Kar4 acts as a Ste12 regulator in Saccharomyces cerevisiae, promoting Ste12 binding to a specific DNA motif genome-wide

Kar4 is a putative transcription factor required for efficient mating in the budding yeast Saccharomyces cerevisiae. Kar4 functions with Ste12, the master transcriptional regulator of the yeast mating pheromone response, to promote the transcription of a subset of Ste12 targets required for mating. However, the mechanism by which Kar4 modulates Ste12 activity has remained uncertain. Here, we examined Kar4s function at the levels of transcription (RNA-seq) and Ste12 DNA binding (ChIP-exo). We show that Kar4 promotes Ste12 binding to nearly all Ste12 DNA-binding sites associated with transcriptionally-upregulated genes, even if their upregulation is not dependent on Kar4. We further found that the majority of Ste12-binding sites have two pheromone response elements (PREs) separated by four nucleotides in a head-to-tail orientation (H-T 4 motif). Sites associated with Kar4-dependent transcription have PREs with more mismatches and substantially lower Ste12 occupancy in kar4{Delta} cells than sites linked to Kar4-independent transcription. During the pheromone response in kar4{Delta} cells, Ste12 exhibits increased binding to non-H-T 4 motifs, particularly T-T 3 motifs, resulting in the abnormal upregulation of many genes not transcribed during the wild-type pheromone response. Therefore, we propose that Kar4 functions by increasing the DNA-binding specificity of Ste12 globally, promoting its binding primarily to H-T 4 motifs. Our model is consistent with previously-observed slower induction kinetics of Kar4-dependent genes via a feed-forward mechanism. Lastly, we uncovered several novel aspects of the pheromone response, including a broad role for the Crz1 transcription factor, induction of stress responses, and the identification of pheromone-responsive intergenic transcripts.