Transcription control reprogramming in genetic backup circuits

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1 Trnscription control reprogrmming in genetic ckup circuits Rn Kfri, Arren Br-Even & Yitzhk Pilpel A key question in moleculr genetics is why severe muttions often do not result in detectly norml phenotype. This roustness ws prtilly scried to redundnt prlogs, tht my provide ckup for one nother in cse of muttion. Mining mutnt viility nd mrna expression in Scchromyces cerevisie, we found tht ckup ws provided predominntly y prlogs tht re expressed dissimilrly in most growth conditions. We considered tht this pprent inconsistency might e resolved y trnscriptionl reprogrmming mechnism tht llows the intct prlog to rescue the orgnism upon muttion of its counterprt. We found tht in wild-type cells, prtil coregultion cross growth conditions predicted the ility of prlogs to lter their trnscription ptterns nd to provide ckup for one nother. Notly, the sets of regultory motifs tht controlled the prlogs with the most efficient ckup ctivity deliertely overlpped only prtilly; prlogs with highly similr or dissimilr sets of motifs hd suoptiml ckup ctivity. Such n rrngement of prtilly shred regultory motifs reconciles the differentil expression of prlogs with their ility to ck ech other up. Functionlly redundnt gene duplictes re inherently evolutionrily unstle; consequently, in mny duplictions, one of the duplictes is silenced,4. Retention of duplictes over long evolutionry time scles ws therefore suggested to require either degenertive sufunctionlizing muttions or introduction of new functions,5 8.Weimhereto understnd oth the relevnce of trnscription regultion to duplicte retention in evolution nd its role in controlling expression of genes tht provide ckup in cse of muttion. Mining single gene knockout phenotype nd nnottions of moleculr functions of ll yest genes, we found high correltion etween the essentility of genes nd the similrity of moleculr function etween themselves nd their prlogs (Supplementry Figs. nd online). We lso found tht only 4% of the dispensle prlogs did not coloclize 9 in the sme orgnelles (Supplementry Fig. online). These oservtions corroorte the notion tht dispensility my e explined y ckup etween prlogs. A priori, it might seem tht ckup requires the prlogs mrnas to e coregulted. To exmine this possiility, we clculted, for ech pir of prlogs, 4 correltion coefficients of mrna expression corresponding to 4 different experiments. We define the mens nd stndrd devitions of such correltions, for ech pir, s their men expression similrity nd prtil coregultion () vlues, respectively. We refer to the stndrd devition of the correltions s ecuse its vlue is high for pirs tht hve interchngely high nd low correltions cross different conditions. Figure shows the proportion of dispensle genes in sets of gene pirs versus their men expression similrity nd. We inspected close nd remote prlogous pirs seprtely nd found mrkedly different trends. Among remote prlogs, we found tht the essentility of coexpressed pirs ws very high, implying tht there is little ckup ctivity mong them. In remote pirs, ckup ws most efficient mong trnscriptionlly noncorrelted pirs, s their essentility ws sustntilly lower thn tht of single genes. Supplementry Figures 4 nd 5 online show the increse in protein-protein interction mong prlogs nd the decrese in similrity of Gene Ontology nnotted moleculr function etween them, respectively, s function of coexpression. These results provide potentil explntion for the oserved decrese in ckup cpcity with incresed coexpression. In contrst to remote pirs, close pirs showed n lmost opposite, more intuitive trend, in which dispensility incresed somewht with expression similrity (in greement with refs.,). Bckup mong nturlly dissimilrly expressed genes A nd B my suggest tht, upon muttion in gene A, expression of gene B is reprogrmmed to cquire profile tht is similr to the wild-type expression profile of gene A. Such reprogrmming hs een experimentlly verified for the Acs nd Acs isoenzymes. Wild-type Acs is suject to glucose repression (Fig. ), ut upon deletion of Acs, the repression of Acs is relieved, nd Acs cquires n Acs-like responsiveness to glucose. Despite dissimilr expression, the two genes shre promoter motif (CSRE) nd lso hve unique motifs.as efits genuine ckup circuit, Acs nd Acs re syntheticlly lethl. Additionl exmples of reprogrmming in response to muttions in prokryotes, yest nd mmmls re given in Supplementry Note online. In serch for mechnism tht my regulte switching etween dissimilr nd similr expression in response to muttion, we exmined the dependence of gene essentility on. We sked whether ckup occurs mong prlogs tht show high in Deprtment of Moleculr Genetics, Weizmnn Institute of Science, Rehovot 76, Isrel. Correspondence should e ddressed to Y.P. (pilpel@weizmnn.c.il). Pulished online Ferury 5; doi:.8/ng5 NATURE GENETICS ADVANCE ONLINE PUBLICATION

2 Figure Dependence of ckup on expression similrity etween prlogs. Proportion of dispensle genes s function of the men expression similrity () nd () in sets of prlogs. Results re shown only for genes hving remote prlogous prtners (K s 4 ). Results for close pirs (K s o ) showed.4 mrginlly significnt opposite trend.4 (Supplementry Figs. nd online). The. red line represents qudrtic regression fit (), scoring n djusted R vlue of, nd logistic regression fit () withp vlue of 5 Men expression similrity. To exclude the possiility tht the trend in reflects tendency for genes tht elong to mjor expression clusters to e essentil, we repeted the nlysis using rndom piring of genes nd oserved nonsignificnt trend (lue). Exmintion of remnnts of whole-genome dupliction 6 showed similr trends to tht oserved with ll remote pirs, ut with mrginl significnce. the wild type. We resoned tht ecuse represents the ility to switch etween similr nd dissimilr expression profiles in condition-dependent mnner, it my e predictive of switching etween similr nd dissimilr expression in response to muttion. We found tht ws very strong predictor of ckup (Fig. ). We next investigted the promoter rchitecture of ckup-providing prlogs. The possiility tht the prtil overlp in the sets of regultory motifs controlling Acs nd Acs ccounts for their wilype differentil expression nd for reprogrmming upon muttion prompted us to inspect the similrity of motif content of ll prlogs. To quntify the extent to which promoters of prlogs re rrnged to otin prtil coexpression, we defined O, normlized mesure of the overlp etween the sets of promoter motifs tht regulte two genes: O ¼ jm \ m j mxðjm j; jm jþ ; where m nd m re the sets of motifs tht regulte genes nd, respectively, nd x isthesizeofsetx. By plotting gene dispensility versus motif-content overlp O, we found tht mximl ckup coincided with intermedite levels of motif shring (Fig. ). Pirs with high or low promoter similrity hd suoptiml ckup ctivity. These oservtions confirm tht optiml ckup is otined when two prlogs shre some, ut not ll, motifs. We propose tht the unique motifs of ech prlog provide differentil expression in the wild type nd tht the shred motifs llow prlogs to respond to the sme conditions. This sitution llows for reprogrmming in response to muttions. We plotted the numer of shred trnscription fctor inding sites ginst the rte of sustitutions per synonymous position, K s ( rough dupliction-ge surrogte), nd found nerly identicl verge numers of shred motifs cross the entire rnge of K s vlues (R ¼.5, P 4.9; Supplementry Figs. 6 nd 7 online). This indictes tht shring of trnscription fctor inding motifs results either from restricted divergence or from convergence nd is not n evolutionry rtifct tht is likely to dissipte on n evolutionry time scle. To corroorte the hypothesis tht underlies reprogrmming nd, ultimtely, ckup, we exmined three predictions. First, one memer of pir with high should e upregulted trnscriptionlly in response to the deletion of its prlog. To investigte this prediction, we used the Rosett Compendium contining genomewide expression response to single-gene deletions. Of the 59 knockouts in the Compendium, 76 hve prlogs in our set. Of these, 8 shre high similrity in moleculr function, nd nother 5 re syntheticlly lethl. We resoned tht if such potentil ckupproviding pirs undergo reprogrmming then the trnscriptionl level Proportion of dispensle genes Proportion of dispensle genes of the intct prlog should increse s function of the pir s. In fct, we found significnt correltion etween nd the logrithm of trnscriptionl response to deletion mong these ckupproviding cndies (R ¼ 7, P ¼.; Fig. ). As negtive control, functionlly similr nonprlogs nd rndom pirs showed no correltion etween nd trnscriptionl response to deletion (R ¼. nd R ¼., respectively). Therefore, we conclude tht mesured cross wild-type conditions predicts ckup cpcity or the ility of gene to respond, y upregultion, to deletion of its counterprt. Our second prediction ddresses 478 prlogs in which only one of the two genes is essentil. We tested our ility to predict which of the two genes in such symmetric pirs is essentil y inspecting their regultory motifs. Our reprogrmming scenrio predicts tht the more motifs control gene, the etter its reprogrmming nd ckup-providing cpcity will e. Therefore, for prlogous pirs, we expect negtive correltion etween numer of motifs controlling gene nd its dispensility. As expected, the more essentil of the two genes tended to hve more motifs (Fig. 4). As negtive control, we repeted the nlysis with rndom piring of the prlogs to determine whether this oservtion merely reflected the potentil is tht essentil genes re regulted y lrger numer of motifs. This nlysis with rndom piring resulted in no signl (Fig. 4). Proportion of dispensle genes Motif-content overlp P =. 4 P =.6 7 Figure Gene dispensility s function of the regultory motif content overlp O etween genes nd their closest prlogs. By fitting these to liner function (not shown), qudrtic function (red) nd the rtionl function (purple; y ¼ (x + )/(x + cx + d)), we otined djusted r vlues of 6, nd, respectively. A inomil test showed tht the proportion of dispensle genes with O vlues etween nd.5 (lue rs) ws significntly higher thn tht of genes with O vluesofeither (P ¼.6 7 )or4.5 (P o. 4 ). ADVANCE ONLINE PUBLICATION NATURE GENETICS

3 Log (Z) Rndom Figure Trnscriptionl response of ckup-providing genes to the deletion of the counterprts. () Trnscriptionl responses of genes to the deletion of their functionlly similr prlogs (red, R ¼ 7, P ¼.) nd functionlly similr nonprlogs (lck, R ¼.) s function of their ( otined from the Rosett Compendium ). Response is depicted s verge log reltive chnge of the expression level of gene in the mutnt strin lcking its prlog divided y the expression level of the gene in the wild type. Decresed reds in response to deletion my result from rtifcts owing to potentil cross-hyridiztion in the wild type. This effect ws excluded y nlyzing only genes tht re upregulted fter the deletion. Only functionlly similr prlogs were nlyzed, defined either s genes encoding enzymes with the sme EC clssifiction or, for nonenzymes, s genes with high Gene Ontology sed semntic similrity 7 (where high similrity indictes similrity exceeding tht oserved t the 9 th percentile of similrities of ll gene pirs in the genome). () Averge upregultion of functionlly similr pirs tht re lso syntheticlly lethl (from the BIND se) compred with rndomly selected gene pirs. Third, our proposed model predicts synthetic lethl interctions. We emedded the prlogous pirs in plne spnned y their men expression similrity nd (Fig. 5). We gthered evidence for synthetic lethlity for certin pirs of prlogs nd oserved tht the prevlence of ckup depended on oth men expression similrity nd score. Bckup ws mximl mong pirs with high nd low coexpression. Physicl interctions etween prlogs showed n opposite trend (Supplementry Fig. 4 online), in greement with previous oservtions 4,5. The model lso includes verified cses of reprogrmming. A crucil question is wht controls reprogrmming of gene upon muttion of its prlog. We propose kinetic model, or reprogrmming switch, consisting of two genes, G nd G, tht encode enzymes Log (Z).5.5 S.L. Normlized difference in numer of motifs Difference in mutnt growth rte Figure 4 Difference in the numer of motifs regulting prlogous pir memers s function of the difference in the growth rtes of mutnts lcking them. For ech pir of prlogs, the numer of motifs contined y the gene with the higher growth rte ws sutrcted from the numer of motifs in the promoter of the gene with the lower growth rte. This difference ws normlized to the size of the lrger of the two motif sets. All prlogs were then grouped into three ctegories on the sis of the solute vlue of the difference etween their growth rtes, nd for ech ctegory, the men normlized difference in the numer of motifs ws clculted. The nlysis ws done seprtely for ll prlogs (lue) nd for prlogs with similr moleculr functions (red; defined s in the legend to Fig. ). E nd E, which interconvert metolite M into metolite M. In the wild type, only E is ctive. Assuming tht the two genes contin inding sites for shred trnscription fctor T tht is induced y M, T reprogrms(i.e., ctivtes) G nd hence mintins the level of M upon knockout of G (Fig. 6). Upon silencing of G, M ccumultes nd the concentrtion of T increses, resulting in more efficient ctivtion of G (Fig. 6). Consequently, the level of E increses nd the level of M returns to its originl vlue fter trnsient decrese (Fig. 6). This model provides pproprite control of ckup s it couples response of G to n environmentl condition (i.e., the ccumultion of M) with response to n internl pertur tion (i.e., silencing of G). The model descries enzymtic rections; enzymes re over-represented in our set (4%; Supplementry Fig. 8 online). Bckup mong prlogous trnscription fctors my use lterntive rchitectures (Supplementry Note online) A B C D Men expression similrity E F Figure 5 Confirmtion nd chrcteriztion of genetic ckup circuits. Prlogous gene pirs re plotted s function of their men expression similrity nd. Pirs re colored red if oth memers re essentil or lue if oth re dispensle. Blck rectngles (A F) enclose sets of genes whose functionl redundncy ws confirmed or disputed using the Proteome nd BIND ses. In this nlysis, pirs were considered to ck ech other up only if they hve similr moleculr ctivities nd re syntheticlly lethl. The numer of such ckup-providing pirs ws divided y the totl numer of functionlly chrcterized pirs in ech of the mrked rectngles individully (A: /9 ¼ 4; B: 4/ ¼.47; C: /5 ¼ 4; D: /45 ¼.; E: 8/ ¼.7; F: /4 ¼.). The highest proility for verified ckup coincides with cses where men expression similrity is B nd 4.4. The plcement of the rectngles reflects our desire to exmine how incidence of ckup depends on the x,y coordintes of the pirs. Four exmples of prlogs tht show trnscriptionl reprogrmming in response to gene deletion re lso shown (green squres): : Acs-Acs (ref. ); : Hxt-Hxt (ref. 8); : Idp-Idp (ref. 9); 4: Fks-Gsc (ref. ). NATURE GENETICS ADVANCE ONLINE PUBLICATION

4 M M E E G G T Concentrtion (ritrry units) Time Figure 6 Schemtic nd dynmics of the reprogrmming switch. () The reprogrmming switch. () Simulted dynmics of the switch efore nd fter knockout, t time point 5. The lue nd green curves represent the concentrtions of E nd E, respectively; the red curve represents the concentrtion of M. The dynmics were clculted from the differentil equtions descriing the system using the ode solver of Mtl s simulink. The different ehvior of close nd remote prlogs proly stems from the profoundly different evolutionry regimens cting on them 5. Focusing first on remote pirs, we propose tht preservtion of high coexpression in suset of these pirs ws predominntly due to evolutionry pressures tht re inconsistent with, nd compromise, ckup. One such effect is evolving protein-protein interctions etween prlogs, which requires coexpression ut precludes ckup (Supplementry Fig. 4 online). Second, sufunctionliztion of proteins my llevite the pressure to diverge in expression, ut tht, too, precludes ckup etween coexpressed pirs (Supplementry Fig. 5 online). Third, quntittive sufunctionliztion 6 tht my result in regultory motif degenertion,7 ccounts for oth coexpression nd lck of ckup (e.g., duetolowdosgeofechofthecoctingprlogs 8,9 ). Why do remote pirs ck ech other up? Although it is difficult to imgine tht ckup y duplictes is evolutionrily selectle, we propose tht ckup-providing duplictes my e retined during evolution if their retention is coupled to other selectle trits, such s cquisition of new regultory cpilities. Such novelties do not preclude ckup, provided tht shred functionlities re preserved. Our finding tht ckup is optiml mong pirs tht mintin high prtil coregultion provides considerle support to this notion. Notwithstnding this, however, ckup hs profound impct on n orgnism s roustness, whether selected for its own ske or not. But pprent dispensility my e prtilly due to limited coverge of growth conditions tested in the lortory, nd recent computtionl study 9 estimted tht this fctor ccounts for 7 68% of dispensle genes, compred with the 5 8% tht re estimted to e compensted y duplicte. Gu et l. estimted similr lower ound of 5% (ref. ). Mny of the close prlogs tht represent recent duplictions 5 re ssumed to e under free selection, mening tht they hve not yet undergone either su- or neofunctionliztion; hence, they re redundntly similrly expressed. This proly explins their somewht more intuitive ehvior (ckup increses with coexpression), which does not depend on evolving reprogrmming. METHODS Set of nlyzed genes nd definition of prlogs. To ensure tht we nlyzed genuine genes, we discrded from the list of S. cerevisie open reding frmes (ORFs) ll entries corresponding to spurious ORFs nd ll trnsposonderived genes s nnotted y Scchromyces Genome Dtse. This resulted in list of 5,86 ORFs. We defined prlogs s pirs of ORFs tht, y BLASTP with stndrd prmeters, hd E vlued o, provided tht the rtio of the length of the long protein to the length of the short protein ws not lrger thn.. For ech pir of prlogs, we clculted the numer of synonymous nd nonsynonymous sustitutions (K s nd K, respectively). We defined remote prlogs s pirs with K s 4 nd close prlogs s pirs with K s r. To void potentil misclssifiction of orderline cses, we lso dopted n lterntive definition in which we regrded remote pirs s those with K s 4. nd close pirs s those with K s o nd found tht the sme trends chrcterized the two sets (Supplementry Fig. 9 online). Supplementry Figure 9 contins dditionl cutoff justifictions including systemtic ssessment of the roustness of the results to chnges of threshold vlue nd to use of lterntive mesures of sequence similrity (e.g., K ). To remove from the set of close pirs (K s o ) ny prlogs tht represent old duplictions, we removed close prlogs in which t lest one of the genes hd low (o) effective numer of codons. Gene essentility. We defined dispensle genes s ll genes with vile gene-deletion phenotype tht were not included in the lists of spurious ORFs or trnsposon-derived ORFs. Additionlly, we otined on growth rtes of mutnts lcking ech of the ORFs in the genome in five different growth medi. mrna expression. We otined whole-genome mrna expression of 4 nturl nd pertured time series nd the Rosett Compendium, which mesures genome-wide trnscription response to gene deletions, from ExpressDB. We normlized ll expression profiles of genes in ech time series with respect to men nd vrince. Detiled descriptions of ll nlyzed conditions is presented on our project wesite (see URL elow). We otined expression from either Affymetrix chips (seven experiments) or PCR product-sed microrrys ( experiments). Becuse the ltter technology is more prone to cross-hyridiztion errors, we used only derived from Affymetrix chips when nlyzing close prlogs. A nonredundnt set of promoter regultory motifs in S. cerevisie. We compiled nonredundnt set of yest regultory motifs, long with their gene ssignments, from three different sources: ChIP-chip (originlly ugmented with phylogenetic conservtion of motifs cross multiple yest species) 4, expression 5 nd phylogenetic conservtion. We included motifs derived from the lst two computtionl methods only if they corresponded to experimentlly known motifs nd hd significnce score higher thn the 9 th percentile in their respective methods. Kinetic nlysis of the reprogrmming switch. We modeled the concentrtion of induced trnscription fctor (T*) nd the frctions of time in which genes G nd G were trnscried, denoted s G* nd G*, respectively, with three sturtion equtions: T ¼ T Tot ðm=k M Þ nm ðm=k M Þ nm + ; G ¼ ðt =K Þ n ðt =K Þ n + ; nd G ¼ ðt =K Þ n ðt =K Þ n + ; where T Tot is the concentrtion of totl trnscription fctor; K M is the ffinity etween the trnscription fctor T nd the inducing metolite M; K nd K re the trnscription fctor s ffinities to G nd G, respectively; nd the powers n M nd n represent inding coopertivity Hill coefficients of M to T nd of T* to the two promoters, respectively. The concentrtion of the enzymes nd the metolites re descried with time-dependent differentil equtions: de ¼ G E; de ¼ G E; dm ¼ M fðe + EÞM; 4 ADVANCE ONLINE PUBLICATION NATURE GENETICS

5 nd dm ¼ fðe + EÞM M M ; where nd M re the mximl production rte of E nd E nd of M, respectively; nd M represent the degrion nd dilution of E nd E nd of M, respectively; nd f represents the conversion rte of M to M. Vlues of the coefficients, the present simultion, re T Tot ¼, K M ¼, K ¼., K ¼, n M ¼ 4, n ¼ (i.e., no coopertivity is ssumed) nd ¼ ¼ M ¼ M ¼ f ¼. There re three resonle ssumptions in the model. First, we ssume tht the inding of M to T nd of T* to G nd G occurs on short time scle compred with the other rections; therefore, these rections re in qusi-stedy stte. Second, we ssume tht M 4 T (the totl numer of free M molecules is roughly the sme s the totl numer of M molecules). Finlly, we ssume tht E nd E work linerly with respect to M s sustrte. Sttisticl nlyses. We computed proportions of dispensle genes s function of men expression similrity, nd motif-content overlp O (Figs. nd ) y inning genes into groups ccording to ech of these three vriles nd then clculting the frequency of vile mutnts in ech in. We counted ech gene in ech in only once to void repetitions cused y one gene hving multiple prlogs. To estlish tht our results re independent of the prticulr choice of inning strtegy, we verified tht the oserved trends were vlid under ny relevnt in-size choice (Supplementry Fig. online). We tested the significnce of trends oserved for the proportions of dispensle genes ginst men expression similrity (Fig. ), (Fig. ) nd motif-content overlp (Fig. ) using logistic regressions nlyses (in Fig., only the declining portion of the curve, with positive expression similrity vlues, ws used). Further sttisticl nlyses of the results re shown in Supplementry Figures 9 nd online. URLs. We downloded gene sequences from the Scchromyces Genome Dtse ( nd retrieved gene knockout phenotype from project/essentil_orfs.txt. Functionl nnottions for ll genes cme from the Gene Ontology nnottion scheme t We downloded synthetic lethl interctions nd physicl interctions etween proteins from the BIND se ( We collected gene expression from ExpressDB ( nd used the Proteome se ( to collect synthetic lethl pirs mnully. Our project wesite is BckUpCircuits/. We otined EC clssifictions from genre/proj/yest/. Note: Supplementry informtion is ville on the Nture Genetics wesite. ACKNOWLEDGMENTS We thnk ll memers of the lortory of Y.P. for discussions; I. Pechersky for computtionl ssistnce; nd Y. Grten, N. Brki, J. Bermn, B. Shilo, A.M. Dudley, I. Yni, O. Mn, S. Shen-Orr, D. Grur, D. Lncet, M. Levy nd D. Artzi for criticl review of the mnuscript. Y.P. is n incument of the Aser Rothstein Creer Development Chir in Genetic Diseses nd is Fellow of the Hurwitz Founion for Complexity Sciences. We thnk the Leo nd Juli Forchheimer Center for Moleculr Genetics nd the Ben My Founion for grnt support. This pper is dedicted to the memory of I. Kfri. COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Received 6 Novemer 4; ccepted 5 Jnury 5 Pulished online t Connt, G.C. & Wgner, A. Duplicte genes nd roustness to trnsient gene knock-downs in Cenorhditis elegns. Proc. R. Soc. Lond. B Biol. Sci. 7, (4).. Gu, Z. et l. Role of duplicte genes in genetic roustness ginst null muttions. Nture 4, 6 66 ().. Nowk, M.A., Boerlijst, M.C., Cooke, J. & Smith, J.M. Evolution of genetic redundncy. Nture 88, 67 7 (997). 4. Lynch, M., O Hely, M., Wlsh, B. & Force, A. The proility of preservtion of newly risen gene duplicte. Genetics 59, (). 5. Lynch, M. & Conery, J.S. The evolutionry fte nd consequences of duplicte genes. Science 9, 5 55 (). 6. Force, A. et l. Preservtion of duplicte genes y complementry, degenertive muttions. Genetics 5, (999). 7. Wgner, A. 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