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

Transcription factors must often transcribe different genes under different conditions. To achieve this, DNA-binding specificity is often constrained by binding to cofactors or other transcription factors. 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 target genes. 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 2 pheromone response elements (PREs) separated by 4 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 kar4Δ cells, than sites linked to Kar4-independent transcription. During the pheromone response in kar4Δ 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 in wild-type. We propose that Kar4 functions by increasing the DNA-binding specificity of Ste12 globally, promoting its binding primarily to H-T 4 motifs. Lastly, we uncovered several novel aspects of the pheromone response, including a broad role for the Crz1 transcription factor, pheromone-responsive intergenic transcripts, and pheromone-induced stress responses.