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.绝大多数真核生物转录因子（transcription factors, TFs）隶属于庞大的蛋白质家族，同一家族的成员（即旁系同源转录因子（paralogous TFs））可识别相似的DNA结合基序，但执行不同的调控功能。众多TF旁系同源物可在细胞内共表达，因此能够在全基因组范围内竞争靶位点。本研究证实，TF旁系同源物之间针对DNA结合的直接竞争，是决定其基因组结合模式的核心因素。本研究以酵母蛋白Cbf1和Pho4为模型系统，开发了一种高通量定量检测方法，可在无细胞体系中捕获竞争状态下TFs的基因组结合谱。实验数据表明，Cbf1与Pho4可通过竞争共有推定基因组结合位点，极大地影响彼此的结合占据情况。这种竞争在不同基因组位点存在差异，具体由TFs的表达水平、以及其DNA结合特异性与亲和力的分化程度所决定。对染色质免疫沉淀测序（ChIP-seq）数据的分析显示，在体外决定竞争性TF结合模式的生物物理规则，在复杂细胞环境的体内条件下同样适用。此外，通过本研究的新检测方法在体外表征的Cbf1-Pho4基因组位点竞争，在细胞内靶基因的特异性激活中发挥关键作用。综上，本研究强调了TF-TF直接竞争对于TF旁系同源物的基因组结合与基因调控的重要性，并提出了一种可定量、高通量研究此类竞争的实验方法。