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 ResearchAs a model of transcriptional network evolution, we exam￾ined 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 pro￾cess 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 con￾servation 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 fre￾quently play important evolutionary roles (Innis et al. 2000). Using this approach, we identified residue 12 of the DNA￾binding domain basic region as the most important evolutionarily divergent posi￾tion 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 prefer￾ence (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 ‘‘half￾sites’’ are positioned in either adjacent or overlapping fashion (Fig. 1A), which we refer to as yAP-1 response element adja￾cent (YRE-A) or yAP-1 response element overlapping (YRE-O), respectively. Pre￾vious analyses of genome-wide chroma￾tin immunoprecipitation with microarray hybridization (ChIP￾chip) data in Sc (Harbison et al. 2004; Tan et al. 2008) have de￾termined 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 (Sup￾plemental 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