Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding [RG_MycMaxMad_v1]

Members of transcription factor (TF) families, i.e. paralogous TFs, are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions in the cell. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein cofactors or the chromatin environment. Contrary to previous assumptions, we find that paralogous TFs have different intrinsic preferences for DNA, not captured by current motif models, and these differences partly explain differential genomic binding and functional specificity. Our finding was possible due to a unique combination of carefully designed high-throughput assays and rigorous computation modeling, integrated into a unified framework called iMADS. We used iMADS to quantity, model, and analyze specificity differences between 11 paralogous TFs from 4 distinct human TF families. Our finding of differential specificity between closely related TFs has important implications for the interpretation of the regulatory effects of non-coding genetic variants. Genomic-context protein binding microarray (gcPBM) experiments were performed for recombinant, full-length, human transcription factors c-Myc, Max, and Mad1. Briefly, the PBMs involved binding of his-tagged transcription factor dimers to double-stranded 180K Agilent microarrays in order to determine their binding specificity 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 gcPBM signal intensity for each spot.

转录因子（Transcription Factor, TF）家族成员，即旁系同源转录因子（paralogous TFs），尽管在细胞中执行截然不同的调控功能，但既往研究常报道它们拥有完全一致的DNA结合基序。一般认为，旁系同源转录因子的基因组靶向差异源于其与蛋白质辅因子的相互作用或染色质微环境的影响。与既往研究假设相悖的是，我们发现旁系同源转录因子对DNA存在独特的内在结合偏好，而当前的基序模型未能捕捉到这类差异，且这些差异可部分解释其基因组结合偏好差异与功能特异性。本研究的发现得益于将精心设计的高通量实验与严谨的计算建模相结合，并将二者整合至一个名为iMADS的统一分析框架中。我们借助iMADS对来自4个不同人类转录因子家族的11种旁系同源转录因子的特异性差异进行了定量分析、建模与解析。我们关于紧密相关转录因子间存在结合特异性差异的发现，对解读非编码遗传变异的调控效应具有重要的科学意义。本研究针对重组全长人类转录因子c-Myc、Max及Mad1开展了基因组语境蛋白质结合微阵列（genomic-context protein binding microarray, gcPBM）实验。简言之，该PBM实验通过将带有组氨酸标签的转录因子二聚体与双链180K安捷伦（Agilent）微阵列结合，以测定其在天然基因组语境下对推定DNA结合位点的结合特异性。微阵列上的每个基因组DNA序列均设置了6个重复斑点。本研究报告了每个斑点的gcPBM信号强度值。