Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding [uPBM_E2f1E2f3E2f]

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. Universal protein-binding microarray (PBM) experiments were performed for recombinant, full-length, human transcription factors E2f1, E2f3, and E2f4. Briefly, universal PBMs involved binding of GST-tagged transcription factors to double-stranded 44K Agilent microarrays containing a DNA library designed to cover all possible 10-bp sequences, with every 8-mer occurring in at least 16 different spots on the array. This design allows comprehensive and unbiased characterization of the binding specificity of transcription factors for all possible 8-bp sequences.

转录因子（Transcription Factor, TF）家族成员，即旁系同源转录因子（paralogous TFs），尽管在细胞内执行截然不同的调控功能，但过往研究常报道其拥有完全一致的DNA结合基序。学界通常认为，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或染色质微环境的相互作用。与既往研究假设相悖，我们发现旁系同源转录因子对DNA存在差异化的内在偏好性，而当前主流的基序模型未能捕捉到这类差异；这类差异可部分解释旁系TFs的基因组结合位点分布差异与功能特异性。本研究的这一发现，得益于将精心设计的高通量实验与严谨的计算建模有机结合，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框架，对来自4个不同人类TF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学术意义。我们针对重组表达的全长人类转录因子E2f1、E2f3及E2f4，开展了通用蛋白质结合微阵列（Universal Protein-binding Microarray, PBM）实验。简言之，通用PBM实验的流程为：将携带谷胱甘肽S-转移酶（Glutathione S-Transferase, GST）标签的转录因子，与双链44K安捷伦（Agilent）微阵列进行结合；该微阵列搭载的DNA文库可覆盖所有可能的10bp序列，且每一种8聚体（8-mer）序列均在阵列上至少存在16个独立点样。该实验设计可实现对转录因子结合所有可能8bp序列的特异性进行全面且无偏的表征。