Comparing DNA binding specificities between orthologous Pho4 transcription factors using extended length probes that include flanking genomic sequences.

Combinatorial control by multiple transcription factors (TFs) is a hallmark of eukaryotic gene regulation. Despite its prevalence and crucial roles in enhancing specificity and integrating information, the mechanisms behind why eukaryotic TFs depend on one another, and whether such interdependence evolves, are not well understood. We exploit natural variation in co-TF dependence in the yeast phosphate starvation (PHO) response to address this question. In the model yeast Saccharomyces cerevisiae, the main TF, Pho4, relies on the co-TF Pho2 to regulate ~28 genes. In a related yeast pathogen, Candida glabrata, its Pho4 exhibits significantly reduced Pho2 dependence and has an expanded target set of ~70 genes. Chomatin IP based motifs for the two orthologous Pho4s are highly similar, with no detectable difference in their core E-box motif. However, past studies found that preference for sequences flanking the core motif can provide additional specificities to TFs in the same family. To determine if ScPho4 and CgPho4 differ in their flanking nucleotide preference, we conducted the genomic context Protein Binding Microarray (gcPBM) using 36 bp oligos centered on E-box motifs from ChIP-identified Pho4 binding locations in both S. cerevisiae and C. glabrata genomes. The results confirmed that the two Pho4 orthologs do not show divergence in their sequence preference even after including the genomic context. Four protein binding microarray (PBM) experiments were performed for recombinant, full-length Pho4 transcription factors from S. cerevisiae and C. glabrata. Briefly, the PBMs measured the binding of GST-tagged Pho4 from either species to double-stranded 60k Agilent microarrays at two different concentrations, for putative DNA binding sites in native genomic context. Each genomic DNA sequence represented on the array is present in 6 replicate spots. We report the PBM signal intensity of both Pho4 orthologs for each spot.