Competition for DNA binding between paralogous transcription factors determines their genomic occupancy and regulatory functions [competition]

Most eukaryotic transcription factors (TFs) are part of large protein families, with members of the same family (i.e. paralogous TFs) recognizing similar DNA-binding motifs but performing different regulatory functions. Many TF paralogs are co-expressed in the cell, and thus can compete for target sites across the genome. Here, we show that direct competition for DNA binding between TF paralogs is a major determinant of their genomic binding patterns. Using yeast proteins Cbf1 and Pho4 as our model system, we designed a high-throughput quantitative assay to capture the genomic binding profiles of competing TFs in a cell-free system. Our data shows that Cbf1 and Pho4 greatly influence each other’s occupancy by competing for their common putative genomic binding sites. The competition is different at different genomic sites, as dictated by the TFs' expression levels and their divergence in DNA-binding specificity and affinity. Analyses of ChIP-seq data show that the biophysical rules that dictate the competitive TF binding patterns in vitro are also followed in vivo, in the complex cellular environment. Furthermore, the Cbf1-Pho4 competition for genomic sites, as characterized in vitro using our new assay, plays a critical role in the specific activation of their target genes in the cell. Overall, our study highlights the importance of direct TF-TF competition for genomic binding and gene regulation by TF paralogs, and proposes an approach for studying this competition in a quantitative and high-throughput manner. Seven competition protein binding microarray (competition PBM) experiments were performed for recombinant, full-length, yeast transcription factors Cbf1 and Pho4. Briefly, the PBMs involved binding of GST-tagged Pho4 and His-tagged Cbf1 to double-stranded 60k Agilent microarrays under direct competition from each other in order to determine their competitive binding patterns 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 Cbf1 and Pho4 respectively for each spot.