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Downloaded from genome. cshlp org on November 3, 2010- Published by Cold Spring Harbor Laboratory Press Research Coevolution within a transcriptional network by compensatory trans and cis mutations Justin Catalana, Timothy Ravasi, Kai Tan, , and Irey ldeker1, g ang> Dwight Kuo, Katherine Licon, Sourav Bandyopadhyay, Ryan Ch ' Departments of Bioengineering and Medicine, University of Califonia, San Diego, La Jolla, California 92093, USA; 'Red Sea Laboratory of Integrative Systems Biology, Division of Chemical and Life Sciences and Engineering, Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia; Departments of Internal Medicine and Biomedical Engineering, University of lowa, lowa City, lowa 52242, USA Transcriptional networks have been shown to evolve very rapidly prompting questions as to how such changes arise and ire tolerated Recent comparisons of transcriptional networks across species have implicated variations in the cis-actin DNA sequences near genes as the main cause of divergence what is less clear is how these changes interact with trans-acting hanges occurring elsewhere in the genetic circuit. Here, we report the discovery of a system of compensatory trans and cis mutations in the yeast AP-l transcriptional network that allows for conserved transcriptional regulation despite continued genetic change We pinpoint a single species, the fungal pathogen Candida glabrata, in which a trans mutation has occurred very recently in a single ap-l family member distinguishing it from its Saccharomyces ortholog. Comparison of chromatin immunoprecipitation profiles between Candida and Saccharomyces shows that despite their different dna-binding domains, the Ap-l orthologs regulate a conserved block of genes. this conservation is enabled by concomitant changes in the cis- regulatory motifs upstream of each gene. Thus, both trans and cis mutations have perturbed the yeast AP-l regulatory ystem in such a way as to compensate for one another this demonstrates an example of "coevolution"between a dna- binding transcription factor and its cis-regulatory site, reminiscent of the coevolution of protein binding partners upplementalmaterialisavailableonlineathttp.www.genome.orgthesequencedatafromthisstudyhavebeen submittedtoheNcblGeneExpressionOmnibus(http://www.ncbi.nlmnihgov/geo/underaccessionno.Gsel5818.] Transcriptional networks are central to understanding both evo- identified transcriptional programs that are dramatically rewired lution and phenotypic diversity among organisms. Of the many over short evolutionary time scales. As with earlier work, many of ways in which transcriptional networks can evolve, much atten- the observed differences in binding and expression have been n has been given to changes in the so-called cis-regulatory re- linked to changes in cis-regulatory regions. For example, Borneman gions of gene promoters (Wray 2007: Wagner and Lynch 2008). et al.(2007)found that the TF Tecl binds only 20% of the same Such changes include gain, loss, or modification of DNA sequence target genes in comparisons between Saccharomyces cerevisiae and motifs(Cliften et al. 2003; Kelliset aL 2003; Gasch et al. 2004; Stark the closely related Saccharomyces bayanus and saccharomyces et al. 2007) as well as alterations in motif spacing relative to the mikatae, and that this difference is due to gain and loss of canonical start of transcription, or to other motifs (Ihmels et al. 2005; Tanay Tecl cis-regulatory motifs. While some recent studies have asso- et al. 2005). In addition to changes in cis, transcriptional networks ciated genetic variants in TEs with gene expression changes ob- can also evolve through alterations to transcription factor (TF) served in interspecies hybrids(wilson et al. 2008; Wittkopp et al. proteins and other trans-acting factors(Wagner and Lynch 2008). 2008; Tirosh et al. 2009; Bullard et al. 2010; Emerson et al. 2010), in trans, potential mechanisms include mutations to protein struc- Gerke et al. 2009; Sung et al. 2009; Zheng et al. 2010), or in human ture impacting transcriptional activation or DNA-binding do- populations(Kasowski et al. 2010), the picture that emerges is that mains(Wagner and Lynch 2008), modulation of TF expression cis-regulatory regions are incredibly plastic over evolutionary time, Sankaran et al. 2009) or post-translational modifications(Holt while TFs(trans)evolve at a comparatively slower rate(Wray 2007) et al. 2009), or gain and loss of protein-protein interactions amo Given the dramatic changes that appear to be occurring in TFs (Tuch et al. 2008; Lavoie et al. 2010). transcriptional networks, a key question is how such systems re- Recently, a number of genome-scale studies have performed tain essential functions over evolutionary time (Wray 2007).One systematic comparisons of TF-binding patterns(Borneman et al. solution is that changes in cis can occur by replacement of one TI 08: Bra Lavoie et al. 2010: cofactor with another, thereby maintaining regulatory control Schmidt et al. 2010)or mRNA expression profiles across species (Tsong et al. 2006). Alternatively, rather than replacing specific Ihmels et al. 2005; Tanayet al 2005; Hogues et al. 2008; Fieldet al. cofactors, it is conceivable that the DNA-binding domains of the 2009; Wapinski et al. 2010). Almost universally, these studies have TFs that bind these cis-regulatory sequences might be altered in lock-step with changes in cis, similarly to the evolution of protein binding partners(Pazos and Valencia 2008). However, such a mechanism of evolution has yet to be observed Here, we present rticle published or Article and publication date are at a direct example of such"coevolution, " where a specific change to http://www.genome.org/cgi/doi/10.1101/gr.111765.110.Freelyavailable DNA-binding transcription factor and its cis-regulatory site have online through the Genome Research Open Access option. occurred in compensatory fashion. 20: 000-000@ 2010 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/10

Research Coevolution within a transcriptional network by compensatory trans and cis mutations Dwight Kuo,1 Katherine Licon,1 Sourav Bandyopadhyay,1 Ryan Chuang,1 Colin Luo,1 Justin Catalana,1 Timothy Ravasi,1,2 Kai Tan,3,4 and Trey Ideker1,4 1 Departments of Bioengineering and Medicine, University of California, San Diego, La Jolla, California 92093, USA; 2 Red Sea Laboratory of Integrative Systems Biology, Division of Chemical and Life Sciences and Engineering, Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia; 3 Departments of Internal Medicine and Biomedical Engineering, University of Iowa, Iowa City, Iowa 52242, USA Transcriptional networks have been shown to evolve very rapidly, prompting questions as to how such changes arise and are tolerated. Recent comparisons of transcriptional networks across species have implicated variations in the cis-acting DNA sequences near genes as the main cause of divergence. What is less clear is how these changes interact with trans-acting changes occurring elsewhere in the genetic circuit. Here, we report the discovery of a system of compensatory trans and cis mutations in the yeast AP-1 transcriptional network that allows for conserved transcriptional regulation despite continued genetic change. We pinpoint a single species, the fungal pathogen Candida glabrata, in which a trans mutation has occurred very recently in a single AP-1 family member, distinguishing it from its Saccharomyces ortholog. Comparison of chromatin immunoprecipitation profiles between Candida and Saccharomyces shows that, despite their different DNA-binding domains, the AP-1 orthologs regulate a conserved block of genes. This conservation is enabled by concomitant changes in the cisregulatory motifs upstream of each gene. Thus, both trans and cis mutations have perturbed the yeast AP-1 regulatory system in such a way as to compensate for one another. This demonstrates an example of ‘‘coevolution’’ between a DNAbinding transcription factor and its cis-regulatory site, reminiscent of the coevolution of protein binding partners. [Supplemental material is available online at http://www.genome.org. The sequence data from this study have been submitted to he NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE15818.] Transcriptional networks are central to understanding both evolution and phenotypic diversity among organisms. Of the many ways in which transcriptional networks can evolve, much attention has been given to changes in the so-called cis-regulatory regions of gene promoters (Wray 2007; Wagner and Lynch 2008). Such changes include gain, loss, or modification of DNA sequence motifs (Cliften et al. 2003; Kellis et al. 2003; Gasch et al. 2004; Stark et al. 2007) as well as alterations in motif spacing relative to the start of transcription, or to other motifs (Ihmels et al. 2005; Tanay et al. 2005). In addition to changes in cis, transcriptional networks can also evolve through alterations to transcription factor (TF) proteins and other trans-acting factors (Wagner and Lynch 2008). Although there have been fewer reports of evolutionary changes in trans, potential mechanisms include mutations to protein structure impacting transcriptional activation or DNA-binding domains (Wagner and Lynch 2008), modulation of TF expression (Sankaran et al. 2009) or post-translational modifications (Holt et al. 2009), or gain and loss of protein–protein interactions among TFs (Tuch et al. 2008; Lavoie et al. 2010). Recently, a number of genome-scale studies have performed systematic comparisons of TF-binding patterns (Borneman et al. 2007; Tuch et al. 2008; Bradley et al. 2010; Lavoie et al. 2010; Schmidt et al. 2010) or mRNA expression profiles across species (Ihmels et al. 2005; Tanay et al. 2005; Hogues et al. 2008; Field et al. 2009; Wapinski et al. 2010). Almost universally, these studies have identified transcriptional programs that are dramatically rewired over short evolutionary time scales. As with earlier work, many of the observed differences in binding and expression have been linked to changes in cis-regulatory regions. For example, Borneman et al. (2007) found that the TF Tec1 binds only 20% of the same target genes in comparisons between Saccharomyces cerevisiae and the closely related Saccharomyces bayanus and Saccharomyces mikatae, and that this difference is due to gain and loss of canonical Tec1 cis-regulatory motifs. While some recent studies have associated genetic variants in TFs with gene expression changes observed in interspecies hybrids (Wilson et al. 2008; Wittkopp et al. 2008; Tirosh et al. 2009; Bullard et al. 2010; Emerson et al. 2010), in outbred crosses (Brem and Kruglyak 2005; Landry et al. 2005; Gerke et al. 2009; Sung et al. 2009; Zheng et al. 2010), or in human populations (Kasowski et al. 2010), the picture that emerges is that cis-regulatory regions are incredibly plastic over evolutionary time, while TFs (trans) evolve at a comparatively slower rate (Wray 2007). Given the dramatic changes that appear to be occurring in transcriptional networks, a key question is how such systems retain essential functions over evolutionary time (Wray 2007). One solution is that changes in cis can occur by replacement of one TF cofactor with another, thereby maintaining regulatory control (Tsong et al. 2006). Alternatively, rather than replacing specific cofactors, it is conceivable that the DNA-binding domains of the TFs that bind these cis-regulatory sequences might be altered in lock-step with changes in cis, similarly to the evolution of proteinbinding partners (Pazos and Valencia 2008). However, such a mechanism of evolution has yet to be observed. Here, we present a direct example of such ‘‘coevolution,’’ where a specific change to a DNA-binding transcription factor and its cis-regulatory site have occurred in compensatory fashion. 4 Corresponding authors. E-mail tideker@ucsd.edu. E-mail kai-tan@uiowa.edu. Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.111765.110. Freely available online through the Genome Research Open Access option. 20:000–000 ! 2010 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/10; www.genome.org Genome Research 1 www.genome.org Downloaded from genome.cshlp.org on November 3, 2010 - Published by Cold Spring Harbor Laboratory Press

ownloaded from genome. cshlp org on November 3, 2010- Published by Cold Spring Harbor Laboratory Press Kuo et al As a model of transcriptional network evolution, we exam- tin immunoprecipitation with microarray hybridization( ChIP- ined the yeast AP-1 (yAP-1)family, which, with a total of eight chip) data in Sc(Harbison et al. 2004; Tan et al. 2008) have de- (Fernandes et al. 1997; Rodrigues-Pousada et al. 2010). Like other Yap8) recognize YRE-O, whereas two family members(Yap4 and paralogous families, AP-1 factors have been born through the pro- Yap6)recognize YRE-A We examined the binding of the remaining cess of gene duplication, which gives rise to multiple copies that Sc AP-1 member Yap3 by ChIP-chip and determined it preferre are free from selective pressure and may functionally diverge from YRE-A sites in both complete media and stress conditions (Sup- their duplicates by sub- or neofunctionalization(Hittinger and plemental Fig. 1). This preference for YRE-o or YRE-A-binding Carroll 2007). AP-1 also provides a classic example of the basic sites in Sc AP-ls correlates precisely with the presence of arginine leucine zipper(bZIP) motif, which is widely conserved across or lysine at residue 12(Fig. 1A) eukaryotes(Tan et al. 2008; Rodrigues-Pousada et al. 2010).In Interestingly, residue 12 is part of an alpha-helical surface that humans, AP-1 TEs have been heavily studied due to their crucial forms multiple contacts to DNA (basic region residues 7-15)(Fig. role in cell proliferation, death, and differentiation(Shaulian and 1B; Fujii et al. 2000). Previously, this residue was predicted as Karin 2002). In yeast, yAP-1-mediated transcriptional networks a likely determinant of DNA half-site spacing preference in Gcn4 carry out overlapping, but distinct biological responses to stress another bZIP family TF(Kim and Struhl 1995). Although in vitro (Tan et al. 2008; Rodrigues-Pousada et al. 2010). In contrast to the testing of Gcn4 mutants was not able to confirm this prediction despread divergence in tF binding that has been demonstrated(Kim and Struhl 1995), it has become apparent that such variations reviously(Borneman et al. 2007; Tuch et al. 2008; Lavoie et al. in half-site recognition are best distinguished in vivo(Suckow and 2010), we show that coupled trans and cis mutations enable con- Hollenberg 1998; Berger et al. 2008; Maerkl and Quake 2009) servation of a subset of genes targeted by yAP-1. These result provide an example of compensatory coevolution of a trans and Residue 12 point mutations cause rewiring of AP-1 regulatory system. transcriptional interactio To further examine the regulatory role of residue 12, we mutated Results this residue in Yapl, a representative YRE-O-binding factor, and a trans mutation is associated with Ap-1 DNA-binding motif specificity A B To identify trans mutations that could be Sc yAP-1 Family ★ NRAAQRAF associated with AP-l-binding preference Yap8(20-40)LRA 9 TTACTAA KNN we pertormed an amino acid sequence AATGATT of all eight AP-l-like TEs in S. cerevisiae cYap2(47-67 (Sc). This alignment and its associated phylogenetic tree(Fig. 1A)were searched CATT Yap Half-site to identify the key polymorphic amino ogen Bond acids whose pattern ScYap1 vs. ScYap1 R79K Binding ScYap4 vS ScYap4 K252R Binding divergence best explain the phylogeny Number of Bound Promoters D Number of Bound Promote (Methods, Evolutionary Trace Ana Such residues have been shown to fre. ■ ScRap4 bound quently play important evolutionary roles YRE-O YRE-O ■ ScRap4.K252 R Bound (Innis et al. 2000). Using this approach, we identified residue 12 of the dna- RE-A a ScYap1. R79K Bound YRE-A binding domain basic region as the most important evolutionarily divergent posi- E ScYap1 vs. ScYap1 R79K mRNA Expression F ScYap4 vs ScYap4 K252R mRNA Expression tion across the yAP-1 family (i. e, the one that was most highly correlated with the Fraction of Promoters with DNA-Binding Motifs Fraction of Promoters with DNA-Binding Motifs phylogeny; Fig 1A) Residue 12 was also predictive of Yap1, R79K AP-1 family DNA-binding motif prefer Background p=24×101 ence(Fig. 1A)(MacIsaac et al. 2006; Tar Background p=1.8x103 et al. 2008). AP-1 family members bind DNA as homo- or heterodimers, where Figure 1. A single residue determines yAP-1 DNA-binding motif specificity.(A) Alignment and each constituent monomer recognize the consensus sequence TTAC (Suckow ar) is predictive of preference for overlapping (YRE-O)or adjacent(YRE-A)DNA-bindi (left) YRE.O et al.1999; Fujii et al. 2000). These"half- 2004). Positions affecting Gcn4 half-site spacing preference(Kim and Struhl 1995)are shown(gray sites"are positioned in either adjacent or stars ). (B) Recognition of the yAP-1 half-site(Fuji et al. 2000).Residue 12(red star)is in close proxin overlapping fashion( Fig. 1A), which we to residues conferring AP-1 sequence specificity. (C, D)ScYap1 R79K and ScYap4 K252R mutants have refer to as yAP-1 response element adja- altered half-site spacing preference as evidenced cent (YRE-A)or yAP-1 response element with either YRE-O or YRE-A sites as assessed by Fishers exact test.(E, F) ScYapl R79 and ScYap4 K252 mRNA expression changes among with YRE-o and YRE-A overlapping (YRE.O), respectively. Pre- sites among the top 50 most differentially expressed genes. P-values denote the significance of YRE-A vious analyses of genome-wide chroma- and YRE-o motifs among gene promoters compared with the genomic background. 2 Genome Research

As a model of transcriptional network evolution, we examined the yeast AP-1 (yAP-1) family, which, with a total of eight members, is one of the largest paralogous TF families in S. cerevisiae (Fernandes et al. 1997; Rodrigues-Pousada et al. 2010). Like other paralogous families, AP-1 factors have been born through the process of gene duplication, which gives rise to multiple copies that are free from selective pressure and may functionally diverge from their duplicates by sub- or neofunctionalization (Hittinger and Carroll 2007). AP-1 also provides a classic example of the basic leucine zipper (bZIP) motif, which is widely conserved across eukaryotes (Tan et al. 2008; Rodrigues-Pousada et al. 2010). In humans, AP-1 TFs have been heavily studied due to their crucial role in cell proliferation, death, and differentiation (Shaulian and Karin 2002). In yeast, yAP-1-mediated transcriptional networks carry out overlapping, but distinct biological responses to stress (Tan et al. 2008; Rodrigues-Pousada et al. 2010). In contrast to the widespread divergence in TF binding that has been demonstrated previously (Borneman et al. 2007; Tuch et al. 2008; Lavoie et al. 2010), we show that coupled trans and cis mutations enable conservation of a subset of genes targeted by yAP-1. These results provide an example of compensatory coevolution of a trans and cis regulatory system. Results A trans mutation is associated with AP-1 DNA-binding motif specificity To identify trans mutations that could be associated with AP-1-binding preference, we performed an amino acid sequence alignment of the DNA-binding domains of all eight AP-1-like TFs in S. cerevisiae (Sc). This alignment and its associated phylogenetic tree (Fig. 1A) were searched to identify the key polymorphic amino acids whose patterns of conservation and divergence best explain the phylogeny (Methods, Evolutionary Trace Analysis). Such residues have been shown to frequently play important evolutionary roles (Innis et al. 2000). Using this approach, we identified residue 12 of the DNAbinding domain basic region as the most important evolutionarily divergent position across the yAP-1 family (i.e., the one that was most highly correlated with the phylogeny; Fig. 1A). Residue 12 was also predictive of AP-1 family DNA-binding motif preference (Fig. 1A) (MacIsaac et al. 2006; Tan et al. 2008). AP-1 family members bind DNA as homo- or heterodimers, where each constituent monomer recognizes the consensus sequence TTAC (Suckow et al. 1999; Fujii et al. 2000). These ‘‘halfsites’’ are positioned in either adjacent or overlapping fashion (Fig. 1A), which we refer to as yAP-1 response element adjacent (YRE-A) or yAP-1 response element overlapping (YRE-O), respectively. Previous analyses of genome-wide chromatin immunoprecipitation with microarray hybridization (ChIPchip) data in Sc (Harbison et al. 2004; Tan et al. 2008) have determined that five AP-1 family members (Yap1, Yap2, Yap5, Yap7, Yap8) recognize YRE-O, whereas two family members (Yap4 and Yap6) recognize YRE-A. We examined the binding of the remaining Sc AP-1 member Yap3 by ChIP-chip and determined it preferred YRE-A sites in both complete media and stress conditions (Supplemental Fig. 1). This preference for YRE-O or YRE-A-binding sites in Sc AP-1s correlates precisely with the presence of arginine or lysine at residue 12 (Fig. 1A). Interestingly, residue 12 is part of an alpha-helical surface that forms multiple contacts to DNA (basic region residues 7–15) (Fig. 1B; Fujii et al. 2000). Previously, this residue was predicted as a likely determinant of DNA half-site spacing preference in Gcn4, another bZIP family TF (Kim and Struhl 1995). Although in vitro testing of Gcn4 mutants was not able to confirm this prediction (Kim and Struhl 1995), it has become apparent that such variations in half-site recognition are best distinguished in vivo (Suckow and Hollenberg 1998; Berger et al. 2008; Maerkl and Quake 2009). Residue 12 point mutations cause rewiring of AP-1 transcriptional interactions To further examine the regulatory role of residue 12, we mutated this residue in Yap1, a representative YRE-O-binding factor, and Figure 1. A single residue determines yAP-1 DNA-binding motif specificity. (A) Alignment and phylogeny of AP-1 DNA-binding domain basic regions (residues 6 to 20 are shown). Residue 12 (red star) is predictive of preference for overlapping (YRE-O) or adjacent (YRE-A) DNA-binding motifs (left). Note that Yap8 possesses an Asp at residue 12 and binds a 2-bp overlapping YRE-O (Harbison et al. 2004). Positions affecting Gcn4 half-site spacing preference (Kim and Struhl 1995) are shown (gray stars). (B) Recognition of the yAP-1 half-site (Fujii et al. 2000). Residue 12 (red star) is in close proximity to residues conferring AP-1 sequence specificity. (C,D) ScYap1.R79K and ScYap4.K252R mutants have altered half-site spacing preference as evidenced by ChIP-chip (Methods). P-values refer to differences in binding to genes with either YRE-O or YRE-A sites as assessed by Fisher’s exact test. (E,F ) ScYap1.R79K and ScYap4.K252R mutations cause mRNA expression changes among genes with YRE-O and YRE-A sites among the top 50 most differentially expressed genes. P-values denote the significance of YRE-A and YRE-O motifs among gene promoters compared with the genomic background. 2 Genome Research www.genome.org Kuo e t al. Downloaded from genome.cshlp.org on November 3, 2010 - Published by Cold Spring Harbor Laboratory Press

ownloaded from genome. cshlp org on November 3, 2010- Published by Cold Spring Harbor Laboratory Press Coevolution within a transcriptional network Yap 4(also known as Cin5), a representative YRE-A-binding fac We used ChIP-chip to determine whether this Lys 12 sub- tor. This process involved generating mutants Yapl. R79K and stitution had a functional effect on CgApl binding (Methods). To Yap4. K252R, changing arginine to lysine in Yapl and lysine to facilitate this assay, we tagged CgApl with the TAP epitope and arginine in Yap4 (Methods). Next, Yapl. R79K binding and designed a custom microarray tiling the C genome(Methods). As Yap4. K252R binding were assayed in vivo using ChIP-chip(Meth- a control on both the TAP construct and the array design, we used ods). Comparison of the top 50 promoters bound by Yap1. R79K ChIP-qPCR to successfully validate a panel of five randomly cho- with the top 50 promoters bound by wild-type Yapl (as de- sen Cg gene promoters that were determined to be bound by termined in Tan et al. 2008)showed that mutation of Yapl CgApl in the ChIP-chip experiment(Supplemental Fig. 5) nificantly altered its preference for YRE-O and YRE-A sites(Fig. 1c; We found that CgApl bound the promoters of a total of 114 Fisher's exact test P=0.0002). Comparison of promoters bound by genes, 90 of which had known orthologs in Sc(Methods). Com- mutant and wild-type Yap 4 also showed the predicted shift in parison of these data with ChIP profiles for each of the AP-1 factors binding preference(Fig. 1D; Fisher's exact test P=0.037). These in Sc grown under the same treatment conditions(as determined results were not dependent on the number of promoters examined in Tan et al. 2008)showed significant overlap between the targets of CgApl and ScYapl(17 genes, P< 10) Overlap with other Sc ext, to assess the functional implications of changes in AP-1 factors was less substantial(Fig 3A). This pattern of overlap yAP-1 binding, we generated genome-wide mRNA expression pro- was reinforced by sequence analysis, in which phylogenetic clus- files for each mutant in comparison to the unmutated parental tering of AP-1 DNA-binding domains places CgApl definitively strain (Methods). Both mutations, Yapl.R79K and Yap4 K252R, with ScYapl and not with other Sc AP-1 sequences(Fig. 2C; altered the expression of genes whose promoters were highly Methods) enriched for AP-1-binding sites (YRE-O and YRE-A, Fig. lE, F; Sup. We were therefore faced with the following conundrum: On plemental Fig 3). These genes were also enriched for Yapl. R79K the one hand, the CgApl sequence diverges from Yapl orthologs at and Yap4 K252R binding(P<10), respectively residue 12, suggesting a shift in dna binding On the other hand, he CgApl-binding profile is quite specifically conserved with that An apparent paradox: Candida AP-l diverges at residue 12, of Yapl, calling into question the importance of residue 12 for but its targets are conserved Based on our observation that residue 12 affects binding of AP-1 paralogs in S. cerevisiae, we next asked whether changes in this Cg apl prefers yre-a rather than YRe-O sites residue could lead to divergent binding of AP-1 orthologs across To investigate this apparent contradiction, we next turned to the pecies. We searched the yeast phylogeny (Wapinski et al. 2007) for gene promoters targeted by CgApl in the ChIP assay. Promoters AP-l orthologs that were anomalous in their use of Arg 12 or Lys targeted by Cg Apl showed a clear preference for YRE-A sites over 12, suggesting lineage-specific mutation(Supplemental Fig. 4). YREO sites(49 vs. four promoters, respectively ) This preference mong TFs orthologous to Sc YAPl, we found that the Candida significantly differs from ScYapl, which prefers YRE-O over YRE-A glabrata( Cg)ortholog CgAPI diverges from other yeasts(Fig. A-C)( Fisher's exact test P= 3.5 X 10-; Fig. 3B, 21 vs. 12 promoter due to the presence of lysine at residue 12, in contrast to other respectively). This preference could not be attributed to threshole yeasts in its clade that possess an arginine. This CgApl amino acid effects on binding-site calls, as direct comparison of motif scores substitution was confirmed by sequencing of genomic DNA from confirmed a preference for YRE-A over YRE-O sites( Mann-Whitney two independent Cg isolates, 2001HTU and NCCLS84( Fig 2B) U test, P=0.0072). This preference was also observed via de novo nd even among the Cg orthologs of all B Species Position Sequence We further analyzed this cis-regula- Soto MMaMlodow tory preference by examining the ortho- logs of genes targeted by both CgApl and Sbayanus ScYapl across 20 sequenced yeast ge- nomes(Wapinski et al. 2007). C glabrata c Phylogeny of Se and Cg yAP-1 DNA Binding Domains stood out clearly as the only species with enrichment for YRE-A sites( Fig. 3D). In contrast. the YRE-o site was enriched in all neighboring species in the yeast other sensu stricto species (S paradoxus S. mikatae, and S. bayanus) as well as the more diverged Saccharomyces castellii, romyces walti, Kluyveromyces lactis Ashbya gosspyii, and Candida tropicalis ocfasporus These results indicate that upstream DNA-binding motifs of CgApl targets Fs (A)CgApl possesses a lysine at residue 12(CgAp1 46) have evolved from YRE-o to YRE-A(Fig. as rtv eals tateterepr and scrap COPhustr. nter al branch paint unders refer to the accompanied by concordant changes in osterior probability a measure of confidence( Drummond and Rambaut 2007) secondary cis-regulatory DNA motifs

Yap4 (also known as Cin5), a representative YRE-A-binding factor. This process involved generating mutants Yap1.R79K and Yap4.K252R, changing arginine to lysine in Yap1 and lysine to arginine in Yap4 (Methods). Next, Yap1.R79K binding and Yap4.K252R binding were assayed in vivo using ChIP-chip (Methods). Comparison of the top 50 promoters bound by Yap1.R79K with the top 50 promoters bound by wild-type Yap1 (as determined in Tan et al. 2008) showed that mutation of Yap1 significantly altered its preference for YRE-O and YRE-A sites (Fig. 1C; Fisher’s exact test P = 0.0002). Comparison of promoters bound by mutant and wild-type Yap4 also showed the predicted shift in binding preference (Fig. 1D; Fisher’s exact test P = 0.037). These results were not dependent on the number of promoters examined (Supplemental Fig. 2). Next, to assess the functional implications of changes in yAP-1 binding, we generated genome-wide mRNA expression profiles for each mutant in comparison to the unmutated parental strain (Methods). Both mutations, Yap1.R79K and Yap4.K252R, altered the expression of genes whose promoters were highly enriched for AP-1-binding sites (YRE-O and YRE-A, Fig. 1E,F; Supplemental Fig. 3). These genes were also enriched for Yap1.R79K and Yap4.K252R binding (P < 10!5 ), respectively. An apparent paradox: Candida AP-1 diverges at residue 12, but its targets are conserved Based on our observation that residue 12 affects binding of AP-1 paralogs in S. cerevisiae, we next asked whether changes in this residue could lead to divergent binding of AP-1 orthologs across species. We searched the yeast phylogeny (Wapinski et al. 2007) for AP-1 orthologs that were anomalous in their use of Arg 12 or Lys 12, suggesting lineage-specific mutation (Supplemental Fig. 4). Among TFs orthologous to Sc YAP1, we found that the Candida glabrata (Cg) ortholog CgAP1 diverges from other yeasts (Fig. A–C) due to the presence of lysine at residue 12, in contrast to other yeasts in its clade that possess an arginine. This CgAp1 amino acid substitution was confirmed by sequencing of genomic DNA from two independent Cg isolates, 2001HTU and NCCLS84 (Fig. 2B). We used ChIP-chip to determine whether this Lys 12 substitution had a functional effect on CgAp1 binding (Methods). To facilitate this assay, we tagged CgAp1 with the TAP epitope and designed a custom microarray tiling the Cg genome (Methods). As a control on both the TAP construct and the array design, we used ChIP-qPCR to successfully validate a panel of five randomly chosen Cg gene promoters that were determined to be bound by CgAp1 in the ChIP-chip experiment (Supplemental Fig. 5). We found that CgAp1 bound the promoters of a total of 114 genes, 90 of which had known orthologs in Sc (Methods). Comparison of these data with ChIP profiles for each of the AP-1 factors in Sc grown under the same treatment conditions (as determined in Tan et al. 2008) showed significant overlap between the targets of CgAp1 and ScYap1 (17 genes, P < 10!17). Overlap with other Sc AP-1 factors was less substantial (Fig. 3A). This pattern of overlap was reinforced by sequence analysis, in which phylogenetic clustering of AP-1 DNA-binding domains places CgAp1 definitively with ScYap1 and not with other Sc AP-1 sequences (Fig. 2C; Methods). We were therefore faced with the following conundrum: On the one hand, the CgAp1 sequence diverges from Yap1 orthologs at residue 12, suggesting a shift in DNA binding. On the other hand, the CgAp1-binding profile is quite specifically conserved with that of Yap1, calling into question the importance of residue 12 for sequence recognition. CgAp1 prefers YRE-A rather than YRE-O sites To investigate this apparent contradiction, we next turned to the gene promoters targeted by CgAp1 in the ChIP assay. Promoters targeted by CgAp1 showed a clear preference for YRE-A sites over YRE-O sites (49 vs. four promoters, respectively). This preference significantly differs from ScYap1, which prefers YRE-O over YRE-A (Fisher’s exact test P = 3.5 3 10!8 ; Fig. 3B, 21 vs. 12 promoters, respectively). This preference could not be attributed to threshold effects on binding-site calls, as direct comparison of motif scores confirmed a preference for YRE-A over YRE-O sites (Mann-Whitney U test, P = 0.0072). This preference was also observed via de novo motif search in these promoters (Fig. 3C) and even among the Cg orthologs of all ScYap1 targets (Q = 0.05). We further analyzed this cis-regulatory preference by examining the orthologs of genes targeted by both CgAp1 and ScYap1 across 20 sequenced yeast genomes (Wapinski et al. 2007). C. glabrata stood out clearly as the only species with enrichment for YRE-A sites (Fig. 3D). In contrast, the YRE-O site was enriched in all neighboring species in the yeast phylogeny, including S. cerevisiae and other sensu stricto species (S. paradoxus, S. mikatae, and S. bayanus) as well as the more diverged Saccharomyces castellii, Kluyveromyces waltii, Kluyveromyces lactis, Ashbya gosspyii, and Candida tropicalis. These results indicate that upstream DNA-binding motifs of CgAp1 targets have evolved from YRE-O to YRE-A (Fig. 3E). Such a switch may have also been accompanied by concordant changes in secondary cis-regulatory DNA motifs Figure 2. Evolution of the yAP-1 TFs. (A) CgAp1 possesses a lysine at residue 12 (CgAp1.46), while most other species possess an arginine. (B) Sequencing of CgAP1 in two unrelated isolates shows complete identity to the Cg reference genome. (C ) Phylogenetic clustering of all Sc and Cg AP-1 DNAbinding domains reveals that CgAp1 and ScYap1 co-cluster. Internal branch point numbers refer to the Bayesian posterior probability, a measure of confidence (Drummond and Rambaut 2007). Coevolu tion wi thin a transcrip tional ne twork Genome Research 3 www.genome.org Downloaded from genome.cshlp.org on November 3, 2010 - Published by Cold Spring Harbor Laboratory Press

ownloaded from genome. cshlp org on November 3, 2010- Published by Cold Spring Harbor Laboratory Press K al D with CgAp1 Overlap Significant 4001 005901 39 ScRap p=002 ScYap3I5 p=0.02 DNA-Binding Motif Preference nidulans CgAp Ancestral Yap1 ScYap2 SeRap ScRap? IrlcTAA TTCH →TSAT ScRap YRE-A Site TTACT TTAC T CATT ScRape has been rewired. (A)F by each yAP.] t site is enriched(star)among in other yeas Compensator i both trans and ) but not Cg(Q-1.0). The YRE-A site is enriched among these targets in Cg(star)but no maintain AP-1 binding (Supplemental material, Yap Cis-Regulatory Motifs Are Coincident Examination of the protein sequences of all AP-1 family with Those of Rtg3 and Aftl; Supplemental Fig. 6) and possible members across 20 available yeast genomes(Wapinski et al. 2007) functional divergence(Supplemental material, Divergence and suggests that mutations in residue 12 have occurred frequently Conservation of Yapl Function). The most plausible explanation is during AP-1 family evolution( Supplemental Fig 4). Interestingly, that these motifs have coevolved with a Lys 12 mutation in CgApl, we found that all yeasts possess at least one AP-l TF with Arg 12 with the result that this transcriptional system has retained reg.(Fig 4). In contrast, several yeasts lack AP-1 TFs with Lys 12, and ulatory control of the same set of target genes over evolutionary these species are the most evolutionarily diverged from Sc. These time results suggest that the common yeast AP-1 ancestor encoded ar- ginine and that the emergence of Tfs using lysine is a more recent n evolutionary innovation(Supplemental material, AP-1 Family Ancestry) Which mutation came first: the cis or trans? It is possible to envi- Within the Candida clade, several species(. tropicalis, C. sion two equally plausible scenarios(Fig. 3E): (1)An initial muta. albicans, C. parapsilosis, and Lodderomyces elongospors) have AP-1 tion in the Yapl tf provided selective pressure for subsequent families based exclusively on Arg 12, while others(C lusitaniae, cis-regulatory changes in Yapl targeted genes;(2)a change from Debaryomyces hanseni, and C guilliermondii (Fig 4)represent both YRE-O to YRE-A-binding site in key Yapl target(s) provided selec- Arg 12 and lys 12 across the AP-1 family. This suggests two equally tive pressure for a mutation in the Yapl TE. In either scenario, plausible scenarios for the emergence of lys 12 in yAP-1 TFs: (1) mutations in trans and cis may have been facilitated by other AP-1 Lysine emerged following the divergence of Yarrowia lipolyt family members. The large size and interconnectivity of the AP-1 from other hemi-ascomycetes, followed by a lineage-specific loss family may serve as a buffer for accumulation of cis and trans within the Candida clade. (2) Lysine emerged following the split of mutations, allowing for highly plastic evolution of the AP-1 reg. the Candida clade from the rest of the hemi-ascomycetes and ulatory network. In support of this hypothesis, several yAP-Is have emerged again within the Candida clade. In either scenario, been shown to bind each other along with common target genes a switch from arginine(coded by AGA or AGG)to lysine( coded by which might compensate for some loss in regulation by paralogs AAA or AAG)could be acc shed by a simple single base-pair (Tan et al. 2008) mutation Genome Research

(Supplemental material, Yap Cis-Regulatory Motifs Are Coincident with Those of Rtg3 and Aft1; Supplemental Fig. 6) and possible functional divergence (Supplemental material, Divergence and Conservation of Yap1 Function). The most plausible explanation is that these motifs have coevolved with a Lys 12 mutation in CgAp1, with the result that this transcriptional system has retained regulatory control of the same set of target genes over evolutionary time. Discussion Which mutation came first: the cis or trans? It is possible to envision two equally plausible scenarios (Fig. 3E): (1) An initial mutation in the Yap1 TF provided selective pressure for subsequent cis-regulatory changes in Yap1 targeted genes; (2) a change from YRE-O to YRE-A-binding site in key Yap1 target(s) provided selective pressure for a mutation in the Yap1 TF. In either scenario, mutations in trans and cis may have been facilitated by other AP-1 family members. The large size and interconnectivity of the AP-1 family may serve as a buffer for accumulation of cis and trans mutations, allowing for highly plastic evolution of the AP-1 regulatory network. In support of this hypothesis, several yAP-1s have been shown to bind each other along with common target genes which might compensate for some loss in regulation by paralogs (Tan et al. 2008). Examination of the protein sequences of all AP-1 family members across 20 available yeast genomes (Wapinski et al. 2007) suggests that mutations in residue 12 have occurred frequently during AP-1 family evolution (Supplemental Fig. 4). Interestingly, we found that all yeasts possess at least one AP-1 TF with Arg 12 (Fig. 4). In contrast, several yeasts lack AP-1 TFs with Lys 12, and these species are the most evolutionarily diverged from Sc. These results suggest that the common yeast AP-1 ancestor encoded arginine and that the emergence of TFs using lysine is a more recent evolutionary innovation (Supplemental material, AP-1 Family Ancestry). Within the Candida clade, several species (C. tropicalis, C. albicans, C. parapsilosis, and Lodderomyces elongosporus) have AP-1 families based exclusively on Arg 12, while others (C. lusitaniae, Debaryomyces hansenii, and C. guilliermondii) (Fig. 4) represent both Arg 12 and Lys 12 across the AP-1 family. This suggests two equally plausible scenarios for the emergence of Lys 12 in yAP-1 TFs: (1) Lysine emerged following the divergence of Yarrowia lipolytica from other hemi-ascomycetes, followed by a lineage-specific loss within the Candida clade. (2) Lysine emerged following the split of the Candida clade from the rest of the hemi-ascomycetes and emerged again within the Candida clade. In either scenario, a switch from arginine (coded by AGA or AGG) to lysine (coded by AAA or AAG) could be accomplished by a simple single base-pair mutation. Figure 3. The CgAp1 transcriptional network has been rewired. (A) For the promoters targeted by each yAP-1 transcription factor in Sc, the overlap with CgAp1 targets is shown (of 90 CgAp1 targets total). (B) CgAp1 prefers YRE-A-binding sites compared with ScYap1 (Fisher’s exact test). (C ) The CgAp1 DNA-binding motif (green) clusters with YRE-A rather than YRE-O motifs. (D) The YRE-O site is enriched (star) among common ScYap1 and CgAp1 targets in other yeasts (hypergeometric test, Q < 0.05), but not Cg (Q ; 1.0). The YRE-A site is enriched among these targets in Cg (star) but not other yeasts. (E ) Compensatory mutations in both trans and cis maintain AP-1 binding. Kuo e t al. 4 Genome Research www.genome.org Downloaded from genome.cshlp.org on November 3, 2010 - Published by Cold Spring Harbor Laboratory Press

ownloaded from genome. cshlp org on November 3, 2010- Published by Cold Spring Harbor Laboratory Press Coevolution within a transcriptional network Number of yAP-1s protein interaction, which have demonstrated cases in which com- Species Lysine( K) Arginine(R) pensatory mutations are required to maintain protein interaction S cerevisiae 3 s paradoxus 3 f transcriptional networks, coevolution gives rise to"regulatory . S. mikatae homeostasis. "in which both mutations in a tf and its dna binding motif occur in compensatory fashion to maintain tran c. glabrata scriptional regulation. This series of compensatory mutations S castellii 4 which maintains both the transcriptional circuit and regulatory logic, parallels that of previous work demonstrating evolution of alternative transcriptional circuits producing identical logic( Tsong et al. 2006). Such systems of tightly coupled compensatory mu- tations might serve to counter the widespread divergence observed in transcriptional networks, and may constitute a general evolu- D hansenii tionary mechanism maintaining the regulation of transcriptional guilliermondii networks Methods L. elongosporus Yeast strains nidulans All immunoprecipitations were performed on strains where the appropriate gene has been endogenously fused to the TAP epitope Rigaut et al. 1999). Sc TAP-tagged strains were obtained from Oper S. octosporus Biosystems In Cg, the 2001HTU strain was used for TAP-tagging Spombe (see below) and deletions. Cg 2001HTU and NCCLS84 were ob- All yeasts in the species phylogeny(Wapinski et al. 2007) tained from atcc nome duplication event (orange star)are noted. Note that the Candida clade does not include Candida glabrate ScYapl. R79K and ScYap4 K252R mutants Endogenously epitope-tagged ScYapl: TAP and ScYap4: TAP strains(Open Biosystems) were used to introduce the appropriate Open questions still remain regarding how arginine and ly. mutation (ScYapl R79K, ScYap4 K252R) via the delitto perfette sine substitutions alter AP-1 DNA-binding motif preference. One method (Storici et al. 2001). In brief, ScYAPI and ScYAP4 were dis. hypothesis is that differences in their electrostatic charges alter the rupted with the URA3 selectable marker from pRS306(Brachman space required to accommodate other positively charged residues et al. 1998)using -100-bp homology Complementary 200-bp of the bZIp nding domain without electrostatic repulsion oligos with a mutation (ScYapl R79K or ScYap4 K252R)were then (Kim and Struhl 1995). This hypothesis suggests that the most transformed by electroporation(Thompson et al. 1998)to remove ine should be associated with YRE.A rather than YREo sites(Kim and Struhl 1995). How ing. This process creates strains possessing endogenous yAP-1 roteins having both the desired mutation and epitope. 11.15)was associated with YRE.A sites An alternative explanation involves a role for AP-1-induced CgApl TAP-tagged strain DNA flexibility. Complexes of yAP-1 protein with the YREO DNa The TAP tag was amplified with-100 otology(CgAP1) from sequence have been associated with an increase in incorporated pFA6a-TAP-HIS3MX6(Longtine et al flexibility (Kim and Struhl 1995)compared with yAP-1/YRE-A media(Amberg et al. 2005). C-terminal integration was verified complexes. A previous report has suggested that changes in dna by PCR and dNA sequencing with protein expression verified flexibility play a key role in determining half-site spacing prefer- by immunoblot (Amberg et al. 2005)with the peroxidase anti- ence and are responsible for differences between in vivo and in peroxidase antibody(Sigma P1291) vitro measurements(Suckow and Hollenberg 1998). Since residue 12 is in close proximity to DNA(Fig. 1B)within the protein-DNA Growth conditions, mRNA expression, and ChIP complex, residue changes may affect the ability of DNa to in- corporate water during binding, thus affecting both yAp-1 DNA Three(CgApl)or two (crapl R7 motif flexibility and binding Dragan et al. 2004b). Interestingly, complete media(Amberg et al. 2005)and treated with 0.03% the higher positive charge of arginine induces a stronger dipole methyl methanesulfonate(Sigma)for I h as performed previously than that of lysine, providing a possible mechanism for the in- (Tan et al. 2008). For mRNA expression analysis, total RNA w crease in the number of incorporated water molecules present at isolated by hot phenol/chloroform extraction and labeled with YRE-O sites and associated changes in DNA flexibility and binding Cy3 or Cys dyes (Invitrogen)(Kuo et al. 2010). Samples were hy- bridized to Agilent expression arrays and washed as recommended In summary, we have shown that conservation of the AP-1 by Agilent(Agilent Technologies regulatory program in yeast occurs through coordinated evolution For ChIP, all TAP-tagged strains were treated as previously of both the sequence of the TF(trans)and in its DNA-binding described (Tan et al. 2008). In brief, cells were fixed with 1% motifs(cis). This finding echoes that of previous studies of protein- formaldehyde for 20 min, inactivated with glycine and washed

Open questions still remain regarding how arginine and lysine substitutions alter AP-1 DNA-binding motif preference. One hypothesis is that differences in their electrostatic charges alter the space required to accommodate other positively charged residues of the bZIP DNA-binding domain without electrostatic repulsion (Kim and Struhl 1995). This hypothesis suggests that the most positively charged residues such as arginine should be associated with YRE-A rather than YRE-O sites (Kim and Struhl 1995). However, in our findings lysine ( pI = 9.59) rather than arginine ( pI = 11.15) was associated with YRE-A sites. An alternative explanation involves a role for AP-1-induced DNA flexibility. Complexes of yAP-1 protein with the YRE-O DNA sequence have been associated with an increase in incorporated water molecules (Dragan et al. 2004a) leading to a decrease in DNA flexibility (Kim and Struhl 1995) compared with yAP-1/YRE-A complexes. A previous report has suggested that changes in DNA flexibility play a key role in determining half-site spacing preference and are responsible for differences between in vivo and in vitro measurements (Suckow and Hollenberg 1998). Since residue 12 is in close proximity to DNA (Fig. 1B) within the protein–DNA complex, residue changes may affect the ability of DNA to incorporate water during binding, thus affecting both yAP-1 DNA motif flexibility and binding (Dragan et al. 2004b). Interestingly, the higher positive charge of arginine induces a stronger dipole than that of lysine, providing a possible mechanism for the increase in the number of incorporated water molecules present at YRE-O sites and associated changes in DNA flexibility and binding preference. In summary, we have shown that conservation of the AP-1 regulatory program in yeast occurs through coordinated evolution of both the sequence of the TF (trans) and in its DNA-binding motifs (cis). This finding echoes that of previous studies of protein– protein interaction, which have demonstrated cases in which compensatory mutations are required to maintain protein interaction over evolutionary time (Pazos and Valencia 2008). In the context of transcriptional networks, coevolution gives rise to ‘‘regulatory homeostasis,’’ in which both mutations in a TF and its DNAbinding motif occur in compensatory fashion to maintain transcriptional regulation. This series of compensatory mutations, which maintains both the transcriptional circuit and regulatory logic, parallels that of previous work demonstrating evolution of alternative transcriptional circuits producing identical logic (Tsong et al. 2006). Such systems of tightly coupled compensatory mutations might serve to counter the widespread divergence observed in transcriptional networks, and may constitute a general evolutionary mechanism maintaining the regulation of transcriptional networks. Methods Yeast strains All immunoprecipitations were performed on strains where the appropriate gene has been endogenously fused to the TAP epitope (Rigaut et al. 1999). Sc TAP-tagged strains were obtained from Open Biosystems. In Cg, the 2001HTU strain was used for TAP-tagging (see below) and deletions. Cg 2001HTU and NCCLS84 were obtained from ATCC. ScYap1.R79K and ScYap4.K252R mutants Endogenously epitope-tagged ScYap1TTAP and ScYap4TTAP strains (Open Biosystems) were used to introduce the appropriate mutation (ScYap1.R79K, ScYap4.K252R) via the delitto perfetto method (Storici et al. 2001). In brief, ScYAP1 and ScYAP4 were disrupted with the URA3 selectable marker from pRS306 (Brachmann et al. 1998) using ;100-bp homology. Complementary 200-bp oligos with a mutation (ScYap1.R79K or ScYap4.K252R) were then transformed by electroporation (Thompson et al. 1998) to remove URA3 by 5-FOA (US Biological) selection and verified by sequencing. This process creates strains possessing endogenous yAP-1 proteins having both the desired mutation and epitope. CgAp1 TAP-tagged strain The TAP tag was amplified with ;100-bp homology (CgAP1) from pFA6a-TAP-HIS3MX6 (Longtine et al. 1998), transformed by electroporation (Thompson et al. 1998), and selected on complete –his media (Amberg et al. 2005). C-terminal integration was verified by PCR and DNA sequencing with protein expression verified by immunoblot (Amberg et al. 2005) with the peroxidase antiperoxidase antibody (Sigma P1291). Growth conditions, mRNA expression, and ChIP Three (CgAp1) or two (ScYap1.R79K, ScYap4.K252R, ScYap3) biological replicates were grown from OD600 0.2 to 0.8 at 30° C in complete media (Amberg et al. 2005) and treated with 0.03% methyl methanesulfonate (Sigma) for 1 h as performed previously (Tan et al. 2008). For mRNA expression analysis, total RNA was isolated by hot phenol/chloroform extraction and labeled with Cy3 or Cy5 dyes (Invitrogen) (Kuo et al. 2010). Samples were hybridized to Agilent expression arrays and washed as recommended by Agilent (Agilent Technologies). For ChIP, all TAP-tagged strains were treated as previously described (Tan et al. 2008). In brief, cells were fixed with 1% formaldehyde for 20 min, inactivated with glycine and washed Figure 4. All yeasts in the species phylogeny (Wapinski et al. 2007) possess an AP-1 with an arginine. The Candida clade (shaded green) and the whole-genome duplication event (orange star) are noted. Note that the Candida clade does not include Candida glabrata. Coevolu tion wi thin a transcrip tional ne twork Genome Research 5 www.genome.org Downloaded from genome.cshlp.org on November 3, 2010 - Published by Cold Spring Harbor Laboratory Press

ownloaded from genome. cshlp org on November 3, 2010- Published by Cold Spring Harbor Laboratory Press Kuo et al with TBS. Cells were lysed for 2 h(Vibrax-VXR 2000)with glass Author contributions: D K, K.T., T.R., and T.I. designed the beads and sonicated for four cycles of 20 sec(+100-sec rest) at study. D K, K.L., S B, R C, J.C., and C L. performed the experi power setting 2 (Misonex Sonicator 3000) on ice. Lysate was in- mental work. D K and K.T. analyzed the data D K and T.I. wrote cubated with Dynabeads M-280 conjugated with anti-TAP anti- the manuscript. K.T. and T I supervised the work. body(Open Biosystems CAB1001)overnight. Cross-link reversal was performed overnight at 65 C with antibody-enriched and References (Invitrogen) with Cy5/Cy3 dyes. Sc and C samples were hybrid Altschul SE, Gish w. 1996. Local alignment statistics Methods Enzymol 266: ized to commercial or custom(see below) Agilent tiling arrays and washed as recommended by Agilent(Agilent Technologies). Amberg dC, Burke D], Strathern JN. 2005. Metl ' ress, Cold spring Harbor Ny. mamual Cold Spring Harbor laboratory Cg tiling microarray design and validation Berger ME, Badis G, Gehrke AR, Talukder S, Philippakis AA, Pena-Castillo L, Alleyne TM, Mnaimneh S, Botvinnik OB, Chan ET, et al. 2008. Variatio resolution with -44,000 60-mer probes designed teat-250-bp We designed a custom microarray tiling the Cg genom in homeodomain DNA binding revealed by high-resolution analysis of Borneman AR, Gianoulis TA, Zhang ZD, Yu H, Rozowsky J, dimerization and variability in melting temperatures (Mfold; Zuker 2003), low-complexity and repetitive sequences(RepeatMasker; http://repeatmasker.org),andcross-hybridization(wUblasT2:Brachman Altschul and Gish 1996). Default settings were used for each pre seful set of strains and plasmids for PCR-mediated gene disruption and (Agilent Technologies). ChIP results were validated by qPCR of five Bradley Rk,Li x-Y, Trapnell C, Davidson S, Pachter L, Chu HC, Tonkin LA, rgets compared against CyACTI(Supplemental Fig. 5) Biggin MD, Eisen MB. 2010. Binding site turnover produces pervasive closely related Drosophila species. PLos Biol 8: e1000343. doi: 10.1371/ journal. pbo 100034 Microarray data processing Brem RB, Kruglyak L. 2005. The landscape of genetic complexity across Intensities were background subtracted and normalized by LOESS 5,700 gene expression traits in yeast. Proc Natl Acad Sci 102: 1572- (Smyth 2005). Expression microarrays were analyzed using the Bullard JH, Mostovoy Y, Dudoit S, Brem RB. 2010. Polygenic and directional linmma package(Smyth 2005)with default parameters. ChIP tiling evolution across pathways in Saccharomyces. Proc Natl Acad Sci array errors were estimated by the Rosetta error model (Weng et a 107:5058-5063 2006)with resulting P-values of binding for each promoter calcu- Cliften P, Sudarsanam P, Desikan A n L Fu B, Majors J, Waterston R, Cohen BA, Johnston M. 2003. Finding lated by combining P-values of adjacent probes as previously de- Saccharomyces genomes by phylogenetic footprinting Science 301: 71- cribed (Tan et al. 2008). EN, Crane-Robinson C privalov 2004a. Thermodynam of GCN4-bzIP binding to DNA Phylogenetic trees, orthologs, and evolutionary trace analysis ruminating between the AP-1 and AtF/ CREB sites. Mol Biol 343: 865-878. agan Al, Liu Y, Makeyeva EN, Privalov PL 2004b DNA-binding domain of with BEAST(Drummond and Rambaut 2007) using default set. DNA. Nndeicesciens ges3 2: 5192tsrs e ATF/CREB and AP-1 binding sites of tings Sequences, species trees, and orthologs were obtained from the Fungal Orthogroups Repository(Wapinskiet al 2007). Multiple- sequence alignment was preformed using MUSCLE (Edgar 2004) nd high throughput. Nucleic Acids Res 32: 1792-179 with default parameters. Evolutionary trace analysis was performed Emerson J, Hsieh LC, Sung HM, Wang TY, Huang C), Lu HH, Lu MY, wu SH, using Trace Suite II (Innis et al. 2000)with default setting H. 2010. Natural selection on cis and trans enome res20:826-836 Motif finding charormyces cerevisiae with distinct biological Cell bio17:69826993 Maclsaac et al. 2006; Tan et al. 2008)using the default settings for is coupled to evolution of DNA-encoded nucleosome organization. Nat et41: STAMP (Mahony et al. 2005 ). Promoters were scanned with the Fujii Y, Shimizu T, Toda T, Yanagida M, Hakoshima T2000.Structural basis default settings for Patser (Hertz and Stormo 1999) for motif en- richment by the hyper netric test with multiple test correction Struct biol 7: 889-893 (Storey and Tibshirani 2003). A motif was considered"present"in Gasch AP, Moses AM, Chiang DY, Fraser HB, Berardini M, Eisen MB. 2004 a promoter for(fraction of maximal information content)20.7 transcription factors cause natural variation in yeast. Science Acknowledgments Hannett NM, Tagne JB, Reynolds DB, You Paul Russell for pFA6a-kanMX6, Nevan Krogan for pFA6a-natMX6, Hertegula stror mode d ggg rdenit ge statistically significant alignments of multiple sequences. Bioinformatics Wapinski, Lorraine Pillus, and members of the T I laboratory for 15:563-5 helpful discussions was supported by the National Science Hittinger CT, Carroll SB. 2007. Gene duplication and the adaptive evolution and Engineering Research Council of Canada. K.T. and T.I. were Hogues H Lavoie H, sellam A, mangos m. Roemer t supported by the David and Lucille Packard Foundation and NIh Whiteway M. 2008. Transcription factor substitution during the grant no. RO1 ESO14811 to T.I. evolution of fungal ribosome regulation. Mol Cell 29: 552-562

with TBS. Cells were lysed for 2 h (Vibrax-VXR 2000) with glass beads and sonicated for four cycles of 20 sec (+100-sec rest) at power setting 2 (Misonex Sonicator 3000) on ice. Lysate was incubated with Dynabeads M-280 conjugated with anti-TAP antibody (Open Biosystems CAB1001) overnight. Cross-link reversal was performed overnight at 65° C with antibody-enriched and unenriched DNA, and amplified (Sigma-Aldrich) and labeled (Invitrogen) with Cy5/Cy3 dyes. Sc and Cg samples were hybridized to commercial or custom (see below) Agilent tiling arrays and washed as recommended by Agilent (Agilent Technologies). Cg tiling microarray design and validation We designed a custom microarray tiling the Cg genome at ;250-bp resolution with ;44,000 60-mer probes designed to avoid selfdimerization and variability in melting temperatures (Mfold; Zuker 2003), low-complexity and repetitive sequences (RepeatMasker; http://repeatmasker.org), and cross-hybridization (WUBLAST2; Altschul and Gish 1996). Default settings were used for each program. Microarrays were manufactured using Agilent technology (Agilent Technologies). ChIP results were validated by qPCR of five targets compared against CgACT1 (Supplemental Fig. 5). Microarray data processing Intensities were background subtracted and normalized by LOESS (Smyth 2005). Expression microarrays were analyzed using the limma package (Smyth 2005) with default parameters. ChIP tiling array errors were estimated by the Rosetta error model (Weng et al. 2006) with resulting P-values of binding for each promoter calculated by combining P-values of adjacent probes as previously described (Tan et al. 2008). Phylogenetic trees, orthologs, and evolutionary trace analysis The yAP-1 DNA-binding domain phylogenetic tree was created with BEAST (Drummond and Rambaut 2007) using default settings. Sequences, species trees, and orthologs were obtained from the Fungal Orthogroups Repository (Wapinski et al. 2007). Multiplesequence alignment was preformed using MUSCLE (Edgar 2004) with default parameters. Evolutionary trace analysis was performed using TraceSuite II (Innis et al. 2000) with default settings. Motif finding De novo motifs were identified by SOMBRERO (Mahony et al. 2005) using default parameters and compared with literature (MacIsaac et al. 2006; Tan et al. 2008) using the default settings for STAMP (Mahony et al. 2005). Promoters were scanned with the default settings for Patser (Hertz and Stormo 1999) for motif enrichment by the hypergeometric test with multiple test correction (Storey and Tibshirani 2003). A motif was considered ‘‘present’’ in a promoter for (fraction of maximal information content) $0.7. Acknowledgments We thank JonathanWeissman for providing pFA6a-TAP-HIS3MX6, Paul Russell for pFA6a-kanMX6, Nevan Krogan for pFA6a-natMX6, and Richard Kolodner for pRS303 and pRS306. We also thank Ilan Wapinski, Lorraine Pillus, and members of the T.I. laboratory for helpful discussions. D.K. was supported by the National Science and Engineering Research Council of Canada. K.T. and T.I. were supported by the David and Lucille Packard Foundation and NIH grant no. R01 ES014811 to T.I. Author contributions: D.K., K.T., T.R., and T.I. designed the study. D.K., K.L., S.B., R.C., J.C., and C.L. performed the experimental work. D.K. and K.T. analyzed the data. D.K. and T.I. wrote the manuscript. K.T. and T.I. supervised the work. References Altschul SF, Gish W. 1996. Local alignment statistics. Methods Enzymol 266: 460–480. Amberg DC, Burke DJ, Strathern JN. 2005. Methods in yeast genetics: A Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Berger MF, Badis G, Gehrke AR, Talukder S, Philippakis AA, Pena-Castillo L, Alleyne TM, Mnaimneh S, Botvinnik OB, Chan ET, et al. 2008. 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