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 motifsYap4 (also known as Cin5), a representative YRE-A-binding fac￾tor. 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 (Meth￾ods). Comparison of the top 50 promoters bound by Yap1.R79K with the top 50 promoters bound by wild-type Yap1 (as de￾termined in Tan et al. 2008) showed that mutation of Yap1 sig￾nificantly 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 pro￾files 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; Sup￾plemental 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 sub￾stitution 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 cho￾sen 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). Com￾parison 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 clus￾tering 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-regula￾tory preference by examining the ortho￾logs of genes targeted by both CgAp1 and ScYap1 across 20 sequenced yeast ge￾nomes (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 DNA￾binding 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