A multistep epigenetic switch enables the stable inheritance of DNA methylation states

Transcription

1 27 Nture ublishing Group A multistep epigenetic switch enbles the stble inheritnce of DNA methyltion sttes Hn N Lim,2 & Alexnder vn Oudenrden In mny prokryotes nd eukryotes, DNA methyltion t cis-regultory sequences determines whether gene expression is on or off. tble inheritnce of these expression sttes is required in bcteril pthogenesis, cncer nd developmentl pthwys,2. Here we delinete the fctors tht control the stbility of these sttes by using the gn3 gene in Escherichi coli s model system. ystemtic disruption of this system shows tht functionl switch requires the presence of severl, rrely occupied, intermedite sttes tht seprte the on nd off sttes. Cells tht leve the on nd off stte enter different intermedite sttes, where there is strong bis tht drives cells bck to their originl stte. The intermedite sttes therefore ct s buffers tht prevent bck nd forth switching. This mechnism of generting multiple sttes is n lterntive to feedbck regultion 3 5, nd its generl principle should be pplicble to the nlysis of other epigenetic switches nd the design of synthetic circuits. Methyltion of DNA t the cis-regultory region of mny prokryotic nd eukryotic genes cts s n epigenetic switch to turn gene expression on or off,2. The on nd off expression sttes cn be inherited for mny genertions, llowing the cretion nd mintennce of differentited cell lineges. The fctors involved in determining whether gene expression is on or off hve been studied in detil for mny epigenetic switches 2,6 ; however, the dynmic process of trnsitioning between the on nd off sttes, which determines the stbility of these sttes, is not well chrcterized. To gin insight into the switching process, we hve used the gn3 epigenetic switch in Escherichi coli s model system becuse it does not involve feedbck regultion 7, which by itself cn generte stble on nd off expression sttes 3 5. The gn3 gene encodes n outer membrne protein, ntigen 3 (refs. 8 ), tht is involved in biofilm formtion. The methyltion stte of three GATC sequences locted in switch region downstrem of the promoter 7, 3 determines whether gn3 expression is on (methylted) or off (unmethylted). witching between the methylted nd unmethylted sttes occurs t rtes of 3 to cells per genertion 3 6. The methyltion stte of the GATC sites is determined by competitive binding between, globl oxidtive stress protein, nd DNA denine methylse (). DNA methyltion by is two-step rection tht first genertes hemimethylted DNA (M H ). The hemimethylted DNA cn then become fully methylted (M F ) when methyltes the complementry strnd. There is no DNA demethyltion rection; therefore, methylted DNA cn be only diluted out of the popultion over mny genertions by the synthesis of unmethylted DNA t DNA repliction, resulting in the conversion of fully methylted to hemimethylted DNA. Hlf of the hemimethylted DNA tht is not methylted by the next round of DNA repliction becomes unmethylted. The unmethylted switch region cn be re-methylted or bound by s tetrmer 7,8, which blocks nd results in off expression. To chrcterize gn3 switching, single-cell mesurements were necessry to distinguish chnges in the expression of gn3 from chnges in the frction of cells in different sttes. A genetic mplifier ws developed to fcilitte single-cell mesurements of trnscription from wek bcteril promoters in their ntive positions (Fig. ). The mplifiction system replced the coding sequence of gn3 in the bcteril chromosome with the coding sequence of T7 RNA polymerse (T7 RNA), resulting in deletion of the Ag3 protein. The expression of T7 RNA, which is under the control of the ntive gn3 regultory region (which includes the first 8 bp of the coding sequence 7 ), ctivtes T7 RNA specific promoter tht regultes expression of the green fluorescent protein (GF) gene locted on plsmid. The cretion of multiple GF trnscripts by ech T7 RNA molecule nd the plcement of GF on multicopy plsmid incresed the fluorescence signl nd improved the signl-to-noise rtio. The mplifiction system enbled colonies nd single cells in the on nd off sttes of the wild-type gn3 strin (chnl35) to be distinguished by fluorescence microscopy nd flow cytometry (Fig. b,c). Overexpression of (chnl) or unopposed ctivity owing to the deletion of (chnl26) shifted the system towrds the methylted sttes (M H nd M F ). The distribution ws unimodl with full trnscription ( on ), indicting the M H nd M F sttes hve the sme expression level nd/or one of the sttes predomintes. In support of the ltter, most hemimethylted GATC sites become fully methylted soon fter pssge of the DNA repliction mchinery 9,2, suggesting tht the on cells re predomintely in the M F stte. The overexpression of (chnl9) or unopposed binding Deprtment of hysics, Msschusetts Institute of Technology, Cmbridge, Msschusetts 239, UA. 2 Deprtment of Integrtive Biology, University of Cliforni, Berkeley, Cliforni 972, UA. Correspondence should be ddressed to H.N.L. ([email protected]). Received 3 August 26; ccepted December 26; published online Jnury 27; doi:.38/ng956 NATURE GENETIC VOLUME 39 [ NUMBER 2 [ FEBRUARY

2 27 Nture ublishing Group b c gn3 regultory region owing to the deletion of (chnl25) prevented methyltion nd cused inctive trnscription ( off ) s expected. The observed behvior of gn3 using the genetic mplifier is consistent with previous LcZ studies,3,8. The deletion of both nd (chnl72) trpped cells in n unmethylted stte without bound to the switch region. This strin hd prtil expression stte tht ws in between the on nd Bckground b c trin chnl72.6 chnl72 dm oxyr.3 chnl25 chnl228 chnl55 chnl288 chnl586 chnl56 T7 RNA polymerse Knmycin Deleted region p5a ori phl32 (Reporter plsmid) Chlormphenicol T7 promoter GF Colonies chnl25 chnl228 chnl55 chnl288 chnl586 chnl56 off sttes (Fig. 2,b). expression in the bsence of nd hs lso been reported in vivo with the LcZ reporter 3 nd with n in vitro trnscription ssy 3. The prtil stte indictes tht expression is not simply switched on nd off by unbinding nd binding to the switch region respectively; otherwise full trnscription would occur in the bsence of. Furthermore it indictes tht DNA methyltion is necessry for the on stte. 2 Totl cell fluorescence (log.u.) Figure 2 Deletions of the gn3 cis-regultory region. () Mutnts strins creted by deleting the upstrem region, promoter () nd/or switch () region (not drwn to scle). The switch contins the three GATC sequences for which nd compete. (b) Histogrms of single-cell fluorescence for ech mutnt strin with gussin fits for the off, prtil nd on expression sttes. The on stte is decresed in the nd chnl586 stins, indicting tht sequences upstrem of the promoter re required for full trnscription. ingle cells hse contrst Fluorescence 3 d equences upstrem of T7 RNA strt codon deleted Totl cell fluorescence (log.u.) Control with no reporter (chnl3) Totl cell fluorescence (log.u.) gn3-t7 RNA chnl gn3-t7 RNA chnl Totl cell fluorescence (log.u.) (c) Deletion of ll gn3 regultory sequences (chnl) results in prtil stte (gry columns) tht hs slightly lower expression thn in the nd double deletion strin, chnl72 (blck line). This lower expression cn be explined by loss of the ribosoml binding site (blck box). The on nd off sttes for the wild-type strin (chnl35) re shown for comprison. (d) Replcement of portion of the upstrem region with n lterntive sequence of identicl size (chnl33). The lterntive sequence (red line) is the residul scr tht follows excision of the knmycin resistnce cssette by the lmbd Red method 3. off (chnl35) on (chnl35) dm (chnl25) + (chnl) oxyr (chnl26) + (chnl9) Figure Genetic mplifiction system for mesuring gn3 expression nd switching. () Genetic mplifiction system. (b) hse-contrst nd fluorescence microscopy imges showing regions of on nd off cells in single wild-type colony (chnl35; top) nd individul on nd off cells in liquid culture (bottom). cle br, B mm; rrows indicte cells tht re off nd therefore not visible in the fluorescence imge. (c) Histogrms of single-cell fluorescence for ech strin (ll crrying the genetic mplifiction system) in M9 medi supplemented with.% glycerol nd mesured by flow cytometry. Left, wild-type () bckground strin derived from two seprte colonies, one of which is minly off nd the other of which is minly on. Blue line indictes the control strin (chnl3) with the phl32 reporter plsmid but no T7 RNA polymerse. Middle nd right, nd deletion strins (top) nd overexpression strins (bottom), respectively VOLUME 39 [ NUMBER 2 [ FEBRUARY 27 NATURE GENETIC

3 27 Nture ublishing Group b Trnscription strt site Upstrem /prtil colony Ribosoml binding site gn3-t7 RNA chnl222 chnl22 chnl226 chnl329 colony (chnl35) chnl chnl chnl chnl Totl cell fluorescence (log.u.) Totl cell fluorescence (log.u.) The origin of the prtil expression stte ws investigted by deleting the promoter, switch region nd the sequence upstrem of the promoter in vrious combintions (Fig. 2). The prtil stte ws observed in mutnts without the promoter (chnl25, chnl228 nd chnl56; Fig. 2b) nd when the entire gn3 cis-regultory region ws deleted (chnl; Fig. 2c), showing tht prtil expression is not specificlly due to the gn3 promoter or to other sequences in this region. In ddition, deleting portion of the upstrem sequence nd replcing it with n equivlently sized lterntive sequence showed tht prtil expression is not due to the fct tht the deletions shift the sequences of upstrem genes closer to gn3 (chnl33; Fig. 2d). Together, these dt suggest tht prtil expression rises from nonspecific trnscription. The prtil expression cn be repressed resulting in the off stte when, the upstrem sequences nd the switch region re present (for exmple, chnl35; Fig. c). The upstrem region is known only to contin the non-essentil Yee pseudogene 2 ; there re no known gn3 regultory sites. We therefore systemticlly deleted the upstrem region, keeping the distl end of the deletion fixed nd extending the proximl end towrds the promoter, in successive mutnts to identify specific sequences necessry for the off stte (Fig. 3). For ech strin, single-cell gn3 expression ws mesured. We observed tht, ech time the deletion ws extended, the frction of cells in the off stte decresed nd the frction in the prtil stte incresed (Fig. 3b,c). The decrese in the off frction ws gretest when the most proximl sequences (B69 to 26 bp upstrem from the center of the switch region) were deleted (Fig. ). The orgniztion of the regultory region with the promoter positioned between two regions required for repression 22 (proximl nd switch), coupled with the observtion tht deletions fr from the c Frction (U N + U O ) (M F + M H ) 222 trins: incresing size of deletion (bp) Figure 3 Deletion mpping of sequences upstrem of the gn3 promoter. () Deletion mpping strtegy showing the promoter () nd switch () regions (not drwn to scle). The ribosoml binding site is represented by blck box; the double-heded rrows indicte the region tht is deleted. (b) Representtive histogrms of cultures derived from off/prtil nd on colonies for ech strin. Gussin fits for the off (red), prtil (blck) nd on (blue) expression sttes were determined for cultures derived from unmethylted colonies. The number of unmethylted cells in the cultures derived from methylted colonies ws insufficient for curve fitting; therefore, curves from the off/prtil cultures were superimposed to show the similrity in the distributions (inset). Owing to the long hlf-life of GF, the prtil expression level is influenced by the mount of time tht cells spend in the off nd prtil sttes. As result, mximum expression of the prtil stte should occur in strin, where cells cnnot switch to the off stte, s ws observed. (c) Reltive frction of cells (± s.e.m.) in the off, prtil nd on sttes in cultures derived from unmethylted colonies for ech upstremdeletion strin. trnscription strt site cn decrese the off frction, suggested tht the repression might involve DNA conformtionl chnge. DNse I footprinting studies hve not identified n -binding site in the proximl region 3 ; therefore, it is unlikely tht DNA looping occurs between molecules bound t the proximl nd switch regions in mnner similr to tht described for the Gl nd Lc repressors 22,23. An lterntive mechnism is tht, which hs DNA-bending properties 2, enbles the switch region to function s hinge tht brings the proximl region in contct with downstrem region (Fig. b). The model predicts tht downstrem sequences between the switch region nd the strt codon re crucil for repressing prtil expression, nd this prediction ws confirmed by loss of the off stte in mutnt in which these sequences were deleted (chnl332; Fig. c). The bove experiments identified two different prtil sttes. e prtil stte occurs when the -binding site nd expression re intct but the upstrem sequences or downstrem re deleted (for exmple, chnl33 nd chnl332; Figs. 2d nd c), which we designte U O (unmethylted plus ). The other prtil stte occurs when there is nked switch region without DNA methyltion or bound (chnl72), which we designte U N (unmethylted plus no ; Fig. 2b). The essentil difference between the U O nd the U N sttes is, respectively, the presence or bsence of binding. Both sttes hve the sme prtil expression, which indictes tht binding by itself does not lter expression. binding does, however, block DNA methyltion. As result, the U O prtil expression stte cn stbly exist in the presence of (, Fig. 2b), wheres the nked switch region (U N stte) is unstble becuse it cnnot prevent DNA methyltion (chnl26, Fig. c). Becuse binding cn occur in the bsence of the repression mechnism, but not vice vers, we propose tht the off stte is chieved in two consecutive steps: the unmethylted switch region (U N ) is first bound by (U O ), nd then undergoes repression (off; Fig. 5). NATURE GENETIC VOLUME 39 [ NUMBER 2 [ FEBRUARY 27 27

4 27 Nture ublishing Group b Frction of unmethylted cells in the off stte chnl35 () ize of the deletion in the upstrem region (bp) roximl X Downstrem expression chnl222 gn3-t7 RNA chnl22 chnl226 chnl329 We constructed kinetic model to determine how trnsitions between the two methylted sttes (M F nd M H ) nd the three unmethylted sttes (U N, U O nd off) regulte gn3 switching (Fig. 5). The frction of hemimethylted cells tht become unmethylted per genertion (M H to U N trnsition), termed methylted loss, is given by.5e kmt,wherek M is the rte t which the hemimethylted switch region becomes fully methylted nd t is the DNA repliction period (upplementry Methods online). The frction of unmethylted cells tht become hemimethylted (U N to M H trnsition), termed unmethylted loss, occurs t rte of k H F UN t cells per genertion, where k H is the rte of U N to M H trnsitions (k H is estimted to be 3 times slower thn k M ; ref. 25) nd F UN is the frction of unmethylted cells in the U N stte. From the expressions for methylted nd unmethylted loss nd reported on-off switching rtes 6,we determined the trnsition rtes nd the distribution of cells between the sttes. The frction of cells in the off nd prtil sttes ws the sme in cultures derived from methylted nd unmethylted colonies, indicting tht trnsitions within the unmethylted sttes hd reched stedy stte (Fig. 3b). The stedy-stte rtio of the U O nd U N sttes provides K O, the equilibrium constnt for binding (k O ) nd unbinding (k O ). imilrly, the rtio of the off nd U O sttes determines K R,the c Normlied cell count Replce the downstrem region Figure 5 Moleculr model of the steps involved in gn3 on-off switching. trnscription rising from non-specific RNA polymerse ctivity is indicted by the purple ellipse. Trnscription in the on stte occurs from the promoter site contining the 35 nd sites for s 7 (blck nd gry ellipses) when the switch region is fully methylted (blue sterisks on both sides of the switch region). At DNA repliction, the fully methylted switch region becomes hemimethylted (blue sterisks on n rbitrry side). The hemimethylted switch cn block binding (less effectively thn full DNA methyltion); it is unknown whether this trnsient stte hs prtil or on expression. The hemimethylted switch cn be converted to the fully methylted stte by ; if this does not occur, then hlf of the hemimethylted switch regions will become unmethylted (U N stte) t the next round of DNA repliction. nd compete for binding to the unmethylted switch region. Binding of to the unmethylted switch region (U O stte) blocks, thereby preventing DNA methyltion. The U O stte cn undergo conformtionl chnge in DNA, resulting in the off stte. 2 3 Totl cell fluorescence (log.u.) roximl trt RB chnl332 Region deleted between cnhl226 nd gn3-t7 RNA expression Figure equences necessry for mintining the off stte. () Frction of unmethylted cells in the off stte s function of the size of the upstrem sequence deleted. Dt re the men ± s.e.m.; the connecting red line is guide for the eye. Deletion of the proximl sequence, B69 to 26 bp upstrem of the center of the switch region (bounded by broken blue lines), cused the gretest reduction in the off frction. (b) roposed mechnism for generting the off stte, involving n interction between the proximl region, downstrem sequence nd (represented by single molecule) tht blocks prtil trnscription (represented by the purple ellipse). X is n unknown protein or fctor tht might medite this interction. (c) The downstrem region (69 86 bp from the center of the switch region) ws replced with the lmbd Red scr to produce chnl332. Red, green nd blck boxes represent the proximl sequence, downstrem sequence nd ribosoml binding site (RB), respectively. Double-heded rrow indictes the region deleted. equilibrium constnt for repression (k R ) nd non-repression (k R )of prtil expression. In the wild-type strin, K O nd K R were 3.7 nd 5.8, respectively, corresponding to cell rtio of bout ::58 for the U N :U O :off sttes. The shift from the off stte to the prtil stte cused by the upstrem deletions (corresponding to decresing K R ) increses the number of cells in the U O nd U N sttes (the U O :U N rtio is unltered becuse the concentrtion nd its binding site re unchnged). As K R decreses, therefore, the frction of unmethylted cells in the U N stte (F UN ) increses from.6% in ttes Nonspecific trnscription (prtil level) witching trnsitions σ 7 -bound RNA polymerse (on level) Upstrem Downstrem Expression Fully methylted (M F ) Upstrem Downstrem Hemimethylted (M H ) Unmethylted no (U N ) Unmethylted + (U O ) Repressed unmethylted + (off) DNA repliction % DNA repliction 5% DNA repliction 5% binding (k O )? DNA conformtion (k R ) (k M ) Upstrem Downstrem (k H ) *** *** *** unbinding (k O ) Upstrem Downstrem Loss of repression (k R ) Upstrem Downstrem? or prtil 272 VOLUME 39 [ NUMBER 2 [ FEBRUARY 27 NATURE GENETIC

5 27 Nture ublishing Group b for ech stte Frction of methylted cells in n unmethylted colony.2 M F..2 M H..8 3 U.5 3 N U O i ii iii Genertions Model Experiment the wild-type strin to 26.8% in the strin without the off stte (). The increse in F UN in the upstrem-deletion strins should increse switching from the unmethylted stte to the methylted stte in ccordnce with the expression for unmethylted loss. To compre this prediction quntittively with the experimentl dt, the dynmics of the gn3 system were simulted with the bove-derived prmeters nd cell growth (Fig. 6). The K R vlue obtined for ech of the upstrem-deletion mutnt strins ws substituted into the simultion to predict the frction of methylted cells tht should rise in n unmethylted colony. The simultions showed tht incresing K R (tht is, n incresing frction of unmethylted cells in the off stte) decreses the frction of methylted cells s predicted (Fig. 6b). In ddition, the simulted nd the experimentlly mesured frction of methylted cells were in quntittive greement (Fig. 6b). This observtion supports the model s prediction tht repressing prtil expression enhnces the stbility of the unmethylted sttes by reducing the frction in the U N stte, which consequently reduces the number of cells tht cn switch to the methylted sttes (Fig. 6c). witching from the methylted to the unmethylted sttes is K R c..2 for ech stte M F M H U N Genertions Bised trnsition rtes distribute cells wy from the intermedite sttes U O U N M H M F i ii iii witching (rre) Figure 6 imultion of gn3 switching. () imulted number of cells (normlized cell count) in ech of the five sttes for synchronously replicting popultion initilized with cells in the off stte. The M H nd M F sttes oscillte, nd the U N stte (green rrow) shows spiking owing to trnsitions tht follow DNA repliction (shown in greter detil for single genertion on the right). The intermedite sttes show low occupncy. (b) Comprison of the simulted (red squres with connecting line to guide the eye) nd observed (blck squres with error brs indicting s.e.m.) frction of methylted cells in n unmethylted culture t the K R vlues mesured in the wild-type () nd mutnt strins. (c) Distribution of cells (ssuming n equl frction of methylted nd unmethylted sttes) towrds the off stte nd the fully methylted stte (M F ) owing to the bis in the trnsition rte. Broken rrows indicte rre trnsitions. similrly reduced by the rpid conversion of hemimethylted DNA to fully methylted DNA such tht few cells re in the M H stte t DNA repliction. The trnsition bis wy from the hemimethylted stte nd the intermedite unmethylted sttes towrds the fully methylted nd off sttes, respectively, cts s buffer tht cptures cells s they leve the on nd off sttes nd returns them to their originl stte. As consequence, cells tht leve the on stte owing to DNA repliction, or tht leve the off stte owing to fluctutions in repression of the prtil stte, rrely switch to the opposite stte. The stbility of the methylted nd unmethylted sttes is lso coupled to the time dely tht occurs between one round of DNA repliction nd the next. Incresing the dely increses the stbility of the methylted sttes becuse the DNA methyltion rections cn proceed for longer (more M H to M F trnsitions). By contrst, decresing the time dely increses the stbility of the unmethylted sttes becuse it decreses the time tht cells in the U N stte cn move to the M H stte. A time dely to regulte the frction of product formed is lso used in kinetic proofreding to increse the fidelity of nucleic cid nd protein synthesis 26. The gn3 switching mechnism hs two key properties tht re bsent in feedbck systems nd my prove dvntgeous in the construction of synthetic circuits. First, cellulr memory is determined by the DNA methyltion stte nd not by the concentrtion of cytoplsmic protein. This property could fcilitte the construction of simpler circuits becuse the protein tht is controlled by the switch does not need to regulte its own expression either directly or indirectly. In ddition, it is theoreticlly possible for expression sttes to be trnsferred to cell by genetic mteril lone, s occurs when virl genomes such s Mu re injected into host cells 27. econd, unlike feedbck regultion, the stte of the system is not determined by the expression level. Therefore, the expression sttes of the gn3 switch could be modified without ffecting switching rtes. For exmple, the system could be engineered such tht the promoter is inctive unless trnscriptionl ctivtor is lso present; in this cse, full expression would occur only if both the switch region ws methylted nd the ctivtor ws present. A similr switching mechnism to gn3 seems to control expression of the bcteriophge Mu mom operon nd lso involves nd competing for switch region with three GATC sequences 27. Other types of repressor or ctivtor protein in E. coli nd other orgnisms tht block DNA methyltion nd whose binding is prevented by DNA methyltion,28 might lso form mechnisticlly similr epigenetic switches. The potentil flexibility of the gn3 switch mkes it n ttrctive strting point for designing rtificil switches nd indictes tht other nturlly occurring, multistep epigenetic switches with bised trnsition rtes my exist in vrious configurtions. NATURE GENETIC VOLUME 39 [ NUMBER 2 [ FEBRUARY

6 27 Nture ublishing Group METHOD Genetic mplifiction system, bcteril strins nd plsmids. A plsmid (phl6) ws constructed contining the gn3 promoter regulting the T7 RNA polymerse gene. The gn3 regultory region nd the gene encoding T7 RNA polymerse were mplified by CR from genomic DNA derived from the E. coli strins MG655 (Coli Genetic tock Center) nd BL2-Gold(DE3) (trtgene), respectively. The chlormphenicol resistnce gene (Cm R )ws mplified by CR from the pza3 plsmid 29 nd inserted djcent to the T7 RNA polymerse gene. The whole phl6 insert (gn3 regultory sequence, T7 RNA polymerse nd Cm R ) ws used to replce the gn3 gene in the MC E. coli strin by mens of the lmbd Red disruption system 3. The T7 RNA polymerse sequence contined two muttions (leding to the mino cid chnges F59 nd 686C) tht were introduced during cloning nd integrtion. All deletion strins were generted with the lmbd Red disruption system nd the deletions were confirmed by CR mplifiction nd DNA sequencing (further detils re provided in upplementry Tbles nd 2 nd upplementry Fig. online). deletion strins were grown on pltes supplemented with ctlse 7. The reporter plsmid (phl32) ws constructed by inserting GF under the control of T7 RNA polymerse specific promoter into pza2 (ref. 29). The T7 RNA polymerse promoter nd GF were mplified by CR from the pet- plsmid (trtgene) nd the pgfmut3. plsmid (Clontech), respectively. The stop codon of GF ws followed by T7 RNA polymerse termintor sequence. The overexpression plsmid (phl3) ws creted by CR mplifiction of the coding region nd inserted into the multiple cloning site of pze under the control of the pteto- promoter 29. Dt cquisition nd nlysis. Cells were streked on LB pltes with pproprite ntibiotics. ingle colonies in the methylted nd unmethylted sttes were selected by fluorescence microscopy nd inoculted into M9 medi with.% glycerol, 3 mg/ml of chlormphenicol nd 5 mg/ml of knmycin. After h of growth, cells were inoculted into fresh medi for further 6 h of growth, nd then single-cell mesurements were mde with Becton- Dickinson FACcn flow cytometer (88-nm excittion lser; 525-nm emission filter). Dt from, individul cells were collected for ech smple. FACcn dt were converted to ACII formt using MFI (E. Mrtz, University of Msschusetts, Amherst) nd nlyzed with MATLAB (MthWorks). Histogrms were fitted to gussin distributions with Origin 7.5 (Microcl oftwre). The growth rtes of on nd off cells, s mesured by the doubling time, were similr in Luri Bertni medi (33. ±.8 min nd 3.5 ±.2 min, respectively); however, in M9 medi plus.% glycerol, the doubling time of the on cells ws longer thn tht of the off cells (9. ±. min nd 82.6 ±. min, respectively). The difference in growth rte is presumbly due to the metbolic burden of high GF expression in the on stte, which becomes pprent in M9 medi owing to the lower nutrient vilbility. Mthemticl model nd simultion. A deterministic model ws used becuse the dt were collected from lrge cell popultion grown over mny genertions. The number of cells in ech stte (M F,M H,U N,U O nd off) is determined by the switching rections, cell growth nd DNA repliction. The switching rections for cells in the M F, M H, U N, U O nd off sttes re consecutive components of d dt xj ðtþ, which describes the chnges in ech stte in genertion j, from the end of one round of DNA repliction (t ¼ ) until the strt of the next (t ¼ t). dx j ðtþ dt ¼ k M M H + g M k H U N k M M H + g H k O U O ðk O + k H ÞU N + g N k R + k O U N ðk O + k R ÞU O + g O k R + k R U O + g R k M, k H, k O, k O, k R nd k R re rte constnts defined s the number of cell trnsitions per genertion. Cell growth is n exponentil process occurring t rte g M, g H, g N, g O nd g R for the M F,M H,U N,U O nd off sttes, respectively, without nutrient depletion. Integrting d dt xj ðtþ t t specifies the number of cells in ech stte immeditely before DNA repliction. Trnsforming x j ðtþ by D, C A mtrix incorporting the DNA repliction ssocited trnsitions, provides the initil conditions for the next genertion j+; tht is, where x j+ ðþ ¼Dx j ðtþ; :5 :5 D ¼ B A : Itertive simultions with Mtlb determined the popultion distribution t 36 genertions (22 genertions on LB gr nd genertions in M9 medium liquid culture), which ws the pproximte number in the experiments (further detils re given in upplementry Methods). Note: upplementry informtion is vilble on the Nture Genetics website. ACKNOWLEDGMENT We thnk E.A. Okenfull for logisticl ssistnce; M. Thtti for discussions nd ssistnce with dt nlysis; M. vn der Woude for bcteril strins nd plsmids; R. Lutz nd H. Bujrd for pz expression plsmids; nd A. Becskei,.V. Godoy nd J.M. edrz for suggestions nd comments. This work ws supported by grnts from the Ntionl cience Foundtion (HY-588) nd the Ntionl Institutes of Helth (R-GM7783). COMETING INTERET TATEMENT The uthors declre tht they hve no competing finncil interests. ublished online t Reprints nd permissions informtion is vilble online t reprintsndpermissions. Jenisch, R. & Bird, A. Epigenetic regultion of gene expression: how the genome integrtes intrinsic nd environmentl signls. Nt. Genet. 33 (uppl.), (23). 2. Low, D.A., Weynd, N.J. & Mhn, M.J. Roles of DNA denine methyltion in regulting bcteril gene expression nd virulence. Infect. Immun. 69, (2). 3. Grdner, T.., Cntor, C.R. & Collins, J.J. Construction of genetic toggle switch in Escherichi coli. Nture 3, (2).. Iscs, F.J., Hsty, J., Cntor, C.R. & Collins, J.J. rediction nd mesurement of n utoregultory genetic module. roc. Ntl. Acd. ci. UA, (23). 5. Xiong, W. & Ferrell, J.E., Jr. A positive-feedbck-bsed bistble memory module tht governs cell fte decision. Nture 26, 6 65 (23). 6. Klose, R.J. & Bird, A.. Genomic DNA methyltion: the mrk nd its meditors. Trends Biochem ci (26). 7. 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