1 rl in 4 ml ol scintittarion tluid using a sc'nriilarion counler, 

approxmalely &(10.1o 11106 cluots were used per /eacrion ro pertom lhe 

ribonuclease protection assay. 

Hybrldizarlon ot F NA transcrlpts 

Th6 li|sr st6p invotved the 

and 5rrgolsample FNA. Th€ RNA sampres wsre isolated dlring diflorenr time 

@urses usng synchronized Hela 

co.precipitation ot probs Oxlo5/readion) 

cells conraining stably integrared H4/CAT 

112

A2iz F: c.u cwL contat al Ha GeN f@diDti@ 

constructs. RNA samples w€.€ supplemenl€d with 1/10th volum€ of 5 M 

NH.OAC and 2.5 vololerhanoland mixed lhoroughlyby vodexlng. Tlie tubes 

were placed in the 20'C lreozer lor 15 minules. Along with A'e expedmenial 

samples, tso control lubes were p/epaed which conlain s rr9 yeast RNA 

(neqallve cofrrol RNA) FNA was prgcipitated by centritlgation at maxi.num 

speed 14,000 rpm in $e microlugo lor 15 mindes at 4'c. Th€ supernabnt 

was caretully aspkated and dried on sofr tissue (Kimwipeg. Tubes werc 

cenrrilug€d brielly to co lect residual ethanoL at rhe bonom oflhe lube, and this 

r€maining fluidwas removed using a pipene man. 

P€llels were dissotued in 20 ,rl oi solu{on A (hybddiz6tion bufie4. 

Samples wero thrcrcughly mixed by vodelng ior 5 to 10 se€onds and then 

centrltuged to colecr the liquid at lhs bottom ol rhe lubes. Tubes were 

translered to 90 - 95'C wate. barh for 3 b 4 minures ro denature and 

solubilize RNA in rhe bufier. Samples were lhen re-vo.texed and brieUy re, 

centriiuged, owed by ncubation at 42 to 45 

'ol 

. C n a waterbath lor at sasr 18 

hours. Ar lhe end oi lhe incubation 2O0 Al of solutio. F+AX (ANass A + 

RNa* f mixlur€ in giycerol and FNA digestion builer) was added to each 

reacton at a ralro ol 1:100 (v/v). ore controLtube which conra ns yeasl FNA 

(r€presenting u.digesred probe) was dissolved in 200 |jl of FNA digesrion 

bufier withoul lhe RNAse enzyme. The second conrrol lube containing yeasr 

FNA was also lncluded wlh oach set ol samp]es (ths sample sholLd yield a 

compl€rely digesred probe), The iubes wer€ vonex6d ce.trlluged and 

incubated ar 37"c lor 30 minlles ro digesl lhe unprotecled po.lio.s ot lhe

Aze F: Cdt Crd. C@t'.t d H1 G@. f@didion 

single stranded nNA, The rcactjon was stopp€d by adding solulion ox to each 

rub€. The samples were mixed and placed at .20'c lor 15 minulos, lollowed 

by conrritugalioo lor 1 5 minutos io recover th6 probecled RNA P.obe lragmeDt. 

Pellets were air dri6d and I !lofsolulion E (g€lloading b!ff60 was added lo 

A 5% 6crylamide denatu.ing gel was prepared lo resolve he proi€cled 

FNA lragm€nt relalive ro the undig€sied prcbe, me gelwas pr€{un tor 15 - 30 

minutss at 200 vons. samplss were heared at 90i95'C prior lo loading. The 

gelwas run lor 3.5 hours aI300 volrs, d €d underth€ vacuum, and a(pos€d 

to x-oMAT AR film ow.nighr ro ouain a pgrman€nt autoradiographic image. 

Dkect counrlng of ih6 fadioactivity ol rhe prorecled BNA tragmenls bands was 

accomplishod using lh€ Selascope, 

114

RESULTS

trr? F: Cen Cyd. Cnn t ot u. G@ ttu@ldttl 

Analysls olthe H4-Slts ll cellcycle domah 

whlch Int€recls wilh multiple tactors 

Results 

As dlscussed in the gon€ral Introduction. lh€ coordinato regulation ol 

multple histone genes in conjuncrion wilh DNA synhesis is r€quned ior lh6 

o.dered packaging ol n€wly replicating DNA inio nLjcbosomes during s 

phase. This organized assembly ol c.hromatin is nec6sssaiy tor maintaining 

genome slruclure and nuclear archiledur€ during succossive cell cyclB 

stages, as well as lor supponlng c€ll and rissue.specitic transcriptional 

requkements. Cell cycle dependent hisbn6 gsnes r€Pre* 6 pa.adigm lor 

lhe study of pfoliferaton-spsclic g€na{egulalofy m€chanisms operative in a 

numbe. of diverso eukaryoles. Histon€ H4 repr8senls lh6 most highly 

mns€Ned nucleosomal prot€in and ls encoded by a multlgens famiLy in 

mammals, Human histone H4 gen€ ta.sciption has been most exl€nsively 

115

trb F: cett clcL cotuot al H4 Gen. fturecnpttfr 

examin€d wirh a hight €xryess€d H4 g3n€ dgsig.at8d Fo108- This gene h4 

been shown to b6 transcrib6d In a csll cycle cpntrolled madner (Rams€y_ 

Ewino el a/., 1994), and is rBgulal€d by a comPl€x atray oi disral and p'oximal 

cls-acling€lem€nts (Kroeqff sla/., 1987i vanwijnsnera/.' 1989i Birnbauner 

a/..1995). 

Ths pronmd prcmotsr ol this H4 9€.6 

conlains two it vivo genomic 

proreln/DNA lntsraciion domains, sltes I and ll (Pauli 6lal ' 1947), €a.h having 

inrricate and overlapping arang€m€nts ot r€cognitjon eleme.ts lor 

inleracuons wiih mlltiple nuclsar tactors in vltro (v€n wln6n el a/' 1991, 

1987). sit6 tl is r€qui.ed and sunicient ior prcm€ration'sp€cific lransqiplio'al 

acliviry. Howev€r, al l6ast lhr€€ dlstinot classos ol tanscipllon iacloE (HINF_ 

M, -P and .D) whose oNA binding activiliss ar6 indgpenddntrv regulaled (van 

wjnen er a/., 1992), inleract with SitB ll H|NF M which r€cently has bee' 

id€ntitied as the oocoprotein IRF-2 (vaughan €t a/., 1995)' and the relal€d 

tumor slppressor protein IFF-1 €ach .ecognizs the same elern€nt (M'bor) 

located in lhe most dislal segmant of H4.site ll. H|NF P/H4TF-2 is a prnatNe 

H4 gene specllic tactor whlch binds prcximal to HiNF M/|FF_2. The H|NF_ 

P/H4-TF2 inle.acrron site (P_box) Pa.iially ov€daps rh€ M-box HiNF_o 

Gprese s a multfcompon€nt plotein comPl6x conlaining lh€ cycln 

dependenl kinase CDCz, cyclin A and an FB r€lat€d Pro€in 

(€n w'lnen eral, 

1994). HiNF.O int€racts wilh th€ 6ntir6 sit6ll, includinglh€ M-and P_boxes as 

well as rh6 proximally locaed TATA box 

Because of lh6 mol€cular cotnpbxnv of site lt on6 key quesrion that 

116

aziz F: c.tl cy.te cotuot al H4 e$e faNnpll.n 

remains to be rssolved is: whd ar6 the Precise tunctional conl.ibutions ol 

indivldualGc€gn ton mollls and cognate lactofs i. del.r Ni.ing the l6velot H4 

gene rransdiplion in mlltiPle cell rypes? ln parucular, we have obserued 

natural s€quenc€ variations in lhe Sit€ ll s€qlences oI diilerenl H4 genes, 

which abolish binding ot speci{ic ranscription lacto|s (van wijnen el a/., 1 992) 

Ihis raises th€ queston: Does th€ absenc€ ol distinct transcriplion facior 

recognilion sites have tunctional consequences for H4 genelransfiplon, and 

could rhis anply heterogoneity in transcriPlional regulalion ol dillered H4 

genes? In this stldy, we address lhss€ quesllons by syslematicaly anaLyzLng 

fie etfecls ol multiple dislinct muiations in each ol lhe Sile ll recognilion motils 

on H4lranscrpUon ir vlvo n several cell lypes, 

Th€ dbral segment oi Slt€ llls esseniial 

lor hlgh levelH4 gens rrarucription 

The sequ€nces spanning l|ls distal segment ol Site ll located 

immediarely upslream ol the TATA-box in th€ H4 gene (Fig. 5) are highly 

consetued among venebrate sp€cies and are also localed in analogols 

regions in rh€ promolers ot several other human hisbn€ H4 genes van 

winen et a/., 1992). Delerion analyses hav6 shown thal ste ll alone can 

suppo.t a low bur signiticanl revel of histooe H4 (Kroeg€r el ar, 1987) or CAf 

(Bamsey er a/., 1994) reporter gen€ lranscriplon n the absence ol sl€ I, 

which when present sl.ongly augments H4 gene r€nscriplion via inleactions 

wirh sPJ and ATF proreins (Bnnbalm dal., 1995). To oblain an €stimale ot 

the maximum extent lo which lh€ consetued elemenl i. site ll conkibutes to 

H4 gene transcription wlhin rhe native context ol lhe entire proximal promoter, 

117

M2F:Co Crrt6 Cdrtot ot H4G.n. fn.piDtion 

we ntrodlced 7 d srinct nlcleo de subslturion mdar ons (mutanl Mc-7) i|to 

Sne rl. These subsriruions are locared ar ksy cdlseMd .ucleotad6s between 

.r .97 and .77, and 6nconpass a seies oI /n vpo G residus proleln/DNA 

conracrs fial hav€ been derecr€d in jn6ct c€lls (Paullel a/., 1987). Mosr ol 

rhese murations oc.ur at methylaiion inrgrlgrence contacls ior HiNF-M/|FF-2, 

HiNF-P/H4.TF2 and/orHiNF-D (Figs.5 and 6). 

Gel shitt compelltion assays show lhat these mdalons when 

i.corporared n a sir€ lr origonlcigoride (Mc.7) abolish bindng ot H NF-M/rFF- 

2 and HiNF P/Ha-TF2, as wellas sev€rely r€duce bindlng o, HjNF.D to Sle ll 

(Frg. 25) Honce. wrren lhis ori9onucl6o da is cron€d inlo a chrmeric H4 

promorer/cAT construct rhe coresponding murant reponer gene consrrlcl 

(MC'7ICAT) repr€sen$ a rripl€ mula wirh respecl to rhe binding o' these 

werL characte zed Sile 'l bind ng prol€ins. For di.6cl companson, we also 

i.corporated a w ldtype Sr€ ll oligonucleotide OM-3; see Mar€ es and 

Melhods) inlo lhe same h4 p.omote./CAT veclor. The wildlype TM.3 plobe 

medrales he exp€cled bindng ol HiNF M,.P and -O in gelshitl assays in Mo 

ditferenl .unnlng condnions (Fig. 24). Oir€ct compaison ol CAT assay resu Is 

with rcporter gen6 consLucts contaiîng the naural hi$one H4 promorer and 

rh€ wildrype TM.3 p.omoter ar6 nor srarisrics ly difisr€nt (dala nol shown). 

Upon transienl translectio. in prolileralirg Heta 53 c€lls, mutanl MC-7/CAT 

dlsplays up to 10 lold reduc€d promoter adivily relatiw to lhe wildlypo 

conslrucl TM.S/CAT (Fig 28). Thls !h6 r€cognirion morls n Site ll which 

118

ldlz F: Cell6d.c@to! otHa 6q.flt@npn@ 

Flg.24. 

- 

F6"6 

Elactrophor€tlc mobillty lhlli assay condltions lor HINF_P' 

M lnd D. (Lstl) HiNF-M and P d6 dstoct€d in a 4% (mr) 

polyacrytamid6 gal run in lx TGE bufld in ih€ ptss€nce of 

salmon sperm non-sp€clffc DM. (Figft) H|NF D and l'llNF_M ar6 

delEcbsd h a 4% (80:1) potyacrylamldo €pl in 0 5X TBE bultbr in 

$s prsencs ol poly dG dC non-sp€cifrc DNA Comp€tition 

anelysis lvas podom€d wlth a pan€l ol wlild lvps and mLrtanl 

oligonucleolid€s which 6re dt6gnoslic for €adl of fie H4 site ll 

119

HiNF-tvl 

HiNF-P 

HiNF-D 

random DNA poly G/C 

rpEsrE 

+ -++ - 

+ ++ - - 

r:l-5-----ft tr=5qi: 

ol-a=z 

+ ++ + 

+ -+ - 

r.. rt t-et 

Yr- -r-- g Ur -v 

lXTGE 

4"k120:1\ 

32P=H+-sit. tt 

rlltr 

0.5 X TBE 

4o,i,(80:1) 

HeLa NE (5 ug)

NE F: Cei Cvclt ctud o! Ha 6qE f4Bctlqd@ 

Flg.25. Murarbnal analyGls ol H4 9lle lt proi€ln-DNA Interaclions. 

- 

Competjtion assays wer6 p€rlormed by using H4 slte ll wildlype 

TM-3 and muianl GT-g, lNsno, SUAnI aftl MC'7 dasmid 

probes(40Kcpm/reac{ion) and competitor oligonucl6otides 

contarning sp€o'fic subsdtution mulstions (1000 lrnob) which aJ€ 

dhgnoslrb lor each lac!o(, $andatd r6adions fo. H|NF o 

d6lec on (to th€ right) w6re p€dorm€d with 5 pg H9La nuclear 

potein in lt|s presencs of poly (dG

andom DNA poly G/C 

competitor 

oligonucleotides: tr>59i 

Site il 

bindingl 

32P-probes: 

iil 

-L 

E" I 

l 

HiNF.I\,1 

HiNF-P 

HiNF.O 

;.";.,:r 

EF25FS 

Y= I-li-- 

Hr 

Y= 

122 

olcDaz 

- + ++ + 

!

Mz F: C.ll Crcl. Cotuat at H4 Gere faNdo an 

mediate binding of HiNF-r',4, -P and -D together arc capablo ormodurating H4 

gene ransiplion by an order ol m6gnitude. 

The HiNF-P/H4-TF2 btnding mo{finlh6 H4 gene t3 

dlspensabls tor H4 gene promo!€r actlvlty/, vtvo 

Located inthe mlddle oi H4-SI€ llis a NGGTCCGNN mori, (Fig. s) that 

represenrs the most highly cons6d6d sequenc€ in hislone H4 genes. For 

examplei this conserved eleme is locaied in bo$ lh6 human H4,FO108 

gene, aswelasttu analogous humanH4-A gene characl€rlzed byHelnrz and 

colleagues. Cross-competition assays have shown rhat borh genes intefact 

wirh HiNF-P/H4-TF2, bur only th3 H4,FO1OA gene appears ro interacr 

addilionally wirh HiNF-M/|RF-2 and HiNF-D_ Ihs NGGTccGNN,motit 

comprises thB main clusrer of m€thylation intedercnce motifs ior HINF, 

P/H4TF-2, and $e ntegity ol lhis recognition motit is €ssenlial ior HiNF- 

Abfogalion ot rhe NGGTCCGNN,moTIf by substirr.rtion oI th€ I 

nucleo d€s cornprlsing rlris motif (mut6 GI.g), aborlshes H NF,P/H4-TF-2 

binding to lhe mutant GI-g promoter, blr has no eiteci on rhe lnteractions of 

H|NF-M/|FF2 or HiNF-O with H4,Sit€ tl (Frg. 25)- Functional activiry ol ths 

mulanl GT 9 promoter is comparabl6 ro ihar of rh€ wildtype TM,3 promot€r 

(F9. 28). Th!s, inlegrlty or the highly conserued NGCTCCGNN motil, and 

recogniuon by ils cognate lactor HiNF-P/H4TF-2, is dspensable lor hiqh tevel 

kanscriprion ol lhe H4,Fo10a gen6 when olher transcri ional morils in sit€ I 

123

a2i2 F: ceu ctcte contol al H4 Gene rAnstlptt@ 

Specitic variailons In the nalurat soqu€nces of anatogous 

human H4 genes alter lranscrtpttonat acttvity 

To assess tho extert to which narurat sequence variation in lhe 

prorfioters or human H4-Fol08 and H4-A gen6s may atf€cl transcfiption, w€ 

d€signed ndo distinct mdatiors dBsignated lNS.10 and SUB-11, The 

conserued region ol Site ll in rhe H4-FO108 gene ditfers trom the H4,A gen€ 

by rwo mura ons (contajned in tNs,10) and six substi(nion mutalions 

'nsedion 

(€pr€senred in mLrlani SUB-11). On€ insenion mlrarion in th€ tNS-jO 

p.omoter is rocaied between nl-87 to .86 in the middt€ ot the HjNF-M/IFF-2 

recognilion site (M,bor), *tereas rhe second insertion is imdediatety 

downsrream or the NGGTccGNN-morit (see Fig. 6); lhe subst'tution nutaiions 

ol th€ SU B'1 1 pfomoler are clustofed botw€en the postt ons of the insentofs 

(Fig.6). Gel shitt analysis shows lhat lhe tNS,10 promote. inreracts wjth HiNF. 

P/H4-TF2 ad HiNF-o, bur due ro ns murarion ot the M-box has lost the abitity 

to bind H|NF-M/IFF-2 (Fig, 25). h conkast, muia oos in !h€ suB-1l promoter 

atred HiNF-D blnding, blt ailow binding (ot hiNF-M/lFF.2 and HiNF-P/H4.TF2. 

Ihese fesulls esrablish that ttr6 tNsno and suB,11 promoters represent 

unique and specilic promoter mutants ior HiNF-M/|RF-2 and HiNF-O, 

/r, v,vo analysis oi hislon€ promoter activity shows that mutant tNS.10 

has 3 fold reduced ac vny. ln conrasli ranscnpdon mediated by muran! SUB- 

I 1 is comparabls lo rha ol wildtypo TM.g (Fig. 28). Ths resutt wirh mutant tNs. 

10 lndicates that natural s€qu€nce variarion has tlnctionatconsoqugnces ior 

124

A21z F: C.tl Cvcte Cdtat at H4 Gqe tu$cti rid 

rh€ e{e.t ro whlch h4 promorers are activ€. B€caus8 the lNs 10 promol€r 

rcpresents a specilic H|NF-M/|FF-2 r€cogniton site mulart, the obserued 

dec.ease in reporler gen€ exprssson wnh lhe INS-1o/CAT conshuct 

sugg€sls that HiNF-r\4/ BF-z is importani ior hlgh level H4 gene lfanscription 

However, fte efect observed wilh lh€ INS-10 promoter is several-told less 

than rhat obrain€d wilh lh€ Mc-7 promotor. This indicales that addi onal site ll 

laciors contribute to lulacrlviry of rh€ H4 promoter. w€ nole that th€ lwo 

inseruons in Sito ll embod ed by mltant INS-10 pol€ aly may aliecl the 

spaiial alignmenr ol orher histone gene promoter lactols interacting wilh Sites I 

and ll. Additional M.box muhnts werc assayed to excrude this contoundidg 

lactor lor a deiinltiv€ conclusion (see b6low). 

absence of a statisll€lly signilica effecl on ranscription lor mllant 

su8-11 suggests rhat the role ot HiNF-o, simjlar to HiNF-P/H4TF-2, in 

mBdiaring high levels ol hisron€ H4 gene kanscripion is limited. The rosults 

obtained wllh the INS-10 and SUB.11 promoter mulants suggests that dist nct 

types ol natural soquence varialion b€tween human H4 genes hav€ dili€rent 

ettecls on H4 gene lranscdption. 

Slmllarllles in muratonal stlacls on H4 genetranscnptlon in dlstlncl 

prollt€rating csu lypes dlsprayrng dliterent cell gfowlh and tissuesp6cnic 

phenotyprc properties 

The MC-7, GT€, rNS-10 and SUBnI muranl CAT construcG wers also 

analyzed in FOS 17/2.8 osteosarcoma c€lls and normal diploid calvadal 

osreoblasrs (FoB) ro assess smilarres in mulalional efiects on H4 gsn€ 

rranscriprion in diiieient cell rypes. similar to HeLa s3 ceruical carcinoma cells, 

125

aziz F: c.ll crd. cdtd 0t H4 Gene tansctibtlon 

lhese cel types contain detedable levels ot HiNF M, -P and -O when aclively 

prolilorating. Tho resuis show that th€ magnitude of mutaliona ofiects lor 

each oi lhe mutants is very similff in each cell type (Fig. 28). Thus, lhe relaljve 

imporlanco ol lranscrlplional recogni on molils and cognate fadors in these 

hree prolileratng cell types is quantitritively similar, rcgatdlsss ot difterences 

in th€ expression of lissLro-sp€cllic and cell growlh r€laled PhenotyPic 

aboltshment ol specitrc H4-slls llfscognltion motrs is dominanlover 

€ltecis on ih9 pulative spatiat gllgnmenls ol H4 iranscrlptlon laclofs 

we tested a set ot lhree additional H4.site ll promoler mulants ocN- 

12, FAM-14 and FAM-15) by gel shitt assays. Mutarn TCN-12 contains $ree 

nuclgotid€ subslitltions al spscific posilio.s in site ll, which do .ot affecl 

blnding or the thfee H4-site ll bndifg proieins (F,9. 26). However, FAIV-14 

conra ns an additiona precise delelion ol nt-80, which resulls n abolishmentof 

HiNF P/84-TF2 bi.dng bur has no eilect on eilher HiNF M/lBF.2 or HiNF-M 

(Fig 26). FAMr 5 contalns an additional short deletion in the distal terminus ol 

H4-Siie ll which abrogales H|NF-M/|FF-2 binding, but does not allecl HiNF 

P/H4'TFz or HiNF-D (Fig. 26). rhus, thess prolein/DNA interaction sludies 

iunhe. delne rhe blnding sits rsquir€ments ol HiNF-M/lRF-z and HNF-P/84' 

TF2, and 6how thar FAMr4 and FAl\,4-15 reprssent iactoFspecilic promoier 

nulants wilh alle€d spacing ol laclors binding at sne ll 

Funclional analvsis ol the FAM-14 promoler shows thal lra.scriptjonal 

adivity ol rh€ FAM-I4ICAT construcl is approximately equivalent lo thal ot 

€onsrucls TM-3/CAT and TCNj2/CAT, This rcsllt indicates lhal abfogation 

126

,'12 E C.t Ctcll4 Cqfrd al Ha eap lwlw, 

ng.2B, Uutatlonal rnalysla or Ha slta ll p.oteln DNA int€ractloG. 

CornPttion analysis v€s perlo.m€d by using H4 sie ll wildtyP€ 

TM3 and mutant TCN-i2, FAMi4, and FAM-15 probes, using 

lhe same amount of prob€, tt6!t nucbar prol€in' compelilor 

DNASand running conditiom es describ€d in Fig l5 

127

compeutor 

oligonucleolides: 

Site ll 

binding: 

eP-probes: 

&o 

t* 

. 

HiNF.I\4 

HiNF-P 

HiNF-D 

random DNA poly GIC 

a i 

h= ;3*d ZoX!. 

; 

Hr I -rJ- .u_!-o 

:z g= il'l- !t - --o 

:= 

i> 

l?8 

l'!_ 

E - *-o

Aziz F: C.tl C1lcte Cotu.t ot H1 C4n6 r6Mbn@ 

ot the HiNF-P/H4 TF2 interaclion silh H4.Sit6 I rogelher with lhe @ncominanl 

ahe,alion of the spacing belr€en the M-box and the TATA-box, does ôt havg 

a signilicant eff€cl on H4 promotff activity (Fig, 29). The simjlarili€s in lhs 

.esulls lor lhe liNF P/H4TF-2 mutani promolers GT-g and FAIV-14 (Figs. 28, 

29) co oborate tiie conclLrson lhat H|NF-P/H4TF-2 do€s not contribute 

siqnilicanty lo lhe level of H4 gene transcription when othef Sts ll taclors 

rcmair capable oi binding. lvor€over, lh€ ditf€r€nce in spacing ol lho lV- and 

TATA-box berween $ese consructs, which could alter possible inreradlons ot 

HiNF.M wirh TATA bjnding protein TFtt-O, does nor significanily afiect rh8 

olncome ol lhe resulls. 

h c$mrasr, the FAM-15/oA1 conslrlcr is expressed ai a 3 lold r€duced 

l€v€l rolative lo the wiidtype TM-3/CAT consl.ucl. Th€ lhrce lold reduction in 

report€r gene expression obtained wth both INSJo/oAT and FAMn5/CAT 

(Figs.3 and 4), whch each repr€s6nl H|NF-M/|BF-2 mltant prornol€rs, 

supponsour modelin which th€ HiNF,M/laF-2 inle.acion wilh H4-sire llplays 

a key roie in determining lhe level ol H4 transcriplion. In addition, the otfocts 

obsetued with the rNs-10 and FAM-15 promoters are quantirively simjlar, 

ahhough lhese conslrucrs diller in the spacing wilhin Sile ll and between Siles 

I and ll which @dd change sparial intoracrions betwee. F_omorer ladors srill 

Taken togelher, ihe s milaril ss n resuts lor GT-g versus FAIV- 1 4 and 

INS-10 v€rsus FAlv 15 in three d fier€.r c6l typ€s ars cons slenl wirh th€ 

concept that difierences in th€ spalial alignmenis oi histone H4 gen€ 

129

A1i2 F: Ceu a:rcte cotuot ot H1 G@ ftqNtioti@ 

transcrip on lactors binding ro mutant prcmot€rs do not hav€ dominant 

efiects on dete.mininq tlre level of reports. geno transcnplion- This tunclional 

flenbilty in nucleotde spacing is consst6nt with sub!€ del€tions and 

insertio.s niat appesr to have occud€d duing €volulion ol th€ cons€v€d 

sequences in sle ll(vanwinen ota/., 1992). 

Ta.gsled murarlon olrhe HINF-M/|nF.2 blnding site reduces H4 

hlroduction of rwo substiturion mutarions {mutanl MsP-16) precisely 

designed in lhe rsgion ol th. M.box lhat doss nol ovenap wilh lhe H|NF. 

P/H4IF 2 binding siie, pre!€nts binding ol HiNF-M/|RF-2 bur nor HiNF- 

P/HaTF-2 o. H|NF O b Sne ll based on g€r shitr analysis (Fig. 27). This 

mltation and additional substitullon ol three nucleotides in lhe NGGTCCGNN 

molif {mutant MPIM-17) abolishes int€raclions of both H|NF lMllFF 2 and 

HiNF-P/H4-TF2 wir.,i Sile ll, whil€ reducifg the HiNF-D/Site Ll inleraction (Fig. 

27). Slbslitution ot rwo guanine rssidles (mutant lGlM-18) representing 

methylation intederence .ontacts lo. both HiNF-M/|FF.2 and HINF-P/H4-TF2 

abolishes HiNF-P/H4-TF2 and rgducss binding of HiNF-rM/lRF-2 and HiNF-O 

ro si16 [ (Fig.27). 

Comparison oJ reponer gen€ erprossion with lhese construds relative 

to the wildlype H4 promoter reveals lhar th€ HiNF M/IRF 2 mdanl MSPno 

conslruct displays 2 10 3 lold reducsd promoter act'vity (Flg. 30). Ihe 

quanlitative resembla.ca of rhs mutarlonal €lrsct wirh ihe 3 fold reducrion 

obserued wllh rhe olher rwo HiNF-M/IFF-2 mutanl conslructs iNs-lo/cAT 

130

MF:@a},d.ttuddluat tn ffil 

ft.n. l|||id.n t ruty.|..of Ha d|. n p.€L|l Da{a In .|cuotla 

Oodtp€Bioo d$tsb u4 p€.lbrtn€d by tr.lno H4 wlld typ. pFP' 

I D|bbe dd dt .s* HINFM b[td.lo d. ,fl.8't ptobr3 MSP_ 

18,MPM-17 trd lGlF18 r,frh t'r .i|tb coftp€[tor Dtl^8 and &. 

rurnhg cadbrt, €3 d*.rlb.d h FE 15. 

131

andom DNA 

poly G/C 

competilor 

oligonucleotides: rpE;FE tr:59 i 

S,le ll 

binding: 

'_P-probes: 

! ; 

E> 

E> 

EJ 

HiNF-M 

HiNF.P 

HiNF-D 

$E j f--- - 

$= 

Y= 

112 

F

azh F: cer cyde c@trot ol H4 G@e tuantdtotbn 

ed FAM'15/CAT, but in the abs€nca of lhe potenlially conlounding 

parameler of binding site spacing, unequivocally establishes rhat the HiNF. 

M/|FF-2 recoqnition elemenl is ossernialior high level H4 transcriplion. 

Muranr [lPM-17 €ipressos th6 CAT r6porter gene ai a morc redlced 

levelthan MSP-16 (6 to s lold reduction) (F g. 30), simlar to mlrant i,IC-7 (s€o 

Fig.28). Thus, while mutalions in the H|NF-P/H4TF-2 and H|NF-D binding 

siles each by itsell do not alf€cr tl4 promoter ac vity (murants GT 9 and SUB- 

1 1 ; see Fig. 28), it appea.s t'at thes€ factors play important auxiliary roles in 

derermining H4 gene rransciplion. 

Mutations in the IGM-18/CAT clnsruct dedeGe reporter gens 

expression approximalely 3 lold relativo to rhe wildrype H4 promoter, but 

expression with IGM-18/CAT r€mains signilic€ntly hgher than that observed 

lor l',4PM.17lCAl As bolh oi thos€ consrrucrs do not bind HNF-P/H4TF,2 

and havc similar redlced afiinity lof HNF-D (Fig. 30), th€se mulants dttief 

only in lhe relarive srrenqth ot t-UNF-lV/tRF-2 binding to Site tt. Th!s, 

quaniitative ditferences n occupancy ol Sit€ ll by HiNF-M/lRF-2 in vitrc appaal 

to resull in conespondlng quantitative difierencas in H4 promots ac-liviry /h 

conlributions ol the H4,slte ll blndlng proiehs ro human 

hislons H4 genelranscripiion dudng the cellcycte 

Studies desc.ibed in pfevlous section indlcate hal marimal 

transcriplion oI celcyce controled histone H4 genes requnes lhe iniegrated 

activities oi at least three dislnct lransdpron taclors, HiNF-1V/tnF,2, HiNF, 

133

EEF:Ce Olcta Cont t ol114 Gene l@nsc ption 

Ftg.28. Transle rranslectlon ot ssveral cell rypes wlth varlous 

mutated H4 promolsr CAT constfuois (A) H4 Slt6 ll wild lyps 

CrM.3) and mulafl promoter CAT construcrs (GT'g, LNsno 

SUB-11 trd MC-7) wef€ assayod by transient lranslod ons ln 

human HeLa 53 ce.vicalcadnoma (HeLa celrs), nomaldlProid 

,at calvarial ost4blsst (BOB) c€lls and rat osleosarcoma 122 8 

(noq cels- The level ol expression for ead' mutant Promoler 

construct was evaluated as p€rc€nt cAT catuersion relati!€ to 

lhe wild lype control conslfud (as described in Marerials and 

Melhods). Black stars, open diamond and black diarnond 

indicate thes€ mutations which r€sullsd in a slg nilicanl decroas€ 

in promorer acrivily clmParcd to th€ wiu lyp€ consruct 

Statis cal signilicanc€ was eslablished by aNovA analysis. (B) 

Fep.esenhlive auloradiog,am ol CAT assays pedomed in A 

(results shown lrom Heta c€lls werB similar ior all thre€ cell hes 

tesred). cM, rcrhloramphsnicol substrate, AccM, aceiylaled 

chloremphenicol. 

134

B 

i= rr ,lt 

a 

o o? o 

o

, F: cal qcb cotfrtl d rh 9.n ftuaf@, 

F9.29. ft-aLt lanalloilcda ol -v3i odl lypla $fn '|do|l 

rr|t.Lr, Ha promory cAT pa[t||cL, (A).H4 Sr.. wld typ€ 

TM-s ard fli.nr p|UnoFr TCl$l2,F ll-14 sd FAll/Fls C.AT 

cq|atrrt! $rn -€ay!d by trr*rt ttrdecrb.rs h tnr€€ 

dt€|rrr cd |h6 HrL!. Bo€ sd noa cdb B d6.rlb€d ln FE 

19. (B) ,B€FE lldv€ euotrdodatEn ot CAT asays 

196

Mulalional ..rly.l3 ol Ha .ll. ll lsino 

H{ PromoLr/C^T lu.loh coturrucrr 

e de 

;= il 

'J 9d 

a 

o 

o a 

? 

I l7 

.; .i 

o 

a 

t

kL E d N. Cdfr.t ol k e.rE ltlr&tu 

Fb.30. TnrEient lranlLclbna ol aavtr.l ctll typelt wnh vailoua 

nrdttad H| ptomotar CAT co[tuct3. (4, +14 9'lo llwlld lyp€ 

Foloa GqT ar|d HINF-M Urihg Bl!6 mt,ati prornot€t MSP-16, 

MPli,l-17 and lci&18 C.AT Con3uuct8 \r€rc ass6y6d by trAndent 

transl6ctions In thrre dlllsr€nt call lln€s H€La, FOS and FOB 

cells as d€s.rlb.d In Flg 19. (B) Boprus€nt ttue 

autoE fiodl4ran ol CAT s3€y3 prrbrm€d h A. 

134

B 

,tt la 

ri 

I t9 

ol H4 promoler CAr 

blndlng slle mulants 

: 

it 

9,i

Mt F: Cett C\.1e Catud cn Ha Ger. I6nsciotion 

O/CDP 1 and HjNF P/H4-TF2 thal bind ro the Sile ll cell cycle domain. 

Howe!€r, th€ contriburions ol lh€se proroios to rhe mdimal level ol H4 gene 

transcriplion during rhe c€ll cycl€ remains io b€ €stabilshed. In the slldies 

d€scribed in rhis chapter, a seloded ser of mutanr H4 promoref reporte.g€n€ 

constucls which were staby lnregrated into the genome of hlman HsLa s3 

c€lls were used, and analyzed N4 prornot€r aclvity during lhe cetcyce n 

synchronized cells. In contast lo prevlous suggesuons, oL/ cellcycls snalysis 

reveals rhal mutarion ol rho H|NF.M/|RF.2 binding sire (mutant Msp-16) which 

dramatically reduces rhe rat6 ot historc g6m iranscripiion, does not atrer ths 

cerl cycle @nvolled enhancemont ol H4 gene promole. aclivily during earty S 

phase. MLta{on ol the HiNF,D/CDP-1 binding sile (murant SUB-j1) or 

combi.€d muHion oi all thro€ hislone gene ranscription faclor bindi.g siles 

{mutanl l\,lPM-17) has subtle bLrt slatislca ty signifcant etiecrs on either tho 

timng or lhe magnirude of H4 g€ne transcfiplion rale. ]t is conctud€ that 

lemporal conrroi ot H4 g6n€ ifanscription dlring the celt cycte apparenly 

rsquires the integrated activilles ot mult ple ranscription factors. 

Ulilization ol RNase proiecrlon assry3 to. studying H4 

gen€ lransc.iprion durrng the celt cycte 

IrasciipUonal control ol lh6 H4 gene ranscription has previously b6€6 

analyzed using run,on rranscriprion anatyses (Ramsey-Ewing er a/., 1994). 

However, it was obsetoed that ihis rechniqus undsr the current erperim€ntal 

condrions does nor represed a r€li€bt€ m€rhod tor moniloring subr6 

vanarons in H4 gene rransc.iplon This s due in pad to ab€tranly targe 

lluclLrations in radiogreph c signals to..ibosomatRNA synlrests which is used 

140

a2i2 F: cdl dcte conrrot o! H1 ctu fdh*tiotlan 

tor normalizing the blors. Dudng this lhesis, hwas eshblished whether FNase 

prolection sssays can be used lo measurs H4 gone Promoter adivily during 

The srabre celrine conralnlng lh€ wildtype H4 prcrnotff (Fo108/cAT) 

was synchronzed and hatuested RNA sampl€s at hourly lime-points lolowing 

release lrom a dooble rhymidine bock, FNA samples were hybrdiz€d to 

inrernally labelled fadiolabelled rlboprcbes speciic for CAT, hislone H4, and 

GAPoH mFNAs, slbjecr ro RNAso digestion and qel eiecrrophoresis of the 

dige$jon-products. 'rhs signal lor CAT mRNA represents a medule ot th6 

extenr ol histone gene promoler aclivily, which is diredly relaled to histone H4 

gen€ t/ansciption. The H4 mRNA leveldelectsd in our assays is lntluenced by 

bolh kanscription oflhe gene and post-kanscriplional regulation oi tho mFNA. 

Histono mRNA is slabitiz€d dudng eady S phase and desrabilizod at rho 

completon of s phase. GAPDH mBNAs encode a consiitutive house- 

keeping'enzyme involved in gycolysis, and can be used lor normalizallon of 

The results ol a iypical analysis lo. the wildtype H4 p.omoter in 

synchronized cells containing lhs H4-Foro8/caT construct (Fig. 31) shows 

lhal CAT mRNAs are mdimal al 2 to 3 hr atler enlry ol cens inio S phaser 

preclsely as prcdicted based on dired measuremenrs ot H4 g€.e lranscriplion 

by run-on anaLysss. ln mnrrast, H4 mRNA conrifue lo .ise du€ lo post. 

lranscrptional evenls thal lac lilate furlh€r accumu ation oi histone mRNA 

levels (F q.32). However, GAPDH mFNAlevels romalnconstanl during th€ cel

A.*F: Cen q.h cdtuotot Ha 6an ranfl4t@ 

Ftg.31. BNase Proteetlon a$ay ot caT mnNA h synchtonlz€d 

cetls. RNas protection assay was psdormed as desc.ibed in 

Materials and Molhods using high sp€cilic acltity lull length CAT 

prot€ (240 bp).LanB 1; mol€€ular we(lhl marke.. Phi X174 FF 

oNA was digested with Ha€ lll and end labeled with !P pATP 

L€n€ 2i conlrol a, Probe alon€ hybtidiBd wih 5 rg ol yeast 

RNA, and not digssted with RNas€. Most ol ihe signal is lull 

l6ngh FNA |ranscriPl, although some small€r hetrogeneous 

radioactivo mateiaL can be sssn- This apparcnt degradanon 

which is pr3sumably dua lo radioactive d€cay ol tho prob€, is 

unavoidable. Lane 3i digestion conlrcl b, prob8 hybiidized with 5 

llg ol Fast FNA digesled wilh statdard amount ol BNasei no 

signal can b€ seen in this lano. lanes 4-19; BNAe prote€lion 

assay ol RNA samdes rrcm Fo1O8 CAT stabt lrensl€€ied HeLa 

c€lls cotlecred at indicated tirnes after release from a double 

lhymidine block. A(ow lo the lett shows lull lengh probe. and 

a(ow to th€ righi shows 1 52 bp of protected cAT t agme.l 

.:

lii> 

r ra 

CAT mRNA hours aller rglgase 

E= 0 1 2 3 4 5 67 89 

!l 

t 

I 

I 

a 

)41

3!j{99!caaw arir c",. r_*rooo 

FI9 32. i aaa tmlaoton -ay ot Ha rr8l,|l| tn 3ynchrciElo catb. 

ft{6a P|frdbn a59Er vr€s psfoned 6 d€sdib.d jn Fb 30, 

o(c€BuE gh €p€dffc rdrv ft{ brElh i&ro€ tt4 Fooc wls 

rr.d. A.row on f|c t€ft |rdbdnE ntl bEdl !4probe d ,rcr, 

dr tE .lSh t rtqdtg probcGd H4 mRNA bat

H4 mRNA hours aller release 

E!0t 23456 7 8I 

- -.ral|atalo- - - 

145

I 

-a2b F: cel cyde c@xol d B1 aa@ ha6ciDtian 

cycle (Fig. 33), and any ilucluations ln the obserued GAPDH L€vels r€ile.t 

spurlous signa s that ar€ not srat stically slgnifcanl. 

Muiatlon ol H4-Sll€ lltranscrlptlon taslor blnding sites has 

subtle etlecls on cellcycl6 contfolol H4g9n0promoter activity 

It was obserued lhal CAT levels are mo(md at 3 hr and lowest al t hr, 

Therclo.e, we simpliii€d our €xp€rim€nlal proiocol to lacilllats p.ocessing of 

muluple sampl€s tom synchronized c6lls io p€rm't proper sratstical analyses 

ol our data. a series ot 8rp€.imonts wer6 pedormsd in which lriplical€ RNA 

ssmples at 3 and t hr after releas€ lrom dolible lhymidine block were isolated 

and subjecled lo FNas6 proteciion analyses (Fig. 34). The resllts show thal 

the wildtyps (H4-FO108) and HiNF-M mutant (MsP-16) cell lin6 hee 

approinaely hree times more CAT mRNA during ea.ly S phase (3 tu after 

release) than duing GzlM (9 ft alt€r release). ln conrasl, this ralo G 

significanrly less in HiNF-D mutant (SUB.11) and lhe HiNF-M and HiNF-P 

double binding site mulant. h each case ot hese mltant promoters a 1.7 iold 

decreasewas obsetued between CAT mANA levels at3 hrand 9 tu (Figs.35, 

36). As conkors T3lT9 rario',s or GAPOH (approximalely 1 told) and histone H4 

mRNA (approximately 10 lod) was measLrred whlch pa.allels llre rale in 

oigoing DNA synlhesis (Fig. 36) Note however lha: lhe 3 h. tme pcinl does 

not coincide with the peak ol histone mFNA expression or DNA replicaton 

which occurs beb,veen 4 and 5 hr n each cel ine. ll was concude that the 

HiNF-M/|RF-2 bl.dng sire is dispensabe lor cell cyce contro and that 

interactions olHiNF.D/CDP.1 and H NF.P/N4.TF2 with Sre ll may conlribute 

to cel cycl€ reg! ar on

- ,dEottq,4ctd/oto,,l/.!'/t'ld[,,&t;n 

Ft to. tff-a ploLalod r...y of OAPDT nRIA h srnctlrorE d 

oL EhF prtadoi ey vi's p€rbrn€d * d€scrbod h 

FC I 56.fa tigh 3p.c{c ir.fvity |l| E tgtt SApDH 

Glyc.r*.ltya.9dt0add. Itdtydtlg€rBse 916 bp) prob€ 

lLd. lnq, dt fie l€fr lrdlc6drE tul t gtt cApDH p.obe ard 

grq oar tE dgtr hdc*tg p.rt€ctad ctapoll mFNA bsr|d. 

147

-= 

GAPDH mRNA 

= = 012 

66 

hours afler release 

3 45 67 89 

.a.flolloa 

fl I 

148

tzE E Cdt atc!. Cotttot o, tU C.@ 7d@.ipti@ 

Flg.34. Anrly3b ot gans sxpre$ton ln synchrontz€d cel ttne3 

dunng S and M-Phase. FNa$ protection assay ot 

synchfonized H€la csls srabt hanshcted wirh witd vp€ Fo1@ 

CAT, mutant Sugnl CAT, MSp-18 CAT and t/ptv-17 cAT was 

p€riomed as d€s.ib€d in t',td3tuts and Merhods. Foltowing 

rcl€$6 lrom s€cond hymidine btock, FltA was isotat€d at 3 

hour and I hour ln tripticale and treatod as indivtduat samptes 

throughoul the experimeniProtociod band of cAT mRNA, H4 

mRNA 6nd GrtrOH mRNA ae shown with €rrows. 

149

Fq *' 

I 

E 

t 

li! 

lil 

tlr 

ilt Frlr 

lill 

lill I 

I 

I 

llr 

rl J; 

tl, ' 

I t: 

rl 

r50 

I T 

wl Foloa 

I I tttlr 

I 

t rtlr 

Mubnl SUB-|1 

liln 

lillr 

liil,l lillr

MF: cd @h oottut &l'to.tl!ialttol4.n 

n$34 

qr.ltron ol T3nf (rt.. Ihr t d. 1nrrEtsd tt 

trntdlcc raylb d t|€ aud*liEphb sigtd dtsh€d h 

BNrrs lrotsc{on trom wld lype snd cl'lf€r6nl mubrt cell lln€3' 

(n *rora ta rutb€. of ayndrorl.! p€.{odt!d.) - 

151

(! 

(! 

t- 

(Y) 

F 

o 

'F (E 

d 

z 

l 

t52 

+ 

ci l!

!d,F,d@rc,rddP,.e"t-'"W 

FEr 94. 

8*tb 

Orap|lc rapaasson ot m^g tn|. The graPh repr636nts 

o|l9 ilHon ol FlNt€e probcrion dat' ol RxA rampt6 coxecred 

ln tlplcalr .t3 erd S hours tirt' pdnt d!'k iEE hdEd€ tlal 

tts df€G.Es hT3/Tg tdc bdn!'n fi' rU ryp3 ttd lwo ol 

aF ntrJant cdtttu.tr (slj&li "d MPri+1? CAT) b ddicdv 

dgnnca P vdlec ero < OIx)5 h bo|h ca6€a' 

't53

E 

Comparison ol TgT9 ratios lor cAT, H4 and GAPDH MBNA 

o 

16 

cAT 

154 

Constructs 

I Fords car 

A su&t 1 car 

I sPr6 car 

E i,Plr!17 ca-r 

GAFDTI

aj| F. Cdt Cttt. C@ud ot H1 cetu rnnsu,p!,@ 

It was furrher €xamined rhat lhe signficance ol reductions in rc/rg 

ratos lor the CAT rnFNAsignats obse.ved with 1wo ot the mutantH4 promotsr 

constucls. Full scale cel cycte synchfonjes with tour difierenr c6I linss wef6 

perlomed (Figs. 3740) lo address whether reduclions in lhe redued T3/m 

fanos ar€ r€Lated to changes in the maqniiude ot hision€ gene irascipnon 

during ea y s phase, or wherher this ratio r€lects a shft in lhe timing oi rh6 

peak ol H4 prcnoter aclivity to tor exampta eartier or tster me-points, The 

RNas6 prot€crion assays show that CAT mRNA tevets diven by r're I!,tSp-16 

mltantH4 promor€r,which can not bind HiNF-M/|FF2. ar€ vory simitar to CAT 

mnNAlevels obssrved lor lho witdryp€ H4 promoter FO1O8 celline (Figs.37 

and 38). In com€st, mutant suBrl which is not capabt€ ot bindtng HiNF-D, 

appears t0 display a modesl delay in the riming of maamat H4 promoier 

adlvity (Fig. 38). Fu.rhermore, rhe muiani MprMrT consruq appears ro navs 

a smilar limlnq in lhe peak ol rnaxlrnat tansc.ipton, but dspays pantaL 

retention ol cell qde dependent etevations in H4 gene ransc.iplton (Fig,4O), 

ll is concluded that cell cycle conl.ol represents a comptex mechanism which 

nvolr'es s-bne co_Lbu0ons oimurripl€di nct pror€,ns 

1!i

Azd F: Cd Ctcle Ca\ttol ot ltt Gtufan*npti@ 

Flg.37. 

R€r"h 

cell cyct6 rnalysls ot thewltd tlyps H4 promoter cAf stabte 

cslt llns. (A) t3Hl Thymidins incorporation was m€asured at th€ 

lndi€at€d Imes lollowing fsleas6 lrom sscord thynidine uock 

as d6scrib6d in Maleials and Merhods B€sults ars €xp'€ssed 

as clunrs per minuls ot acid prcdPitat€d pn1 t'vmloi* 1e) 

RNass proteclion alsay ol H4 CAT and GAPDH RNA as 

described in Figw€s 30'32 (C) Ouantitatlon of th€ assays in pad 

A ushg a Belascop€ analvz6r and Prssented as perc€nl 

maximum oata points a'6 the mean valu€s trom M0 

independent expedments and th€ ba6 repcsent the differences 

betu€en each pan ol r€spectivs values The signal associated 

with caPDH mBNA did not aler signilicantly throughoul lne 

156

M9n!*s!Fon4t 

6 

e 012 3a5a?!e 

| ---.;...-;.*"*^ 

dq-,4- d s rr*dt 

t-------- -|--'"-, 

| .....---.. 

I 

t51 

| ' GAF.H.'M

:tE &I qiE{, ccaoi oftaa Cer tir fiFdh, 

Eg.98. c.r oyc. .'|.rrd. & !1. u8p.ta (]0xF-u u!.rg H4 

promorar caT cdt In . (A) l+I-Trrymidf€ hcorodion. (B] 

AN6€ p.lilctioo essly. (C) Ouerifradon of BNAS6 prd€ctbn 

osay. 3€6 ddaE h F9. 35. 

158

t- 

t----. .. 

f. e.o.oo _ oo 

r59 

c.qar'*dmlllqfudrr 

rl 

'l 

I

kEE@q.l.co''o,a,tl,od,',rutg/bt!,. 

Flg.99. c.N o!,c|o .idyc! ot th. 

promote. cal cefi In6, (a) 

Frlase p.oHor| -say. (C) 

a&ay. s€€ d6iak h F|g. 95. 

r60 

Slr}l t (tlNF.D mutrn0 H4 

PH]-Thrnkjho 'Incqpodton. (B) 

Ouantdion of FN*a polsrdo|l

I 

c 

q.*+drr!d€a 

l-..tnr.{.*-, 

l---.-----1 

. *''* 

l-ro.r.q16 | . n rot "o, 

l5l

ibEc|,Otd.c!,'t,dlHao'tllep,rcl 

Fg. {O, Cla QyC. an W ol IP|}17 (t|i|fD|| .rd P sbrn) H' 

profiror.. Clr cdl lln . (a) itl_'niymldln lncorporulion (B) 

RN!3. proi.cticn a$ay. (c) ouatnrdon ol iNAte Fd€cbn 

a6say. S€€ dabl! h Fq, 35. 

162

*'fr-[-94'q' 

holr3a6at 

I 

caqd.*-d*F*-t 

l"'.-...- l'*'* 

t---------- | - crr's'rr 

I rrn1ar l'*-."* 

163

DrscussroN

I 

Atz F: c.u crcte cdnrol ot H't Gd. ftur@dpndn 

iscussion 

This study was aimed ai dissectlon ol th€ complex molecuEr 

organization ol ih3 H4 gene promote. sns ll sequences which are indtumental 

in mediating prolilera on-spscliic transcridional comp€tency a.d encods lhe 

crucial iniorna on loi H4 gene c€ll cycle rcgulation (Fa'isey_Ewmg el €/- 

1994). The rosuns unequivocally establish thai lhe multiple ovenapping 

recognirion s€quences lor cognate vaGcription laclors in Site ll logether 

modulate H4 gene t€nsc.iption levels by al leasl an order ol magnitude we 

have also shown lhat lhs r€cognilion soqLrence oi H|NF_M/|FF_2' which 

coincldes wirh lhe H4 cell cycle regLrlatory element is the dominant 

component and nodulates H4 gene transciplion levels by 2 io 3 lold. ou. 

dala slggest thal HNF-M/|RF.2 as wellas perhaps lRFn which recognizes 

thesame sequence, is involved in delerminng both basallevels as wellas lhe 

16.1

I 

azz F Con CYb Cnror ot H4 G@e ntns.ldion 

cell cycLe enhanc€d transc ption al the G1/S lransiton Thus, at app€ars that 

HiNF-M/|RF-2 is analogous to oTF-1/ocTn which pedorms a simil* dual 

lunction in cel cycle regulalion ol human histone H2B transc ption (Flelcher el 

a/.,1987). 

Ou rcsulls also rcpr€s€nt the li.sl direct indication that natural 

sequ€nc€ vaiation in the 5 regions ol dislinct H4 96n€s 

has lunctional 

consequences lor lh€ exent to which human H4 promoteE are aciive This 

observation is rolevanl to the overall expression ol hislono H4 genes' bocauss 

hi$one H4 proleins are lranslaled trom mLrltiple mFNAs encodlng very smilal 

or idernicsl proteins and tunclional H4 mRNAs are lranscribed lrom distincl H4 

genes displaying considerable variation in |he organizallon ol s' ilanking 

sequences. This promoter heterogeneity is .eflect€d by ditierences in lhe 

presence, number ol copies, relalive location spacing' and orientaton or 

consensus ta.scriptional elemenls. Forexample, the sit6llregion olih€ H4_A 

gene and rhe H4 (FO1O8) gene dille. al seve.al kev nucleotides .esulting in 

absenc€ of bindlng sites lor HINF-!|/IRF_2 and HiNF-O (COC2/cvcln A/RB' 

related complex) in the H4.A gene. oirect jncorporation oi one set ol 

nucleotide !€riations (mutant INS'10) inthe promoler ol lhe H4 {FO108) gene 

results in a 3 iold decrease in the evel oi lranscripton The posslblity arises 

thal diilerenl H4 P.omole. organizaiions may have evolved to accomodale 

deleopmenta and homeostalc responsive.ess ro a brcad spectlm ol 

slgnaling pathways that med ate competency 

r65 

'ar 

prolleral on and ce I cycle

Atiz F:Cettat -aCtuot o! Ha Gq.r'?Nnpti' 

DiscNo 

o|Jl mutalonal anayses also show thal th€ rBcognition el€menls lor 

HiNF P/H4TF-2, HiNF-O (CDc2/cydin A/RB_r6laled comPlex) or boih al€ 

only detectable as tatelimiing lor basal lranscriplion in th6 abs€nc€ ol a 

lunctional H|NF-M/|RF.2 bind;ig sl€ 11 ls Possibl€ 

that €ach ol th6s€ taclors 

operates via a simpla independenl and ps.haps mutlslly erclugve 

transcriptional nechanism al Site ll, with €ach providing only a quantftanvely 

mode$co ribution (10%ro 30%) lottu oveialll€v€lol H4 gene lransc.r on 

Howsver, equally importanl is rh€ possibilily $at She ll proteins tundion in 

conc€rl a.d that rhe rolative convibutions ol lh6s€ iransciptional acivities is 

slroigly cell cycle $ag€ or cell typ€ d€Pendent Disctiminerion b6tween hes€ 

and o|her .elinemsrns ol models lor H4 gens lransdiption €quned tunh€r 

experimenlalion However, the resLlLls clearly slgggst thar lh€ lundamenta 

basis lor H4 qene regllarron by si€ ll €n noi b€ a$ribul€d io |he aclon ol 

one panicula. histon€ subtype_specilic lador al a singular €rement' as h6s 

been proposed lor seve.a other human hislone genes (Heinlz, 1991) Falh6r' 

the integrated aclivities oi mulliple transc.ipnon laclors at a comoos'le 

regulatory doman logether elgvale H4 gene vansc ption in difierenl 

proliierating cells expressing ditinct tissue_specilic and cell Orov{h 'elaied 

phenovpic Prope.lLes 

The experiments described ln this hesis were airned at understanding 

lhe comibuton ol H4 gene promoier sile ll sequenes and lhe cognare 

iactors io cell cycle comroLbd rranscridion ol the H4 ge's arhouqh our 

reslLtsclearly mdicale dramatic qlantita ve efiecls ol HNF M/|FF'2 o' Site ll

,@2 F. Cdt CEle Cotud .l U1 G.N fansdicrid 

rnedlared transcriplionaL mechanlsms, ihe cognate elem€nl appeaB to be 

dispensable loi cell cfcle conlrolled enhancement during early S phase. 

FudhemoG,lh€ HiNF-D/CDP-1 and H|NF-P binding siles topr€senl aunlary 

elements lhat conlribLte io ov6rall levels ol H4 promoter aclivity, as well as 

have slblle ellects on H4 cell cycle contrcl, Tsken togslh€f, th€ results 

p.es€ded in lhis dissertaiion suggests lhal call cyc{e control ol H4 g€no 

transcription is not medial€d by a single laclor binding lo a single element 

within Sits ll. Ralher, the results of ths mutant H4 promoter constructs I hav6 

analyzed suggest lhat rcgulalion ol H4 gen€ transcriptjon is nediated by an 

inrricale mechanism and raquir€s rhe intsglated acliviliss ol laclo.s thal 

inilenc€ bolh lh€ timing and magnitudo ot H4 gens transcnpnon 

167

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l NE P C.l qE/€ Cotud d H4 Gw f@fiPt@ 

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