Genes, Genomes a,td Genomics aznt ourot s"i,on r**"

Transition from DNA Looping to Simple Binding or DNAPairing in GeneRegulation and Replication:

A Matter of Numbers for tbe Cell

I. FROM T1I[ NIIMBER OF L{C OPERITIOR SITES AND REPRI]SSOR MOLECULES TO DNA LOOPINC AND TO MTII Il'

MichèleÂmouyal

. --,s;;'"ffJi:i:#Jl'i"ij.' -.ABSTRACT

RèpÈssion of ùe l. .dli læros opercn is æhi.ved rhough DNA loôping erd rh@ ope6to6 at the phlsiolo8icâl .epressr @ncenràrion.ln smins ole.prod$ing the rcpese. or ûth plænids wi(h ! hieh opy nmbê.. lhe coilpe.drile mode of.epresion is naked b) orh.rmôdes 1, erd. trten Éveral DNA molæuls æ pÉsem DNA loop fomation is Fgta.ed br intê|mlsule dscialions slill mediaredb) the lac leprcssr ln b€r|ria such associarion\ kmm æ 'hùd@fling' ùd redialal bI tlæ innidor prctcin. &e obsèûed in;Dlicarion of rne ilmn-clds of plavnids Wh€n no<terôle mours of ininalor ând its binding lo the Eplicalion ongin (æhieved in enteiftnrn.es. b) DNA lmpin8) allow Eplicarion ro prlcréd. high ænertrations prevenr Èpli@rio. od l@d to hsndcùfnt8 thal ænÈlls rhcnùnbe. ofpldmids. ID principl€. wh€n DNA looping is f6ible. DNA poidns is âie Possible ifnorc thù one DNÀ moleÙle is Pl*nrin rhe etl ln eut3ryotes, rhe action ofrh€ C.TCF prcr.in is paniolùly rcpèentalile ofthis situâtio.. nris key @nF)nènr of elemnrsùal insulde gene èxp6sio. from th€ slmuding 8Énomic effects in von€brales. âle æts 6 ù oryani2rr of higb€rcnlcr chremannslfuc{lm al lhe Èglobin ed /g,?H/9 lei- Ar this lsdet l()s, qlaF @ntrcls Eenonic impnnlit8 bl DN^ lmpin8 Reent dlia sug8slrhar gcmmic imprinrin8 dd momÀllelic expNion niphl sie be 6n1rcll.d lhruugh.hromoemc Pairing

X.,\Tonh: cl Cl'. lâ.1o* op€o oligomsutiot plMid eplicarioo

CONTENTS

oPERATOR REPRESSTON Ot THE E. COI1LlCOPERON.......-............. 1052. RÊPLICATION OF THE ITERON{I-ASS OF PLASMIDS AND CONTROL OF'HI]IR COPY NIJMBT]R IN BAC'I'ERIA ".' IO8J CONTROI- OF CCNOMIC IMPRINTINC BY DNA T-OOPING OR CHROMOSOMf, PA]RING IN MAMMAI,S ' .,'.'-." ".. ' IO8

Molsular biolos, has been firsl and nonnally interEsted inidentiryins the componcnts that arc involvcd iî the bsiccellulâr processes. Whcn it was not€d lhal somc elcrnentswcr. in vârioùs conccntmlions, narurally or âniticially. ducto i rodùced genebc mutâtiods, it was css€ntiâlly dêscrip-tve. Thus. for DNA-protcin intetrations involvcd in g€neexpr€ssion and thei. regulalion, the genersl idcâ w&s thâlthe DroÈin had to be in sufficient âmount in th€ ccll to cn-sure binding to the coresponding site and gencnte ûe pm-c€ss of inlerest. ln bactcria for instânc€, it seem.d unimDor-t nt to us€ strâins ovcrproducing a traft.ription factorinstead offte wild-typ€ slrain and physiologicâl ârnounts ofprorein. And b€câuse it is eâsier technically, multi-copyDlâsnids are ofien used in Dlace of chromosomâl consEucts.

Sinc€ then. il has appeâred in sev€ral instaîces that thenumber of copies of a gene or of â protein wss eblc tochùge lh€ regulation process-

One enmpl€ is pmvided by .he t. colt n'rlti-site /acrepression. From th€ approach eûploy€d to demonsFat€ th€fêÀsibiliù of DNA looping betwc€n /cc rÊplecror and two/r. opeEton on thê sâme moleculc, it wes cld thâr DNAlooping could t'€ concentralion-depend€nl (lkâmer el a/.1987: Amoùyal 1991). When mediated by /æ rcpr€ssor,D^_A læpina is lost with increased conceû'ariôns of reFressor ând DNA ûrolecul€s to the benefit of other int€mct-lions. independenl binding to the op€rators or inlermolec-

ular associations. This findinr was â decisive elemenrtowards th€ uncov€ring of the ii;octiôùâliry oflhe secondâry

DNA looDins is nor restricred lo Fânsffiplion. ll i< âl$involved in ieolierion. to initiâte it for the bacterial RoxDlâsmid {Muci.h€ri€t er a./. l98Er Viron g/ r'/ lqell or onitre commn ro sto-o ir for rne / . .o/i F-factor md to limtr ,tsnumber to ône coiy (cf. zuma, ând Bastia 2005). The ini-tiâror Drotein bridaci lh€s. loops. Hoqevêr. the cop) num_brr odthe itercn+lass ofplÀsmids is generall) controlled b'-hândcumnp-, i.e. intermolecular açsociâlions bridged b'the initiator-prot€in (Palând Chanoraj 1988; McEachem eral- l9E9l.

Int€moleculâr àssociations are âlso r€present€d by theDairins of chrcmosomes for rhe @otdinated resularion orihei r ;enes. lhc CTCF Drote in i5 in lo l \ed in Êercmrc rm-prinriig of rhe nalnmalian td2 Hta locus lt does so b!ionlro ing lhe targel of lhe enhâncer shæd b) lhe r*o im _prinæd genes rlmugtr attemate D\A looping 4d meth'l-;dôn ôfihe CrCF bindinq sne lMunell cr J. 20M1. lt alsoDaniciDâtes in chmmoso;al âçsociâlions (Ling er d/. 2006:zhg.o tt al.?.006\.

The inrerDlat between DNA lmping. simple bindingand DNA pairinB i5 de!elop€d for lhese thrÊe specrfic situ-

I. FROI THÊ iIUiIBER OF LAC OPERAÎORSITES A}ID REPRESSOR OLËCULËS 10 DT{ALOOPIIIG AND TO i'ULIIOPERATORREPRESSDI{ OF THÊ E. COL' LAC OPÊROI{

It has beo knom since the origin of rnol€.ular biology(Jacob ând Monod 196l) that the /ac op€rDn is repress€dfiom a 2l bp operator sequence, O./. locât€d on th€ promo-ter rÊgion and ceniered at I I I with r€sp€ci io ihe tr|ns.rip-tion siân. A proteiî. Lâcl. is responsible for ùis repressjonwhen it binds the operator (Gilb€n e, dl 1966, in a hypo'thesis formulated by the forme. ones). A simplified or8ùi-zalion ofthe /dc op€ron is shown in flg. r.

In lhe s€venties, two homologous sequences, O2 c€n-tered at +412 and OJ, c€nt€r€d at -82, werc discovered(Reaikotre, a/. 1974: Gilb.net al.1975t.

During nenrly 20 years, these siles hâve bêen r€gardedâs cryptic sites, like the two promoterlike elements, P2 a'dPl. ofrhe /& operon IXiong er r/. Icgl ). In facr. repressionin the âbsênc€ ofthe s€côndar) op€rârors is efricient due lothe strons amniD olreprcser for O,l (K l0''M . âcco'-ding ro winter €t a./. l98l). Thus it was not clerr why rep-ression had to be assisted. Moreover. the constitutive mut-alions *erê all mapped in OI and f,one exclGively in theO? or OJ rcgions.

This ooint wâs coroborât€d b! tbe wea* afiinilies ofthe repressor for 02 and OJ. ()2 has a -Io-lold r€duced af-finiry mmpared ro O,l (Pfahl et al. 1979; wiîrel et al.l98la) ând OJ an âi leasr 100-fold reduced aflinity (Pfâhlet al. 1979]. Wiîter et û1. l9Ela). A fragmenl catrying allthree op€lalols has nrly the sârn€ âmnity fo.lhe repress-sor than a hâgrnenl c{rryins the sôle 01 op€ràtor (Pfahl e/d/. 1979: O Corman et a/- 1980).

Fo.lhese r€asons. lhe Dossible conùibution ofo2 or OJwàs disrârd€d until the nexr decade. and a first decisive im-puls€ wift ihe dis€overy ofthe eukaryotic cnhânce6 (MGreau e/ a/. l98li Banedi er a/. 1981). Soon aft€r, in I9E3-19t4. somewhst analoSous s€quenc€s were found nec€s-sffy for repr€ssion of two prckaryotic operoos, th€ arâbin-os€ and galactose op€rons, in addition to a fi$t operclorsequence locaæd in rhe vicinity of the pmmoter (Imoi e, d.l9El : Dlm"rd l . 1984).

As a .çsul! two groups in 1984-1985 tried ùo establish amùlti-site .epression in an artificiâl /ac systens of eJçres-sion lâcking lhe nâtive 1dc operon (Henin er a/. 1984;8e:!€ e, al l9E6). These works lumnârize the el€nentsthat *eR supposed to bê nec.ssary for multi-site rEprcssionh the mid-€ishti€s.

Since nulti-sil€ repression .|l/as obs€rved in tnc a@ opc-rcn wilh â control €lernent wirhin â strùc1ùlâl g.R rclhin-dinS of /æO2, bùt not in the tac op€mn, lhe gercmic andbiochemical orgânizâtion of rhe gar contml region s€cmedof first importanoe. For this reason, the artificial rcgLrlarionbuilt by Herin and Bennet 1984 mimick / thc ga, opemnwirh a hybrid locarp ptotn'otet corftolling aala.rokinase

. ln the native /ac operon, /acol overlaps wirh tbe prûmoÈr. ln rhe gal opero4 the galo and gal0e opêûotrssuround the pro.noter, allowing the RNA polymetas€lo sil on it in the presence ofthe rêpressor Thùs, a l,'ppmmoie. was flank€d by two /acot operarors. The/oco] op€Étor wâs employed as a s€quenc€ with suf-fici€nt aflinity to ênsure op€raror occupâncy. in a w)sinilat .ô aaloe ^nd galoi when binding the gdl rep-

. The comtructs were cani€d by a 2otopy plasmid.

. An effect was only_pbserved when ûe reprÊssor ùasprovided by a laclw slrain, produciog l0-fold morËrepressor than the nalive sEain (Mùller-Hill el a/. 1968).It was the first report lhat two /rc op€rttom call c.op€r-

ate in repression. How€v€r. interpr€tation of the dstâ issonewhâr complicâled by the interference with tp .epres-

The anificial /ac reaulation built by Muller-Hill's gmupùâs i'rsoired b! llenin and Bennel\ wort and also d;s-

plâyed some coopemtivit). lt is closer lo â ldc .egulationsinc€ a /d. p.omoter controlling /acz expr€s,lion .Êplacedthe r/p pmnot€r-op€ralor on â plâsmid. However, lhe /dc02and /æOJ sites \rer€ noi rÉmoved, since lhey w€rÊ lhouShlto b€ inactive. Tbe /nc promoter was surroûnded by two"ideâI" la? opentor shes with a ten-fold incrêaçêd atrniiytor the r€pressor. as compârEd !o lhe /.rr r/ site (Sadler el a/.l98J). tT€ study wô câried out in€ /r'c,1" strain produc'n8€v€. mo.€ reDr€ssôr thân the /der? strâin. about 100-foldmore than the *1 strain (Câlos and Mill€r I 98 | ).

Two 'clichés" would hav€ to be overcom€ lo show thecontribution of OJ ând O,? to /dc rep.ession under ûâtivcconditions. They aæ complementâry becâuse of their rela-tion to the law of març-action. First, ten copies of rep.essorare not sufficienl lo hâve the secondâry sites occupied.Thes€ ten copies aæ the number of tetramers p.oduced bythe lr, cell (Cilb€n and Mûlle.-Hill 1966). Thus. lhe con-cênt.âtion of reoressor in lh€ cell has to b€ increased ùoforce the oc.upancy of the sit€s. Second, th€ affinity of 02for rhe repressor, wilhout sp€âking of that of OJ, is looweak to allow their occupùcy. Thus, their alfinity has to beincreased to forc€ rhe otrupânc,

In anv casê. in 198.t-1985. wh€thet for lhe natural aEb-inoæ and galactose regulations, or for the artificial /4. re8-ulations (fof lâck of tuodionâl secondary operatoF), twoopêrâ1or sites either separated by llo-llsbp (lrÂni ?r dl1983; HeFin and Bennet 1984) or 220-240 bp (Dunn "r dr.1984: Besse et at. 1986), wer€ rcquired to hav€ tull repress-sion. How they coop€r.te for repression at these dislanc€s inf,. !oli. hâd to b€ exolaiæd.

DNA l@ping sanelaied by the simùltat€ous binding ofthe .epressor to lhe lwo distant sites was one possible model,bur wÀ resÂrded as mosl unlilely before 1986. The oppos-hion to this model wâs strong. as R. Scfile;t recalls h(Schl€if2003).

In fac! conceming lâcl specificâllt som€ early ù vir.odata ôlher s€€med to favor othe. models, such as sliding ofrhe reprcssor along DNA uniil the -phyriologicâl O/ siretwinl€r ?r al. l98lb). Orher /, t/rro preliminât) dâr.a in1985 indicat€d that the reprEssr-op€mtor complex was notrelained by niùocellulos€ ir filær binding âssâys wn€n 220bp s€psrated the rwo opelators. A nuclmsome-qle sùuctuFwhcre DN.A is $rapped iwice around Lâcl. could explainrhis unusuâl silùation (B€sse el a/. 1986). Olh€r nuclecsom€-tlpe models we.e proposed later on, such as the 'reP'

ressosome" thal would form between the lwo aal op€mtorssepaiâted by ll4 bp, the Aal repressor and vanoùs olhêrproteins, th€ RNA polymerase, the CRP or HU pmteins (s€€lor e\ânple Kuhnk€ ", d/ 1989: Al; et dl. 1996).

Thus D\A loopins for /ac repr€ssron specificâlly andlor oùer resuldions in senelâl. wâs not obvious and its f€â-sibility had lo be demonstrôtÊd. The /dc reprcssor was ùideal pDtein for lhat pùrpos€ tecaus€ of its strùstur€. Thist€traffer is organized inio two dime6 that bind DNA, asdet€rnined by vârious techniques (Kâniâ Ând Bmwn 1976:o'cofrnan er a/. 1980; culâd and Maurizot 1981). ll couldthen in principle inducc DNA looping when mix€d with âftâgm€nt cârryirg two /ac op€râtor sites.

In fact, according to the concentrations of repr€ssot andDNA, variou! conplexes, apân th€ loop. could be predicledfor a mixrure of repressor ând fragment cârrying rwo distânloperâior shes (Kraner ?, cl. lq8TlAmoulal l99lr Fi&2).

Sùfiiciently low conc€nô_atiôns of DNA and rep.essorwere €xp€cted to favor the siûple binding (A) to on€ oPer-ato. site. tf fl€xibility of DNA allowed it, sinultaneous bin-ding to the second operaror site alld looping ofth€ interven-

' "ln 1984. t @ csÛdins sslely tlÉ fomation of DNA-proler l@ps

beuF I wr.d to Drek th. s@l6c inle@tion betlÉ rhe cRP ælr-laror dd RNA pdFæ ,'r dE læ pmmoler (s omlusio. ôr a'lu$land Bùc 1937) by ltrtis €eqindts Ths ndl makna lmps b.t1"6( RP&d R\A polymæ I lJæpGed lhb poj(t o rh. lr Ep|N inI 985, fôrgdring th. lie'iio. slç fo. *ôich M mt Éluind The miriâlpm|d1, a wll æ I bnsp6ni6 |o the lac t@6so( s *dely spsdb€làre n s?s èv6 qrn.d -

105

D N ^ | q + | t l d D N A p . û i 8 ' n Ù É < . 1 | M i . h . h \ m ! r |

promoter Fi& I S.h.mli. mp ol lt. 6- .di r.c-ro.. op.Éi (rot b r.l.I opsrbsrt6 ù blÉ. struclonl 8aB oi th€ opêM f ycnoq ponor.r ESrd n 3Én.brndrng srté fd |j1e cRP dlivalor n ad

TCRP

n-// -"7n

wA

Repregsion mode

sinple

Ft 2 Tt. v..iod Ép,scop.nro. inl.nctor oLa.ir.d b.tra. ,.. '.nrts.n.l . lrrgn !t qrryi.C lro,4. op.drûr 3itd (frr .ùluùr) rtih i..É.i!g .Gc.lrndon! ôr ÉpEls ...1DNÂ{Ford colnml (A)Afdrg to only oe op.rdùsirei (a) sihultsnæt biful'n3ofùe rêpts to th. t*ô oÈ'6tt tit6 with lo.ging of {h.inlePding oNA (C) t"ndm bind'.8lû tlE tK op.-nb.snÊsoftn sæ Êtg-ngt. (D) intmolæulùâscciâtiùs. Th€ thrrd olmn trnimt6 fi€ 6p61et Epl6rd

0

A -\ v sRep (++)DilA (+)

Cooperation

Superposition otindeoende|lt î|odes

Sharing of oîe nodebetvreen molecules

Rep (++)Dt{A (r+)

t Th€ DÉlinD.ry d.t sth rhè ELrdatiù s€l drqinats wc @_nu.'€ied b tE Bslin Sunme Schml ù DNA sdrdE in 1986

- : _ - ê N oA A - *-sffi -s-g-

"U

inq DNA {Bl *ould occur. For incEâç€d concenEatioru ofreircssor, ttre shes would be indep.ndently occupied in â'Iandem' struaure (C), and for both incr€$€d rEprÊssorand DNA concentmtions, int€rmoleculù species {D) whereih€ repressor is in sandwich betu€€n two DNA molecul€s,

The Kram€r er dt (19t7) stùdy is the exacr translâlionot rhis conc€ntralion sch€me and of oùer tniiciPated loopte.lurÊs. The Erârdârion gel assrys' were performed so asrhe Dmo€rties of thÊ loops ùe dirÊcrl' r.sd on the gelswithoui an) effon: conlinuity ofDNA loopinS-ovcr â lùger.nce of dist nces. a ooposed to punc$al fonndion of"*ie"tome-tree srruclures at 210 bp or oiher punctualsùuctur€s, concenEalion effect on DNA loop fonnarion.identification ol intermolecular adducts, phasing st shondistânc€ a! a oons€quence of DN\ loopin& ifnor a pmofÈlælron micmscoDi \râs rescrved to ihe obs€r ion oflaree sizr loops anàio the dernonst arion ofù oplimal dis'lâni€ for "DNA cyclisation- âround 50o bP {ùe "cycliz'

tion_ aDDroâch is detâilcd in Amouyal ls9l).TT; study cteely showed lhat DNA looping is fâvocd

D vito by ihe corcentmtions thst do dot saturate the op€r-âtors rÀ,iih reDrêssôr ln ihe wt. stnin, th€re is oûly onecoDy of DNA r'er cell, rhe cbomosomc. ùrd d|€ cell prcd-ucé only l0 cnpies of t€Fâmêric represrôr (Gilben andMuller'tiill 1966)- The concentrÂlion of reprcssor can beincrcased lo-fold in â /dclv st$in (Câlos ând Miller lc8 l)and aboul l00-fold in a /dcl' strain (M0ilcr-Hill €, d/.1968)- Th€ numbe. otoperator copies was 20 in the H€mtùd Bennet (1984) study ând can reaah 500_700 copies p€r

Concentr.tion

ReP (+)DIA (+]

cell for ùe ouc.based Dlasmids Thur. the use of slrainsovemrodùciric rfie reonssor and oI mulri-coP) plasmidsmiclir hav€ conc€aled D\A loopins in rhe fomer ânificial/dc-resulâtion. To dêi€c1 DNA iooPin& if il did Gcur. ùeassavilaa ro se caflled out as clos€ a5 possible to !fie ndiveconditions. with $e ihrce oDerÀtors on the chrcmosome (orver) low copy plamids. al rnoso ând siù the repressorproducÊd by ùe chDmosomal /dct gene

The s€cond -clichê- was r€lârcd lo the suppo5€dl' toolow âffiniry ofthe s€€ondff) rites for them io be implied inrcorÊssion. ll waç relyina on a fâcl lnom for long and @n-firmed bv s€venl qrcuDs: lhe association of the reprcssor"irh a Ésnent carrvini all thrÉe operaùors is hardl, stron-eer tlouriola or lessl thô with a hâgmenl carr) ing the sin-;le O, oDeEtor (Pfahl er d/. l97c; O Connan er d/ 1980)b\A sudercoiling bmke lhis cliché" v) work of trârsi-lion between Chemistry and DNA looping (Amouyal ândBuc 1987) hâd made me â*âre lhat D\A is sup€rcoiled in Ecol'. lf D\A loooinq wa fe&rible on â linear remplale.D\A suDer$ilina co;ld D€duô h, or on rhe conhr) arsisrn. Thus. Êom ù;beginning of m) worl on DI\A loopin8 inl9E4-1q85. a second steD Ms Dlanned çiLh â supercoilêdremplâte (cf. Klëmer ?r d/ 1988). In facr. /u vl.o. DNAloo;ins is st onclv stâbilized b! DNA supêrcoiling: lhehaliliie oflh€ o/-'ol-OJ comple). on â plàsmid is 2t h in-stead of 7 min on a linear template (Whitson er d/. 1987)On a otasmid, Éle s€condq operalors do$ Iâcl dissoci-arion i0o-fotd accordinÊ lo Whjrsôn Pr d/ (1q87). ftom 7'fold ro J2-fold a.cordinq to Eismann and MÛller'Hill (1990).whên comDùi's the ùlf-lives of $e Ol'O2'Ol a'd Olcomol."esi oirc"r - .'o aDprcâches hâve ako unrav€ll€dù€ influence of DNA sup€rcoiling /, vivo D\A fooFrilcinc of the O/-O, rEsion by various chemical âgenB showsiût oJ is only Gcupi.d when DNA is 5upercoiled (Boro-

r06

L.vt. rw\ od.nNù,. I(t ). l

A

Roglic.tion Initirûon

Porltivs control

Control of plaEmid copy numbor

l{€gaiive coikol

Fi& I R.plic.rion ofô. ii.m}.Lr olDLmid& Pr*l À: cù4!@irve bindiq of lhe 'nitratd rôthe ft.mns for æphcalio. inititiid

Gencol cs) Below. ponrcurùsrrù€tion of th€ R6K pl6m|d ulnDNA læprng lù mûrakù of R6Kat lhe r (or P) onsin. P.!.1 Bl''hùdcùmn8 for lhe conhl ofùe Dlomid mpy nùnbd (g6Falæ) B€lN, pan'culù srtuarioofoÉ F factor *th DN looprngfor lniuiion of its op) nùmbd to

lnitlaùol

O

tllonomel

Di|nêr

lrrcC/ r'j--v'-?-\

: KotiF

wiec er d,r. 1987. see also Flashn€r and Gralla 1988 for lhei, vilro Ol-O2 oc.up ncy). From th€se assays, Grnlla andcGworlers hâve deduced thar OJ binds thc r.prersor ,,rivo oniy l0-fold less tiShtl) th8n O/, whercby i, vtlo in'deD€ndeitly on a linear template, Ol binds ûe npressor aLteâsr 100-fold less tighlly lPfaÏl "r a./. lc79l Wi er e/ o/lcSla). DNA sup€rcoilins matcs DNA loop fomat'on soasv thar the Éûuiftm€nl for relatile onenhtion of two faco;Ébrs that ii obvious betw€en 153 and 168 bp on alin€ar or rÊlâxed DNA lemplâte. is no longer legible on asuD€rcoiled one. hstead. loops ârÊ formed for all distÂncesând only rheir stâbilii), instead of their existence. is nows€nsitive to helical ôrientâtion of the sites (X-ramer er d/.1988).

ln sDite of thes€ r€sults. the clichés took some lime aodisapæ:r sbce all the ld. rÊgùlations p€rfoEn€d before1990 still included rhe Ol site âs â crlpric sit€ thoughtina.tive, or wer€ run with stains ovearoducing dle re'pcs{or and plesnids sith a hiSh level of copies. *hichiôhêwhâr ærtùt6s the int€mrctation of thcir dalâ

Finâlly, th€ inactivation of ea.h sile by mutagen€sis,includine tbe OJ site under conditioûs Às clos€ &s Dossibleto ùe w:. ones. with tlrc chmmosomal repressor aène andrhe rbre€ siles oD thÊ chromolorne, deternined lhe contri-burion of eâch opemror \ire to rÊpression (Oehl6 el a/1990, 1994). Ih€ pictùr€ of /ac rÊpression was definilel)môdified. h âpDcârÈd thal repression of lhe læ op€mn re_lied on th€ ùË operâûors and not exclusively on O/. Thisconùibutiotr deDdds on the intracellulâr €oncenûalion of/ac rcDr€ssor. wh€n the three ooerators âr€ on the chromo_some. rpDression enhance.ent;t o/ by bolh 02 and OJ.dettr; Ëom 72-fold to 48-fold and 6-fold *h€n lhenumber of lac reoressor tetraners incr€es€s Èom l0 io 50ând q)0, r€spectùly. In fac,t, reprËssion issued Fom o,i-02 ot OI-OJ oôoperation is replaccd by rept€ssion fiomindependent binding âr Oi, O? and Or.

This view is supponed by the rcsult5 obrâined wilh thesiosl€ oDeÉto.s Ând âlso b\ rhe usê of reDressor mutantsû'aiæ;liners unable ro forin le ramers ind ùo induæ DNAloooine (Alb€nipr dl. l9q l: Chakerian e, al. l99l).ThusarreËrivèlv bw r€Dre5sor concentration (200 subunhs ofeither di;êr or teiimerl. lhe dimer induc€s dl€ sâme levelof reor€ssion ftorn ihe three oD€râtors lha'| Ih€ tc6am€rfiom ùc single o/ opemtor toéhler e/ a./. t990: o€hler pra/. 1c9.1). This is the indication ùar. *hen th€ reprcssor rsunâble to folm DNA loops, it batdly biods 02 and oJ allow' n€âr-.o-physiological intrâcellulat repr€ssor concentra-

This 's an over-all effect rçsulting from altema.ive looFins between eilher o/ and 02. or 01 and OJ. This conc€n'triion effecr is moE cleârly followed $hen onlj one loop isDr€s€nl, eilher ùe O/-O2 loop or ùe O/-OJ looP ùd shenônly t*o opemtors atÊ prcsenl and th€ rhird one s tnac-

In a siluation wher! only lhe O? aûd O.1 op€latols âi€pressnt on the chmmosome, tbe r€pression enhâncemenl ofO? versus O/. falls fiom e l0/l I.s-fold effect to none (l l/| .5 fold effect) with an increasing aûount of tetEmers Êoln50 to 90o nlnolecules (Oehlû et al. tqq),1994). The influ-enc€ of O? is also lost with elevated reprÊssor concentrâ_tions and wh€n 02 or 03 r.place O/ ott the promolcrroehl€r ?t d/. 1994). On ùe @ntrâD, qhèn lh€ dimer rePirês rhe natral teù-ane.ic reDressor. i.e. in lhe absence ofD\A looDinr" rh€re is no influence ofO? 41400 bp ofâ firstoperator bveilæping the promote( and lhis situat;on is nolconcentralion{eocndent.

The ()/-oJ interætion is less simDl€ rhan the Ot'o2int€.action. tnd€€d, the ptotêin r€sponsible lor âctivalion ofthe /d. oDeron, th€ cR? pmlein. binds a site cenerÊd âr-ô1.5 clôae to ùe OJ sit C82). ls CRP r€rponsible for ùer.oression observed from oJ, $hether by competing wilhre;ressor for bindina ât lhe \ame locaiion or by âssininsrcbressor binding tF;ea a"a Huason lee6: Penos andst€irz 1996)?

In lhe ircz consûùcts of Amouyal and von Wilckeo-Bersmann l t992), ther€ is no sire for CRP binding and âni-fici;l lacrlltsiks hav€ b€en used either on the promler. âlthe Oi locstion. or uDstrEai:, atthe 03 location. The lacwMsitcs do not allow itpEssion by th€mselv€s when locatedeirher on the DromolB or utslream. Howeler. when two/dcn M op.É6r siies are in ilæa o1 Ot ând OJ. lhe Fgal-actosidase âctivity is rËpEssed fi-om E' ùo ls-fol4 depên-dine on ùËir rclâtive oricntation ând on whether !h€ rePreesor-e€n€ and oDeldot sites are c-ânied by lhe chmmosomeor s--coov rlas;ids. This siBnificânl level of coopemtionwa! reoipaucea wi|h two o.l;peralors (oehler e, d,/. 1994)Funheimore, rhe moærarion r€sul!;ng tïom the exchdgeofO/ b! OJ in lhe oriÊinâlly O/-O2 construcr (Oehler e, d/1994; is âeain an indlcdiôn that DNA loopina inlolves oJwithour ù; n€€d for CRP. Thu!, unassisted DNA looping isa tru€ comDonent of the nâtùrâlly obsewêd repression be-tween O/ ;nd oJ. Th€ concentmtion €ffect h also consis-renr çith D\A loopina since the effect ofOJ with respe.t loo/ frtts fto'n .28-fôldio s.ô-fold fiom 200 to J600 subunits

t0?

R6K T it€rcns

t__ -,, -R6K orlo

Dr-'a lqiq ùd DNA Fi;nÊ i! rh. c.ll Michal. ÂDryl

of telramer. Wiih the dimer. this effecr Etnains in th€ lowertange. The4 DNA looping competes with the activationproc€ss, leâding to ar âdditional level of Eprcsion (Oehleret al. 1994t.

Note that in the artificial regulation descaibed inAmouyal and von Wilcken-Bergmânn (1992) or Perez el .1/.(2000), there is no n€€d for âssistânce, excepi for nâlumlsup€rcoilin& whelhe. two "poor" /rc op€rator sites côop'erare (Amouyal and von Wilcken-B€rgmann 1992) or twoproteins with good affinity for their sires. inte.nct weâkl!,lik€ the two galaciose repressor dime6 (Perez er c./- 2000).

Accordi.g fo a recent work by Zhane et al. (2m6\,DNA looping b€iween two /ac operâtors s€pfl{€d by 100bp would rÊquire the nucleoid HU protein. It should b€noted dlat interpetation of the data obtained with Hu-def-ici€nt sûains is uneasy becâu.se ofvarious mutarions gene-Er€d b) lhe bacrerium lo compensare for ihis deficienc). aspointed out in Perez er dl (2000) and in Arlouyal (2005).Second, DNA sup€.coiling is sufficienr to €xplâin DNAlooping with two /ac operâtors âs above delailed (s€€ alsoPmhil ed Nelson 2006).

2. RÊPUCAIIOI{ OF THE ITERON4LASS OFPLASIIIOS AND CONTROL OF THEIR COPYiIUIBÊR IN BACÎERIA

Al an inlehâtional confer€nc€ wher€ I wâs pr€s€nting th€fils1 da(a oD DNA l@pins with the /ac repressor, sp€cialistsofplâsmid replication came to m€ and mad€ the remârk thatlhe concentràtion scheme displayed in Fig. 3 c-ould s wellb€ applied io the control of plâsmid copy number (ct Palmd Ctattoraj 1988; Nordstrom 1990).

In facr the Dunber of Dlasnids, lhcse €xtrâ-€hromoso-msl generic elemenE in bacleriâ- is tepr siihin preciselimit! in lhe cell. There a.e lor exÂmple I to 2 Pl plÀsmidsp€r c€ll (ct DÀs e, a/. 2005), abour l0 R6K plâsmids (cf.Muckergeê e, a/. 1988), about I 5 colE I plâsm'ds (ct TwigSand Shenâtt 1980) a'd only one F-faclor {cf. Zzâmm andBastia 2005) in E. coli, l5 to | 8 pPSl0 plâsmids in Psetdôûonas ætusinosa (cî. Gi.aldo and Femandez-TrÊsslelres2004).

The us€ of multiple shoft repets of DNA (of abour 20pb), or itemns, is a major wây of controlling the nùmber ofplasmids in Crm-negârive bâcr€ria. This fâmily includesth€ R6K, PI, F and psclo plasmids, among oth€rs. Tbeseiterons ar€ prEsent al the r€plicârio't origin. Their .eplic-âtion pr€sents som€ analogy with thc tnnscripfionâl rÊprÊs-

To control the number ofrhese plasmids onc€ a specificnumb€r of copies hâs been reached, the bactêrium prÊvenis

Replica.ion is inhibited by plasmid pairing ar the originof rcplicaiio', called "handcuffing" (Pal ând Cha$oBj1988; McEâchem et a/. 1989; Fig. 38) as delccled by ligâ-tion €xpenments (Kunnimâllaiyaân er a/. 2005), the actualbasis for th€ lC rechniqu€ widely us€d in eukaryotic cellsto det€ct DNA looping or pairing (s€e section l).

DNA pairing is bridged by â k€y compoûe ofnplica-tion- lhe iditidor brotein. The iniriator is inde€d firsr re-quired for initiation ofreplicariôn at the ircrons, by cooper-ative binding to th€ itcrons (ri& 3A). Hândcufiing pre-vents new munds ofplasmid r€plication ând is li'ked to theincr€asins concenùâtion of initialor with the number ofolâsmids:Thus, th€ number of Pi dim€rs, rh€ initiâlor olR6K rÊplicâtion, can reâch about 6000 mol€cules p€r cell(Filutovicz e, al. 1986).

For some plasmids of lhe iteron ianily, such as th€R6K and Pl plâsmids, there is another s€t ofDNA repeats,ât \ôme distârce fiom lh€ origin of Rplicârion. in a regionùat cùses incompatibility, i.e. the impossibility for twogoups of plasmids to co-exist in the sam€ cell. This distantregion is nâmed i/,c for Pl, ard Tfor R6K. tn fârr, thebacterium applies lhe sâm€ proc€dur€, handcufrn& whenânother plâsmid carrying the same replicario. elements isinlroduced and inùrÊâses thè global nunber of pl6rDids ;r

In case of lhe ,. .o/t F-factor, handcùffing is replacedby DNA loopina b€rween lhe ongin ofreplicalion and ir.{,al âbout 1.5 kb (ri& 3B). DNA looping inhibib strand s€p-amtion required for iniiiation of rEp'icâlior at the onein(Zz man ând Bastia 2005). This allows the c€llto linir thenumber ofcopies to â single one in case ofthe F-faclor-

Thus, the iniriaror is boih involv€d in the positive andthe neeative codtrol of replicâtion, like th€ fâns.riplionalactivator ând repressor protein, AraC (Schleif2003). And ina similar wây, conformational ûansitions modiry the fùnc-tion of the protein. Additionally ùo a r€p€rtoire of mulatiofffor âll kûown initiator.. thrÈe structures have beel| solved:the iteron-R€pE monomer conplex from F-factor (Komoriet al. t999). the din€rizatioû domâin of pPSl0 RepA(Oiraldo ard Fernandez-Tr€sgùefÎês 2004) and lhe il€ron-Pimonom€r comple)( (Swan er dl. 2006). ThÈ provides a*eallh of information and a glob.l view of how the initiâtor

In the solutioq lhe initialor €xisls predominanlly undêrth€ d;meric form deprived ofiûitiâtioû actiliry In this fom,il binds porly rh€ iterons ând cannot activât€ rcplicalionfiom the ite.ons. It d, however, bind ân inven€d.epeat, inwhich tbe two half-sites ofthe iterod âr€ in opposite dirc-tions. when lhis inv€rted rcp€at is presenl, âdjac€nt to lhe;t€rcns ofth€ origin ofreplication, the initiator is âlso âbleto repress ils oùrl synthesis (see fo. example replicâtionconlrol region of the pPslo plâsinid! Ginldo ând Femsn-dez-Treseu€res 2004).

A chàperone system gcnenlly mediates th€ conveB'otof the inâctiv€ dimeric foIm into an active one for initialion(Zzaman et al.20O4l. the active fonn is a monomer actu-ally olisonenzed by its binding to th€ iterons (Germino andBa$ia 1983).

Th€ two foms ôf the protein. monomeric d dimeric.are involv€d in DNA looping or handcuffing. Co mry tothe &ans€ripiional repressors, tbe mommer has th€ p4ùli-arity ro bind the teo half-sires oflh€ iteron both through lheN-|Éminal ed th€ C-terminal domains. a: obsen€d for rheF RepE-ire.oû (KoNri et a/. I 999) and R6K PÈiteron crys-lals {Swan er dr. 2006).

For some pl&smids. such as the R6K family. this is mrthe end of ihe siory a.d replicâtion pmvides an example ofth€ glmnastics that the same moleculâr el€menls mn p€Ffom to âchi€ve differenl soal! in the cell rhh ân ecooom)of meôm (cf Schleif20ol). Indeed, fôr lhe R6K plasmidscârrying the disbnr T s€l of iterons, replication from the sol€orisin ofrcplicalion is not eflicienr. Replicalion .ËquiB lhêdis-tant y itérons to procced. From lhis poinl of vièw. the Iirerons arc also rEDlicâlion eûhânc€rs.

Vor€ prtciscÛ. ttle replicâtion proceeds trom two ori-sins, û and 0. tÏe I element contains an amy ol *vetr :lbp rep€ars rhât can bind the inirialor protein. The r origincônuinr a binding sile for the iniliator. bul il binds the prot-ein poorly by iéIÎ tnitialion at this origin can onl] startwhen th€ seven 1 ilemns, about 4 kb away (the largest dis-tance obseFed ôr ethâncer âction in t. cor), âr€ physicÂllylir*ed ro the û sire thmugh tite initiato. (FiF 3A). Thiscôntact stabilizes lhe protein al the r origin. In the same wây,the Ê origin contains a half-itemn only invôlved in iniliationwhen aa the sam€ 1ime, lhe initiator occupies the T ilerons,about 1.2 kb awây (Muckherjee "/ al 1988; Mimn e, al1992: Swan el aI2006).

3, CONTROL OF GET{OIIC IT|iPRII{TIT{G BY DNALOOPI'{G OR CHROflOSOITiE PAlRll{G lNfitAit[ALs

F.om lh€ above êxamDles relaled to bacteri4 it is clear thâlwhen DNA looping is possible /r crr, mediated by a protein,rraln-associatiods arE also Dossible with the sane molecuhrelements wten severâl DNÀ molecules are presed, in orderto achieve the sane biolosical process. Recent findinss r€l-ated ûo genomic imprinting of the r.i,al]]'û]' lian I8l2 HIglôcu( and CICI d ica le thal l l , is i ' ro l r rs l r ic led lu r . . , ' / i

t0E

6e' ti.1,æt c"d &nam6 ltt l. l

A

cB

?r"

w2l|atemal allolo P.ternal allela

l * r l x u l

{ù/"

f ig ' .P . . . |Âs ih ' l i6e{ tvEwof lh€doerg / } ' / , | sThc@wsind '€ l . lh€p lmorgsof t tE lwsæ. /gem$eI€n ' r / l9mtn .nd4 iny€io* Thê drM slrar€d by th. rm sq6 @ iûticed b, a cd E-ette, lh€ ,ipri.tsi 61rot cgiôn (rcR), In frediun blw. rh. difdol'sfly

;.drytstqt doddB, DMRo, DMR ldd DMR2. in tsù! ùe naFix srllclmoi ftgion. MARI, m dârt 6lùe P'r.l B th€ spânal @hltætw indûld bv

crc; (.d p6ôl! où* p-Li.t wh6 lt bi,ns io its ùnn tnyb€d sii6 d rhc rcR rcsid âtd m th. DMRI ,.gor\ ù rhe tutùmÂl alele & à @l(

oc <rrwJ m oty onræt ara switcfi d rh. Hl9 aæ ù16@ thers,? sFe, trappcd in æ of rrr ro.Ds. is tqéeed p.,!t c on dF patsn l arr.te,

dE c.rcF siù6 æ;.tnyrdd TlE 6lsisls rddtirc ùe ndhyrsrion slatùs orDNA the ICR-DMRI-MÂRI inæraciion ûftne nd.ml .U.le. ir

qlæed h-! s ICR- DMfo, |llt.'rà.tion. britlin8 ÛE 6h{É clôs to tne /sA llmdç Ûd swahins 6 tn' /ee gdq stEr6 DNA nerlrylatid

æ rh. t/9 l'motd

The CICF (CCCTC-binding fâclor) proteit l/âs firstfourd as oân ofthe 5'-HS4 insularor elem€tu ofthe chickenFslobin locN. sepùsring the active globio domsin Êomthe h€ærochromâlic ômain. Thê s'-Hy elem€m proleclsqen€ expression agâinst enhancers wheo plac€d b€lweenrhe scne and eîhancer. as well as asÂinsl position êtr cts onthe

-chmmosome tsee for examplé Bù'gess-Beusse Pt a/

2002). CTCF is requirEd for the €nhanccr blocking activity(Bell et ol- 1999'r.

This activity is â reneBl f€slure of ùe CTCF proleinihâr is hist'ly ;nservèd In higher eukaryolcs. from Dtos-orr'la lo humahs { Moone et a/. 2005).

Thê ll zinc fineers oflhe prolein Âllow iis binding to âmultiDlicitt ofD\À krsels (cf. Ohlsson 3r a.L 20Ol) CTCFbinds rcpe;lÊd sites in ;hishly cooperativ€ mânner tParlr sr,/ 2{XXr- ln the akenc€ ofDNA. it cân âlso dimerize witbhelp oftûe zinc fingers or lhe C-tedinal pan offÊ pmtein.Pânr et al.2m4\.

lD rnous€ eD'throid cells, the CTcF-binditg sitês pârti-ciDatc in sDariai interactions betw€m the activ€ ÈSlobingËncs and. âbour 50 kb amy. th€ LCR rÈquir€d for theirhieh level exDr.ssion. This rEsuhs in whd was lÊimed byrtrË aurtron air Aclive Chmmâtin Hub wiih seveml âctivelooDs a.tins in conc€rt over 200 kb (Tolhuis er d/ 2002).Th; cTcF bindins sires seem to snuciure an inâcrile chrû.malin ore-franew;rk. âs wa! found fi'om ônâlysis ofihc 0_globin locus in prceedtor c€lls, wher€ lh€ globin senes arenor vet acriv€ {SDlint€r?/ a/.2006).

îtCn atso i"em' to organirÊ spatially the coordinated

resuldion of ditrercnl cenes ând loops at the mammâliantsh Htq locw, eNl'ô to tonFot rheir Senomic imprintins inrhi. *ar ts€e forexamole. Reik and Waher 2001)

This. dre insulin ùowth Èclor. lGF2. is ê rÊSulator oflelal growth. The con€sponding eene is imprinted. i.e ii isonh ;xDressed fi'om on€ of the two parcntal alleles. |ts ex-preisioi is coordinaad wilh lhal o1ânother imprinled 8en€.Hi9. fTe t*o senes âte locsled s0 kb apan on mous€ chcmosôm€ 7 (Fi; 4A). On lhe mâl€mâl chromosome. H/9 isexpressed whereâs /&/2 is repress€d Lhmughou develoÈm;nr. lhe ooDosire is found on fte pa|€mal allel€ ln mous€neonaral livè: râtlscriplion ofihê rqo Senes is regulâtêd bvan enhâncei located do*nstr€ah offll9 (Fig. 4A), âbout l0kb from 919 ând about 120 kb from 1&2. A clusler of fou'bindinc sites for CTCF is locâted between th€ two genes' inùe lCn imprinting conFol region lhal regulates lhe methyl-arion ofdilïerÊnt sitrs wilhin th€ cluster and is locarcd 2 tbupstr€an of â,19. CTCF carttot bind to its sites wh€d they

cx rle maæmal inherited chromosome, th€ ditrerer!tiallv merh! târed resion. DMRI, also cônrains binding sitesfor aTcF. ir Dhysicàlly inrcrÀcls with the unmerhylaGd lcRlhouqh CTCF ând porsibly other proteins (Knluti ?r al.200o-), a\ dêtected by ùe lC technique (Dekker er 4/ 2002).The ICR also ohvsic{llv int€mcts wilh th€ maFii arlâch-mem resion VÀJù bdled nen ùo lhe DMRI dom.;n. l&2is rappfo in a 20 kb loop (Fi& 4B). This is sêemingly sùÊfici;fto orevenl ûe enhanc€r to âctivate th€ mat€mal /&2promoler (Kurukuti e, a/. 2006)-

t09

PrevioEçll lhe sâne group hâd shown lhal on the pater-nal chromosome, the methylated ICR physicâlly idemctswith one of the diferÊ'tiâlly rnêlhylâ1ed rÊgion, DMR2, oftd2 ll./wll et ol. 2004\. This brings th€ /e/2 promoterinto coniact wiih the €nhancer in a ll0 kb DNA looD. thusl€âding to ,eP e),pression (Fig. 4C). As the enhancer is oc-cupi€d else*here, it cârnot activar€ l'.19. Th€ pmteins res-oonsible fo. the ICR-DMR2 iflter.ction hav€ not b€€n idcn-tified yet. Some zinc-fin8er proteins bind m€rhylat d DNA.Some ofthem cofocalize fi rhe Hlg/tgl2 tCR lFill;on ela/. 2006)- Thes€ proteins have b€en found to Àssist r€pr€ss-siotr of lr.l 9. ln ânalogy with CTCF, they might also bridgethe tcR-DMR2 inrerâction.

thus CTcF-m€dial€d DNA looping and methylationjointly di.ect the €nhancer to a d;fferEnt proErot€r t!'get onth€ Datemal and îtaremal chromosomes.

Since CTCF m€diâtes DNA loopin& n is nor surprising.at l€ast with th€ vi€wDoint oflhis review. thâr CTCF ;s âlsoinvolvcd in intemôleoulôr âssociatiods.

Accordingly, Ling e, (r/. (2006) have rec€nlly found thatthe 1&2/}/.19 bcus on nouse chromosome 7 was physicallylinked to ânolher locus, t/J ,r/rvt, on chrcmosome I I viaCTCF bound to the Imprinting Conlrol Regioo. This alsoci-ation wâs foùnd by scrcening for all CTcT-mediâled asso-ciarions by th€ 3C t.chnique (or a variârt) ând by fluo-.es.€nr ir rrr! hybridizalion (FISH)- This is ân intriguingfact becâùse only the pâtemal allel€ of chromosome I I isfoùnd thoùgh this locus is nol imp.int€d. D€l€tion of lhehalehal ICR câhcelled the association. Tfte silencing ofCTCF by RNA int€rfe.Ence disrupted the coordinâre reg-ulation of the two loci. only expr€ssion of tfi€ pat€malWsbl,Nfllocrs is alt€red. Thui it was conclud€d thai theâssociation and CTCF allow fd',r-regulation of the aÊnesor âltemâtively lhal the two chromosomes share separatelythe same elenents côncentrrted !t a tnnsc.iption fâctory(Hugùes et al. | 995i Chakalov^ et al - 2U)5 )-

Since the association is restrict€d to th€ Datemâl alleleof chronosomel I, it might be involv€d in the impri inSpncess of ùÊ Ig2/H l9locus on chonosome 7 (Ling e, aI.2006), o. in a tnnsicnt m€thylation and silencing of lheltrô./ gene a! the lvsbl/Nfl lo.tts (Krueger ed Gbôrne2æ61.

similar dala hâv€ b€€n quali&tively ob!âined by zhaoer o/. (2m6) with a 3c-bâs€d techniqtæ, exccpt thar rh€yfind I 14 inter-or intra-chrcmosomal $socistions inslead of3 for Ling s, d./. (2006). Up io foirr chromosomes converg€to tie ICR of lhe /&2 H/9 locus. Cl@l), rficir Ê:p€rimen-tal conditions incr€as€ the sensitivity of thc tÊchniq're. It isalso cl€ar thei the uncovered inter.crions are pr€fcrôntiâl]ylinted to imprinting. Th€y aæ âlso sp€ciffc of a certainstage of gmwth ard depend on th€ reprogrâmning of lhe

l er-chmmosomal âssociations might prEsent somesinilarity with tmnsveciion. s phenomenm knos since1954 in Drosophila. Transvection is an altcration of gene€xpEssior thât dcp€nds upon whether or not gÊne! arepair€d with 6eir homolôgs (Wù 1993; Doncan 2002). Itger€rÀlly involvcs th€ action ofcnharcers in t dÆ.

CTCF binding sites s€nsitive ro DNA meûylatiof, haveâlso been found near the 3'end ofxisr, ihe gene on the X-chromosôme rhat triggers lhe ina.tivation of a single X-ckornosome in X-x fernale manmals (Chao e, al 2002),as well a5 at the boundâ.ies ofdomaids which escâp€ x-in-activation (Filipova s/ .r/. 2005). Th€ precise mle ofCTcFin both cas€s is not known. However, il recently appear€dfton ttro r€€ent studies (B^chet et al.2006: hr et al.20061that the two X-chromosomes ar€ ù"ârsiertly pair€d, $assess€d by RNA"FISH analysis. This pâiring prÊcedes X-chmmosome inactivation. Binding competiiion assays witl,sub-fi'agments of the X ina.tivâtion center region. disruptsX-inactivation al|d confirms this associarion. ComDetitionwith DNA fiasments cootaioins rhe CTCF biûdnrg sit. alsodisrupts X-iBctivqtion (Donoho€ er a1. 2007). Like for the/gz,l1l9 ICR associarions, this suggesls a role tor CTCF âsa bridging prorei'r. aloo8 w;lh cofacrùrs su.h æ the Yll

DNA roa9i'{ Dd DNÀ piims ii rrr

protein (Donoho€ e, ar. 2007).Anolher L/rrs-chrcmosomal regulation was prêviously

described by Spilianalis e/ a/. (2005). Thb group has foundthat th€ interferon'T pmnoter regioD on mous€ chroînsomel0 was j uxtâpos€d with lhe locus control region ofthe idr€r-'eukine sen€s otr chmmosome l1 in rhymoc)'tes precursoEofth€ imlnune sysl€m T-cells, both CD4-help€r T-cells andCD8-killer T-cells. It now aDD€ars thât this inlcr-ch.omoso-lnâl âssociation is lost when naive T-cells differentiâte inlo1,,1 or TH2 cells. On the cont âry, in activated Tt2 cells. thepmduction of interleukin is associat€d with the folding ofûe interleukin gene into sv€râl loops with coordinaled €x-pression, due ro a potein, SATBI, only found in thymo-claes. A câgelike distibution of SATa I surounds the nuc-leus ând anchors th€ loops io tie chromosomal soaffold (Csier d/. 2006). As th€ nâive T-c€lls eith€r do nol €xp.ess th€cyokines or Fom onl) one allele under sperific condirions.Spilinatis ?, d/. (2005) sùssest rhat pâiring is âssociaredwith silencing of the allele.

Thus in mâminals. chromosomal associarior s€em iopromoLe silencing and monoâllelic expression. *hercbyD\A l@pins is morÊ ùnbivalenr. This is âlso *har emersesliom bâcte.ial replicâtion, where handcufiing is exclEivelydevoled !o silencins ùd to lhe control of plasmid copyrumbea whereby DNA loopins can be us€d for borh posi.liv€ (cf R6K) and n€gative c-{'ntrol (cf. F-fâcùor) of replic-ation, Iike in o-ànscriptionâl regulation.

4. OLIGONÊR|ZATION OF THE PROTEIN

S€vdâl p.oteins involved in DNA loopins âre nalurally oli-soderized, sômetimes to a high degree.

Thânls to a leucine zipper, two subunits of/ac reprEssorinteractins side-by-side as a dim€r, can also int€.acl heâd toheâd as a tetlâmer wilh two binding sites for DNA (Alb€ni?l d/. l99l; Chakaheriân e, a,I l99l).

Th€ deo .ÊprÊssor is a tripl€ dimêt with thr€e biodiogsites fo. DNA (Monensên el a/. 1989), rcsultitg in doùbleloop formâlior on binding with nativ€ DNA (Amouyâl e, a/.1989).

The )cl r€oressor is a dimer caoable of tetmm€rizâliorand oclaineriâioo in d|e abGenc€ of DNA (Senear el dl1993: B€ll el dl. 2000: B€ll ard L€wis 2001). The octïner-iztion allows DNA looping b€twee. the O/ ând Oraîaysofrcp€Âts (Dodd er a/. 2001).

The record of oligomerizâtion pertains to thê I 86CI repressor that compris€s 14 sùbunits âs â h6ptame. of dimersaîanged like a wùe€l widr seven possible binding sites lorDNA (Pinkett e, or. 2006). Electrcstâtic forces mther thansoecific seouence dêteminanli âre resoonsihle for this oli_gbmerizatidn. This otiSomerizâlion exdlains well rhe âll{r-non€ occupancy of the thr€€ | 86cl rep€âls of the phaSe PRprcmot€r (IHd "r or. 1996; Pi'kett e/ a/. 2006). Th€ arraysot sires a.e wrapp€d ffound this "wh€el', making the sutFùnit orgsnizâtion pârticularly suit€d to the tandem alrange-ment ofthe siles (cf. Amouyal el lrl 1998). For û!e l86clr€pressor (ând lô â less€r extenl, for th€ lcl .ep.essôr), its€€ms that rhe cell has s€l€cted both site and prolein orgânÈzâdor, in a way lhey ar€ best fitted for âdjâcent bioding.

Now. apân fiom açsisting repressor binding to DNArep€at!. oligomerizarion ofthe prolein "h€n il pmvides anal.€ady assembled protein bndge, cm also siabilize lhe loop,as is the cas€ for lie /ac reDEssor However. more lhan wirha long range diiarc€ aclion, oligomerizâlion in lhe absencêof D\A seems ro b€ associaled with lhe fa.ilitated r€(og-nirion of nultiple loci scâter€d on a preci* potion oftheg€nom€. as app€ars liom the /o.. deo or /E6( / rÊgulations.Ind€€d, if th€ two arnys of Or ând Or sit€s of ihe lphage,are 2.3 kb disrânq the mosl dista sit€s for lhe lt6CI pro-lein, F. ând Fr, âre "only" 300 bp distânt, 8nd betw€€n lhes€two sites. ihere arE at le€sr four olher operator sites, clos€ loPL and Pi, 62 bp distânt. the lhee /dc operato.s are like-wise scanerÊd over 500 bp ând the thr€€ d"o op€ralots ôver880 bo.

In t. coli. the lârSesr d;sranc€ involled ir DNA lopins

1 1 0

a.E,c.ffiardctu tO), l

is that obs€rved fo. replication initiatioû of plasmid R6K.SDecificallv. the t oriein il€on and the $vcn Y ilcronsb;dsed b] Pi, are sepÀmred by 4 kb (Fig .lÀ) wiÈh lheoresér sàre ofknowiecge. il is dimcull ro sp€ciry lhe pre-;isc shrctûe ofthe protein bridge responsible for this longdisrance action. Ho*.vêr, DNA looPinB migl'l requir€ aso€.ific iditiâror imerfacê, differenl ftom lhe contâcts thatarÊ âlrlady lstovn betw€en two initialor ûoleculcs (Sranet ol.2006).

llandcotfing se€ms to require rÊp€ated itemns, fiorn 3(DSClol) lo 7 (R6K-t) repeÂts, as *tll À5 stroflg inErac-rions ihar ar€ provided by a highly coop€rrtive orSarizadonof th€ oroùein rr ûe iteronq whh r laver of dimeric ptoreurholdiîi aDan iqo lÂyels of D\A-boùnd nonome$ (Tout-darian-anà Helins*i 1998i Zzaman ând aadia 2005: Flt.3B).

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ct r.bt" r- n nnnd tr Mikùtù JÀ o.bo.* cq Fr.s ? (2m5) R.Pldi)o !.d llwiFid: 3haphg tlE lort .F olt[.8m ^/tu Xet_tM Gd66.669477

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clbrn R Mflrùlt JC (l93il Læ RepM&. @.itor inr.rr.nôtt oEÙ_br didroi$ sùly. NElei. ædr Âalu4h 9.517t\tu

Di ||( rtlic4È.ir! ia B..uEy 41. Ftte nÀ È!D t?. trulotr J.(l.tlodi D( r2&5, Muh'pk h.trEctû( Gùll's Ù .h. @ml olPl" nra ràs'm P6e.d'"2! ût tk \tdtùl/td'd.qot &tqrc\ rxl

lm. 2856-2361D.kLr J. &Dt K I).|&r Xl,l, X}.tar N (X02) CtD(Ûine ch|r'lr|)'@

enfomarid &taY 295, 136lll ID.dd IB. P.rliE Àt ttdi!&i. D, Es.n Ja (200I ) (xrandizaiÙ of rcl

EplNr 6 n .d.d fù.iÊdiE rw*iù ôf PRM !d eftci.i sd'n'nBÂ'on begFy Cæ d Ds'.l"æt |5,1013-1022

ooaa n, rr- .n Oçcl oNe Unaoe bv rhe @liph.s! 136 eÉ.3s bndingûottincl Jùùn d Bralqiùn Clcddtvz1l. ttsl2'll54l)

Domùæ Mq Zb.{ Lt, X. N, Sù| Y' r- JT i2OCr?) lthdifioriû ôf !cTcF coitu Yvl ld ù. x ôtmme binary sqtch ,&t .rdr t'zl, 25.

Dulot IW l20(Tl) Tdvedrù etus i lt@lÀIn ÀNdl kerb- ol(;u'

Ihnn Ît! tur! s, O!É.r g sbGl RF 0 934) Àn o!.dd , -:30 be F|urhn ù Eqùnd for qr6in of @r,rD op€M p@@r .ddidd of DNAb€licll n8 bd€ lù. ôD-û a.d D.mû{s cvolic.llv hitdm Ear<jdP@.arrys ôl tfu Ndtûel r.rti.Ày,lt.1cleL4r US4 31. 5017-5m0

Dl'û.m En .!d Mdlrda a ( l99O) /d. ieD(6s forc shbl. looF ' v'!D

wid sDèctLd d d ræ DNA@biniIg ![ llllæ nû]r.l opsàG ./d/zJôt |rol ardû anlosl 21 3. 1 6 -n 5

Fitipor cN, cr.q ù|( MoE Jr Trung ff, E{ YJ' NFv.. DK Ts.ti- xO. rcr. <n t:oo:l e-od.t6 b.@ cbrMtl ftdt@ôr ii'mtùd od egæ bid (-IC[ srÈ lrr CpC ùdh)ldh dÛngeth d.wl@@ D.*tqtu 1 LJI A, ll-,1ù2

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F udld n|. M. FlLn MJ. E li-Li DR (1936) P6iriw Ù'd egl rcrol. oaû ûitidd FtÉô d ù mgjf 01Êplicdi@ Pmeedryt oItl":u-,ôttl ^cabtu d*t.M LISÀA3, 15-9(a9

nr.ùnd Y. OÉ!. JD I l9A0 Dd @h.rsm of r.'Nim !t â ditu in

ttq ùr .îm P@tt"g of tk NaûMl .4caleù, d S'1tu2' ItSÀ 6

r.i.d M r'd rudta JM O96) DNA l@ping ând ,?. rcPEsr4AP intænù.,L,æ2?a. l9:lGl93l

G.mim.r, s.!ô D (1933) Iredriôô of th. pldsnd R6Kdc'd'd tqleid innitdpreir sirh È bhding tites m DNA t--t'lf,4 l2rll4

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rtû i! i;oMining b.c.nd d@tds tldtl t.69-*1s.rin cI, 8..ùd GN (l93,r) RoL of DNA Egiors tlùlirg dE tvÈoprt

vturû nî Es.i.r'dtu @û rl bstd of ,s op.rat@ tlaMd (;æ t2.

Euru. TÀ Pùùo À M.Mtrs J. fl@L P. Jrctlo! It|" Co'r Pn I lçr)À *'n. !|lfu or nuo- na *n?\t6 tttsil Jtntd 4(clt s I

.nc. SlNlffi 19. t945Iàni r'9. Ot@ t" Adrtr s (1931) À cffil €ldal sirÀin ' $ÙcÛtl

@ x@lor.moaI.a/, (?l/ J,, ?3-l_733ù;.r' R Mûloi J ( leôl ' adEt. Égùler! É4hm'ru 'n th. s06is or

.'titins Jdttul 4 LhLdlrù Rtol!'g 3 313'356ie.tr J. B.ûi DT (1916) nE ûD.ridtl Fp{est Frre ot ' ffic 6

Gpl"r|s+g.lnttdæ chiJllâ æ s9iûize,J in ùtffi Pæ'aitas {ttPN d t aûl /@&,ty of 5. EB IÂA 73, 3 529'3 5 13

Korori f,, M.eilr E aisûli Y, Ltni 14 w.d. c, Mili x ( lry) ctvebi idG of . ÛÛr.rvdic i.oridid iÛri.ld prdeit bdrd ro DNA a2.6A @ttniû. lir EwOJNTat ra,15q- 4aJ

Xnn.r B. Nld.r|.' i4 A.dti i4 nÀd t' rn W0cli.ûD'r!'r' ÀMrs.Ftlil s (l9AD ræ Epl6s foms l@pé ùlh li@ DNA drvi'at*o eiflbb 9...d &. otdrG fltt titrrBo Joe1n|6.l4alrlcl

xnn.. !- ^r{rd iL Nddr.it À M.|lFai Ella$) DNA cuDos'l-

Ds cns*É th. FdB Equ@d{ of M,a. op.rd,F lû DNA loop rs'mûù qtfi /d r4rs tr l'r6oJtunl1 \41'556

xffiC.O.n ECS{2Û06)R ising !b. cùlln on nLicbonlrxld 'M-at* r'.r* - r;æ* n.dlltlC

Kuhnb q Aù.€ C, friÈ |tt' Ahrig r 0939) RNA po!]tæ d 3t! e

ocs ùr'n rbul|@dt ttd wt' DNA hdrns ro rh€ ctul r'sim of

if* bÈJ.&t.td.,l, e.rdc 4.Ù nPl vauJ'ùnna 1211 1155KuriE.Ltt !r S ldr. x& r.rôsùi SÀ ribrÙi@ M ('06) RoL of

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(u;bd s. ÎÈ.d vIç L'tilbr q P'4rctd D' M!ÉI À zrto 7-

rrù.6rov v tdr w, oùrr.t lt (2006) cTcF biiding .r rn' t'9 D-pnDong cttul tlgtd 'rd|s ÈÈntd[ 'nnêntd hrgrradPr *mutiiefotuio b r.sd dùd€ qÉ b t4l2 Ptu'cÀ'F: dttt \:awa,l.ad.tu df.9t.d t^,, 1lB, 1063{-10639

Litrs J(). û i. ur JP, r,| rù. ab IIL Qlù xw ft.rrv ri'L uorr' ÀR,irxx-' crcr .'"a'æ,nqgt*l):@lolodl'dd bdÉ /9,? n/o

st]d ïsbI NI SeIas 312,1&212

l

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M.Xxùd ii, aon MÀ Tm*.r PA, HdiËri o|t11939) NegltrE @iûolof pl6ni R6K retli@im: pcsible rk ôa idmkolù opt'nB orrettiÉid cisi6 Pu?srrrx dtlc Ndtohi 'l.d.ry aIS.@M t$a

Mim A, Mu.lda q Bdù D (199t) ^clivûo of dsd r('Ùcdion ùi-

si6 '' ve b9 DNA loping 6 ev€aled by ! mvd mrûr aod ôfd mili-.rd ,.æin &f.dit. i. co.!€.n,vily âi â ditu flt t: 4x, tnfln ll,

ùlc l|, FilinFvr q ld|||trd D, Pùr-r.rr E, Ctô Q, s|ritn Sr,Mùrù.[ À G|tt À B.rtlùbr 14 AEold & i.rl U.I-lttif}Do n oth.d n zbù J. n nlrir, & Lrrdr@ v (2û5) crcF is(jo@Éd ûd lrnsdt /d to huDæ rri dfers dhlE Udbng of rbetb* iÉllas E)tt8o Rù\ 6. 165-t 70

M.€. P. Bd R lvlyltl B, Ev.r.rt & Gùb MP, CLnbd P ( l98l | 11ÉSv,tO 2ù9 ED.d t6 ô sking cfiæt d g@ .xF6id borh i. Svro oddù6 cùlmic Mbirot! lr'!.&o !ët& R.t@t 9.û114.xa

Moriær L D.nd.æ[ q llrûed ( ( 1939) Plmficdio !n d.ltlm_rq ofdÉ rf.oR r!îN ofr'. .,l lhe EMBo JMtô,32r]ir

MrdL.iÉ S, Erird E, a.tih D (1933) E âÉdei. tn'æûm nplsùt R6K inwlB â DNA lmp ûniared Ùr iniùd F@in ael 9.l7J-333

Md.'-tli! B, CnDo l. Cilù. W (l%3) Mulùs $,. nlrê rR ræt stæ IMrÊt ô[ thè N.ttml ,14'deûr .t &1æs lN1a9, 1259'

M..Ell À ûd| S ndl w (2004) I'tedm bdw di|f.dri'Iv ne_thyl.r.d Egtm p6itids $o inpd €ng@lg2dnHI9itlopoÊnsrec'6c clEddjn IæF xdrE d.,sr.r J6, 3t9191

|lo.idm x (1990) Cdtbl of pl6md repliciicH* do i|ms sl $.EglElno ftqsx' (.ir63, Il2l-1124

o. sS. En .n f,L t(rn.r H. MrllæE a(!99o)rù.rhÉop.rdGofth. læ ooem æo!êtu tn rctc$on 11t EI RO JN.\C 9.913 C19

6r.. S ^.dyd M. |(oltùol R trtr Wikré8.r!!rù B, Mdh._ûiù I( tqS4) (àEliry arld postn@ of dæ ùÈ /e o!.Etqs of E cdl d.fiæ .6ciæ or @iû fr. EMltt).loutùn 1J,L41vu55

o o.Dn Ra, DùMny iq l9Lttr*. I(s (1980) DNA bùdi4 ch',dF-t$6 ot la_bs r.oGsû ad dE typ$çrdic.lt æ Eûs I . J@_ùt o{ Btolqt.d I lLnii4 8,lotao l01c6

otbd l( ltEb*nz & hb.n..Ld v (2lnl ) cTcF is ! ûiqalt wlilctffiiÉim r.sdrnd linrd b ctigeÉtd ïù diæ IP'À r' rLæ,.t

?.1SK (L|Mi DX (1933) Fl pleid nptùrt inh:de s.qrcsÙ.rim isiMd.qrd. ro dDlâô @trcl hy iniliûdt riÀing nra Jtu n of a.ùcr'

È!r V, Kimrdi g P|! .bd L ShtEtldtli. S it ri.m P, Ralqrit, &Krd@. È rd|mrw V On&4s R (2c04) }r|iitt or. nnd. C:TCFÈrSd siÈ wirhir dF â/9 imFinting @hl Eerd kd rô 16 of /eA ih'Dnûns dd c.intlex rd.ns ofdt ,aru dùyldio l+d n5.6d ùbsi-@ t M a1d celrkn Biolos) 4.3491-3tv

È€ lr. f,.r..|r M, Àndy.l M (?040) ̂ fdit6l 6ev in Fs.*.a.rzcoû b dd.cl nùé'sèd i æno bw e.læ@ cFÉs dlm.uNulen ldû RevaÊh 24, lf.lo-an/B

P.fu i,l, $.it TÂ (1996) DNA lmping rnn ,u. r4rM{ÀP 'niqicl'dS.@ttl, l9]9-193O

Ë.ùl i,l, Cdld. v, BqrsFoir S (19æ) ssdd ed ûi'd olfd ofl6.lr..'t mr s rnEtigdio of rhdr rck m tÉ Ég e.y D..ùûtBi Jdrr,r a/t oleùû Ad.rg) lr1,31r344

Piklr ulv, stànir xI,g.yûr s, Irodd IB ùÉ T, Ech..ùild ÂE!r. Jq lÂb M i2oo6) The sr.4tuol bi! of @<'oqtiw Egû.tù aù .&drrê sÉû€.ic swilci n&ledrr. c.l] 2, @55t5

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