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

Kar4 is a putative transcription factor required for efficient mating in the budding yeast <i>Saccharomyces cerevisiae</i>. 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 Kar4's 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 <i>kar4</i>Δ cells than sites linked to Kar4-independent transcription. During the pheromone response in <i>kar4</i>Δ 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.