conserved , highly pheromone - induced genes were identified by comparing previously published expression data from s. cerevisiae and c. albicans . we combined results from three independent ortholog maps to find pheromone response genes that were present in most hemiascomycete yeast species . general pheromone - activated genes were defined as genes that were among the top ten most highly pheromone - activated genes in s. cerevisiae or c. albicans , present in the genomes of most hemiascomycete species , and are not an asg in s. cerevisiae , k. lactis , or c. albicans . the asgs were defined as showing differential expression between a and  cells in s. cerevisiae , k. lactis , or c. albicans . the protein sequences of these genes were used as a psi - blast query to identify orthologs in species whose genomes were not included in any of the ortholog maps . mfa1 genes , which encode for the a - factor pheromone , are too short to be annotated by automated bioinformatics techniques , so they were identified manually in most species using tblastn . ste12 motifs were generated from general pheromone response gene intergenic regions pooled from all the species in a given clade . each intergenic region was considered up to 600bp upstream of the start codon , a distance that is likely to capture functional ste12 cis - regulatory sites . the program meme was used to find overrepresented sequences in these sets of intergenic regions , under conditions assuming zero to one binding site per intergenic region . because the motifs generated for ste12 from the kluyveromyces , saccharomyces , and candida clades were highly similar , we pooled intergenic regions from all three clades and generated a 7bp motif to score individual gene regulatory regions across the hemiascomycetes . the upstream 600bp of intergenic regions were scored as described for the number of ste12 motifs above a given cutoff . for  consensus sites , the cutoff only accepted a single sequence : tgaaaca . for mismatched sites , the cutoff allowed deviation from the consensus sequence of a single nucleotide in any position in the motif . for genes that were duplicated in a species ( i.e. contained more than one copy ) the copy with the largest number of ste12 sites was taken , because this was the most stringent criterion to test our hypothesis that the a - specific genes lacked ste12 sites in the kluyveromyces and candida clades . mfa1 genes often are found in multiple copies in each species ( i.e. mfa1 and mfa2 ) so two copies were included when present . ste12 motifs were also enumerated in the rest of the intergenic regions in each species . enrichment of the ste12 motif in either the a - specific genes or the general pheromone response genes with respect to the rest of the genome was calculated using the hypergeometric distribution at all possible cutoffs of number of ste12 motifs . the cutoff giving the most significant p - value was chosen , because this represented the most stringent test of our hypothesis that the a - specific genes lacked ste12 sites in the kluyveromyces and candida clades . sequence data were obtained from the yeast gene order browser ( ygob ) and the joint genomes institute ( jgi ) websites ( http://ygob.ucd.ie and http://genome.jgi.doe.gov , respectively ) . constructs for gene disruption , epitope tagging , and promoter replacement were generated by fusion pcr . three to five individual pcr components were amplified using extaq ( takara ) , and 50 ng of each were combined into a single 50 l reaction along wth 0.5 l extaq , 0.4 l 25 mm dntps , 0.1 l of each 100 m primer . the reactions were incubated in a thermocycler : 95c 3:00 , [ 95 0:30 , 55 0:30 , 72 1:00/kb ] x 4 , [ 95 0:30 , 5872 0:30 ( depending on the annealing temperature of the primers ) , 72 1:00/kb ] x 30 , 72 10:00 . the gfp reporters for k. lactis , l. kluyveri , and s. cerevisiae were constructed from the original vector that was made for use in k. lactis . pts12 was made from a 5-piece fusion pcr , digested with kasi and hindiii ( new england biolabs ) and ligated into puc19 using fast - link ligase ( epicentre biotechnologies ) . vectors were treated with antarctic phosphatase ( new england biolabs ) for 30 minutes prior to ligation . the reporter consisted of the upstream flanking region of the k. lactis trp1 gene , the s. cerevisiae trp1 gene , the entire s. cerevisiae cyc1 upstream intergenic region , the cagfp and act1 terminator , and the downstream flanking region of k. lactis trp1 . the s. cerevisiae trp1 gene was replaced with the hygromycin resistance marker from pfa6-htb - hphmx4 by digestion with bsiwi and pmli and ligated to make pts16 . dna oligos with sites for noti and bamhi sites were annealed in fast - link ligase buffer ( epicentre biotechnologies ) and ligated in a 50:1 insert to vector ratio to make pts26 . this vector was used to make the other reporters for use in k. lactis by ligating annealed oligos or digested pcr products into the noti and xhoi sites . vectors incorporating an entire intergenic region of a gene were constructed by ligating digested pcr products the sites saci and agei . the l. kluyveri reporter was made by cloning upstream and downstream homology to the trp1 locus into the ngomiv / bsteii and blpi / hindiii sites , respectively . these homology regions were then swapped out for homology to the ura3 locus to improve the efficiency of integration of the reporter by selection with 5-foa . the altered regions of the l. kluyveri ste2 promoter were cloned as with the k. lactis reporter . the s. cerevisiae reporter was made by cloning the ste2 promoter fused to gfp into a centromeric reporter , prs412 , using the kpni and saci sites . to measure the pheromone response in a strain that lacks mata2 , we replaced the promoter of ste2 with that of a general pheromone response gene that is not dependent on a2 . strain yts43 in which the ste2 promoter was replaced by the sst2 promoter was made in the mata2 background . this replacement construct consisted of a 4-piece fusion pcr that contained 5 homology , the ura3 marker , the sst2-promoter , and 3 homology . k. lactis and l. kluyveri were transformed with 1 g dna according to published protocols , and s. cerevisiae was transformed using a standard lithium acetate protocol . k. lactis cells were grown overnight for ~14h , then starved in sd medium lacking phosphate as previously described . cells were induced after 6h with 6.25 m -factor pheromone ( wswitlrpgqpif > 95% purity , 100mg / ml in 100% dmso ; genemed synthesis ) or an equivalent amount of dmso . cells were removed at several time points and were pelleted , washed , and frozen in liquid nitrogen . rna was extracted using the ribopure rna purification kit ( ambion ) . the rna was reverse transcribed into cdna using superscript ii as described previously . transcript levels were measured using sybr green on a steponeplus rt pcr machine ( applied biosystems ) . transcripts were normalized to that of vps4 , a gene that was found to be highly consistent across conditions in previous expression experiments . strains yts314 and yts315 were grown using a phosphate starvation protocol modified from our previously published protocol . yeast were grown overnight for ~14h , then pelleted , resuspended in phosphate starvation media , then diluted in 100200ml phosphate starvation media to od600 = 0.25  0.3 . these cultures were grown for 2h , then 13-mer -factor pheromone was added to 6.25 m , and the cultures were grown for an additional 2h . cells were crosslinked with 1% formaldehyde for 15 minutes , quenched with glycine , pelleted , washed , and frozen in liquid nitrogen . cells were lysed in 300700l lysis buffer ( 50 mm hepes / koh ph 7.5 , 140 mm nacl , 1 mm edta , 1% triton x-100 , 0.1% na - deoxycholate ) with added edta - free protease inhibitor tablet ( roche ) . cells were transferred to a fresh tube with 500l 0.4 mm glass beads , and were lysed for ~45min on a vortex genie with tube adaptor ( labrepco ) . lysate was recovered by centrifugation , and sonicated 34x on a diagenode bioruptor ( level 5 , 30s on , 1min off ) . lysate was cleared by centrifugation at max speed in a table - top centrifuge at 4 c . immunoprecipitation was carried out overnight with 300l cleared lysate , 200l fresh lysis buffer , and 10l 200g / ml anti - c - myc antibody ( invitrogen ) . 50l of washed protein g sepharose beads 50% slurry was added and incubated for 2h . crosslinking was reversed by incubating 16h at 65 c , and immunoprecipitated dna was recovered using the minelute kit ( qiagen ) . chip - seq libraries were prepared using the nebnext chip - seq library prep kit for illumina ( new england biolabs ) and ampure xp magnetic beads ( beckman coulter , inc ) . libraries were pooled and sequenced on a hiseq 2500 ( illumina ) through the ucsf center for advanced technology ( cat.ucsf.edu ) . the chip experiment was performed with a total of three replicates on two separate days , and a single replicate was performed of the untagged control . k. lactis reporter strains were grown as 1ml cultures in sd medium overnight for ~14h in a 96 well plate . cells were pelleted , resuspended in phosphate starvation medium , and diluted to od600 = 0.25 . cells were grown 6h , then induced with 6.25m -factor ( or dmso for control cells ) for 4h . cells were diluted 10-fold into the same media before measuring gfp fluorescence on a bd lsr ii flow cytometer . cells were gated to exclude debris and the mean of the cell population was used for further analysis . the autofluorescence level of a cell containing no reporter was subtracted from each reporter strain , and the standard deviation of each reporter strain was added to the standard deviation of the autofluorescence strain . values were divided by the mean autofluorescence to give an indication of the signal relative to noise . multiple independent isolates were tested for each reporter strain , and each experiment was performed separately two or more times . in rare cases where one isolate displayed a drastic difference in expression from the others , it was tested to see whether it was a statistical outlier , and if so , was removed for subsequent experimental repetitions . statistics for the reporter assays were conducted using an anova followed by tukey s hsd test . the intergenic sequences for the a - specific genes were obtained from ygob for the species s. cerevisiae , s. paradoxus , s. mikatae , s. uvarum , and s. kudriavzevii . 19 k. lactis strains were obtained from the phaff yeast culture collection ( davis , california ) . from these and 3 additional k. lactis strains , a - specific gene intergenic sequences were amplified from both directions using extaq ( takara ) and sanger sequenced . the intergenic regions from the saccharomyces sensu stricto and k. lactis species complex were then analyzed separately as follows . for each species or isolate , the a - specific gene intergenic regions were scored for the presence of strong and weak ste12 cis - regulatory regions . the appropriate nucleotide evolution model was selected as previously described ; the hky+g model was selected for the sensu stricto species , and the gtr+g model was selected for k. lactis . these models output the estimated evolutionary rate for each site in the alignment and each site was categorized as being part of a consensus or mismatched ste12 cis - regulatory site in at least one species , or no site at all . then , the trees generated by this method were used as input to test for different evolutionary rates among the different categories using the rphast package . the null model was generated using phylofit on the intergenic regions lacking mismatched and consensus ste12 sites using the model for the sensu stricto species and the  rev  model for k. lactis . then , the mismatched and consensus ste12 sites were tested for increased conservation in comparison the null model using phylop . conserved , highly pheromone - induced genes were identified by comparing previously published expression data from s. cerevisiae and c. albicans . we combined results from three independent ortholog maps to find pheromone response genes that were present in most hemiascomycete yeast species . general pheromone - activated genes were defined as genes that were among the top ten most highly pheromone - activated genes in s. cerevisiae or c. albicans , present in the genomes of most hemiascomycete species , and are not an asg in s. cerevisiae , k. lactis , or c. albicans . the asgs were defined as showing differential expression between a and  cells in s. cerevisiae , k. lactis , or c. albicans . the protein sequences of these genes were used as a psi - blast query to identify orthologs in species whose genomes were not included in any of the ortholog maps . mfa1 genes , which encode for the a - factor pheromone , are too short to be annotated by automated bioinformatics techniques , so they were identified manually in most species using tblastn . ste12 motifs were generated from general pheromone response gene intergenic regions pooled from all the species in a given clade . each intergenic region was considered up to 600bp upstream of the start codon , a distance that is likely to capture functional ste12 cis - regulatory sites . the program meme was used to find overrepresented sequences in these sets of intergenic regions , under conditions assuming zero to one binding site per intergenic region . because the motifs generated for ste12 from the kluyveromyces , saccharomyces , and candida clades were highly similar , we pooled intergenic regions from all three clades and generated a 7bp motif to score individual gene regulatory regions across the hemiascomycetes . the upstream 600bp of intergenic regions were scored as described for the number of ste12 motifs above a given cutoff . for  consensus sites , the cutoff only accepted a single sequence : tgaaaca . for mismatched sites , the cutoff allowed deviation from the consensus sequence of a single nucleotide in any position in the motif . for genes that were duplicated in a species ( i.e. contained more than one copy ) the copy with the largest number of ste12 sites was taken , because this was the most stringent criterion to test our hypothesis that the a - specific genes lacked ste12 sites in the kluyveromyces and candida clades . mfa1 genes often are found in multiple copies in each species ( i.e. mfa1 and mfa2 ) so two copies were included when present . ste12 motifs were also enumerated in the rest of the intergenic regions in each species . enrichment of the ste12 motif in either the a - specific genes or the general pheromone response genes with respect to the rest of the genome was calculated using the hypergeometric distribution at all possible cutoffs of number of ste12 motifs . the cutoff giving the most significant p - value was chosen , because this represented the most stringent test of our hypothesis that the a - specific genes lacked ste12 sites in the kluyveromyces and candida clades . sequence data were obtained from the yeast gene order browser ( ygob ) and the joint genomes institute ( jgi ) websites ( http://ygob.ucd.ie and http://genome.jgi.doe.gov , respectively ) . constructs for gene disruption , epitope tagging , and promoter replacement were generated by fusion pcr . three to five individual pcr components were amplified using extaq ( takara ) , and 50 ng of each were combined into a single 50 l reaction along wth 0.5 l extaq , 0.4 l 25 mm dntps , 0.1 l of each 100 m primer . the reactions were incubated in a thermocycler : 95c 3:00 , [ 95 0:30 , 55 0:30 , 72 1:00/kb ] x 4 , [ 95 0:30 , 5872 0:30 ( depending on the annealing temperature of the primers ) , 72 1:00/kb ] x 30 , 72 10:00 . the gfp reporters for k. lactis , l. kluyveri , and s. cerevisiae were constructed from the original vector that was made for use in k. lactis . pts12 was made from a 5-piece fusion pcr , digested with kasi and hindiii ( new england biolabs ) and ligated into puc19 using fast - link ligase ( epicentre biotechnologies ) . vectors were treated with antarctic phosphatase ( new england biolabs ) for 30 minutes prior to ligation . the reporter consisted of the upstream flanking region of the k. lactis trp1 gene , the s. cerevisiae trp1 gene , the entire s. cerevisiae cyc1 upstream intergenic region , the cagfp and act1 terminator , and the downstream flanking region of k. lactis trp1 . the s. cerevisiae trp1 gene was replaced with the hygromycin resistance marker from pfa6-htb - hphmx4 by digestion with bsiwi and pmli and ligated to make pts16 . dna oligos with sites for noti and bamhi sites were annealed in fast - link ligase buffer ( epicentre biotechnologies ) and ligated in a 50:1 insert to vector ratio to make pts26 . this vector was used to make the other reporters for use in k. lactis by ligating annealed oligos or digested pcr products into the noti and xhoi sites . vectors incorporating an entire intergenic region of a gene were constructed by ligating digested pcr products the sites saci and agei . the l. kluyveri reporter was made by cloning upstream and downstream homology to the trp1 locus into the ngomiv / bsteii and blpi / hindiii sites , respectively . these homology regions were then swapped out for homology to the ura3 locus to improve the efficiency of integration of the reporter by selection with 5-foa . the altered regions of the l. kluyveri ste2 promoter were cloned as with the k. lactis reporter . the s. cerevisiae reporter was made by cloning the ste2 promoter fused to gfp into a centromeric reporter , prs412 , using the kpni and saci sites . to measure the pheromone response in a strain that lacks mata2 , we replaced the promoter of ste2 with that of a general pheromone response gene that is not dependent on a2 . strain yts43 in which the ste2 promoter was replaced by the sst2 promoter was made in the mata2 background . this replacement construct consisted of a 4-piece fusion pcr that contained 5 homology , the ura3 marker , the sst2-promoter , and 3 homology . k. lactis and l. kluyveri were transformed with 1 g dna according to published protocols , and s. cerevisiae was transformed using a standard lithium acetate protocol . k. lactis cells were grown overnight for ~14h , then starved in sd medium lacking phosphate as previously described . cells were induced after 6h with 6.25 m -factor pheromone ( wswitlrpgqpif > 95% purity , 100mg / ml in 100% dmso ; genemed synthesis ) or an equivalent amount of dmso . cells were removed at several time points and were pelleted , washed , and frozen in liquid nitrogen . rna was extracted using the ribopure rna purification kit ( ambion ) . the rna was reverse transcribed into cdna using superscript ii as described previously . transcript levels were measured using sybr green on a steponeplus rt pcr machine ( applied biosystems ) . transcripts were normalized to that of vps4 , a gene that was found to be highly consistent across conditions in previous expression experiments . strains yts314 and yts315 were grown using a phosphate starvation protocol modified from our previously published protocol . yeast were grown overnight for ~14h , then pelleted , resuspended in phosphate starvation media , then diluted in 100200ml phosphate starvation media to od600 = 0.25  0.3 . these cultures were grown for 2h , then 13-mer -factor pheromone was added to 6.25 m , and the cultures were grown for an additional 2h . cells were crosslinked with 1% formaldehyde for 15 minutes , quenched with glycine , pelleted , washed , and frozen in liquid nitrogen . cells were lysed in 300700l lysis buffer ( 50 mm hepes / koh ph 7.5 , 140 mm nacl , 1 mm edta , 1% triton x-100 , 0.1% na - deoxycholate ) with added edta - free protease inhibitor tablet ( roche ) . cells were transferred to a fresh tube with 500l 0.4 mm glass beads , and were lysed for ~45min on a vortex genie with tube adaptor ( labrepco ) . lysate was recovered by centrifugation , and sonicated 34x on a diagenode bioruptor ( level 5 , 30s on , 1min off ) . lysate was cleared by centrifugation at max speed in a table - top centrifuge at 4 c . immunoprecipitation was carried out overnight with 300l cleared lysate , 200l fresh lysis buffer , and 10l 200g / ml anti - c - myc antibody ( invitrogen ) . 50l of washed protein g sepharose beads 50% slurry was added and incubated for 2h . crosslinking was reversed by incubating 16h at 65 c , and immunoprecipitated dna was recovered using the minelute kit ( qiagen ) . chip - seq libraries were prepared using the nebnext chip - seq library prep kit for illumina ( new england biolabs ) and ampure xp magnetic beads ( beckman coulter , inc ) . libraries were pooled and sequenced on a hiseq 2500 ( illumina ) through the ucsf center for advanced technology ( cat.ucsf.edu ) . file types were manipulated using samtools , and peaks were called using macs . coverage and peak the chip experiment was performed with a total of three replicates on two separate days , and a single replicate was performed of the untagged control . k. lactis reporter strains were grown as 1ml cultures in sd medium overnight for ~14h in a 96 well plate . cells were pelleted , resuspended in phosphate starvation medium , and diluted to od600 = 0.25 . cells were grown 6h , then induced with 6.25m -factor ( or dmso for control cells ) for 4h . cells were diluted 10-fold into the same media before measuring gfp fluorescence on a bd lsr ii flow cytometer . cells were gated to exclude debris and the mean of the cell population was used for further analysis . the autofluorescence level of a cell containing no reporter was subtracted from each reporter strain , and the standard deviation of each reporter strain was added to the standard deviation of the autofluorescence strain . values were divided by the mean autofluorescence to give an indication of the signal relative to noise . multiple independent isolates were tested for each reporter strain , and each experiment was performed separately two or more times . in rare cases where one isolate displayed a drastic difference in expression from the others , it was tested to see whether it was a statistical outlier , and if so , was removed for subsequent experimental repetitions . statistics for the reporter assays were conducted using an anova followed by tukey s hsd test . the intergenic sequences for the a - specific genes were obtained from ygob for the species s. cerevisiae , s. paradoxus , s. mikatae , s. uvarum , and s. kudriavzevii . 19 k. lactis strains were obtained from the phaff yeast culture collection ( davis , california ) . from these and 3 additional k. lactis strains , a - specific gene intergenic sequences were amplified from both directions using extaq ( takara ) and sanger sequenced . the intergenic regions from the saccharomyces sensu stricto and k. lactis species complex were then analyzed separately as follows . for each species or isolate , the a - specific gene intergenic regions were scored for the presence of strong and weak ste12 cis - regulatory regions . the appropriate nucleotide evolution model was selected as previously described ; the hky+g model was selected for the sensu stricto species , and the gtr+g model was selected for k. lactis . these models output the estimated evolutionary rate for each site in the alignment and each site was categorized as being part of a consensus or mismatched ste12 cis - regulatory site in at least one species , or no site at all . then , the trees generated by this method were used as input to test for different evolutionary rates among the different categories using the rphast package . the null model was generated using phylofit on the intergenic regions lacking mismatched and consensus ste12 sites using the model for the sensu stricto species and the  rev  model for k. lactis . then , the mismatched and consensus ste12 sites were tested for increased conservation in comparison the null model using phylop . expression of the asg ste2 and the general pheromone - activated gene fus3 in the presence of k. lactis ste12 , in the absence of ste12 , and in the presence of s. cerevsiae ste12 . expression under uninduced and pheromone - induced conditions are shown as mean fluorescence of three independent genetic isolates + / s.d . individual a - specific genes and general pheromone - activated genes were scored for the presence of ste12 cis - regulatory motifs in their upstream regulatory regions . shown is the number of consensus ste12 sites within 600bp of the translation start site . the enrichment for the ste12 motif in each set of genes in each species is shown in figure 1c . asgs were scored for the presence of weak ste12 binding sites in their upstream regulatory regions . the enrichment of the ste12 site in the asgs in each species is shown in the bottom row . the s. cerevisiae ste2 promoter was fused to gfp and the role of ste12 cis - regulatory sites in expression . expression under uninduced and pheromone - induced conditions are shown as mean fluorescence of three independent genetic isolates + / s.d . the k. lactis ste2 gfp reporter and heterologous reporter containing the a2-mcm1 binding site were transformed into wild type and ste12 cells . the k. lactis ste2 gfp reporter was tested with a mutated mcm1 cis - regulatory site . shown is mean fluorescence of three independent genetic isolates + / s.d . a , the presence of the mcm1 binding site is unchanged across the candida , kluyveromyces , and saccharomyces clades . asgs were scored for the strength of the mcm1 binding site in their upstream regulatory regions . the enrichment of the strength of the mcm1 site in the asgs in each species is shown in the bottom row . b , a construct with increased mcm1 binding site strength was tested for its ability to compensate for the deletion of a2 . the yeast phylogeny along with the number of ste12 binding sites in extant species was used to estimate the gain and loss rate of the sites over evolutionary time . the saccharomyces clade was allowed to have different rates ( orange ) ( x = 49.33 , df = 2 , p = 1.94e-11 ) . the gain and loss rate units are cis - regulatory sites per amino acid substitution in the species phylogeny . the evolutionary rate of nucleotides in ste12 binding sites was compared to rates in the rest of the upstream regulatory regions of the a - specific genes , shown as violin plots . promoters between closely related species were aligned and the evolutionary rate of each basepair in the alignment was determined after model selection .
epistasis  the non - additive interactions between different genetic loci  constrains evolutionary pathways , blocking some and permitting others18 . for biological networks such as transcription circuits , 
 the nature of these constraints and their consequences are largely unknown . here 
 we describe the evolutionary pathways of a transcription network that controls the response to mating pheromone in yeasts9 . 
 a component of this network , the transcription regulator ste12 , has evolved two different modes of binding to a set of its target genes . in one group of species , 
 ste12 binds to specific dna binding sites , while in another lineage it occupies dna indirectly , relying on a second transcription regulator to recognize dna . 
 we show , through the construction of various possible evolutionary intermediates , that evolution of the direct mode of dna binding was not directly accessible to the ancestor . instead , it was contingent on a lineage - specific change to an overlapping transcription network with a different function , the specification of cell type . 
 these results show that analyzing and predicting the evolution of cis - regulatory regions requires an understanding of their positions in overlapping networks , as this placement constrains the available evolutionary pathways .