Widespread and distinct sequence signatures of combinatorial transcriptional regulation. M Kazemian¹, H Pham², M Brodsky², S Sinha¹. 1) U of Illinois, Urbana, IL; 2) UMASS Med School, Worcester, MA.

   There is a growing realization that transcriptional gene regulation is often combinatorial, with multiple transcription factors (TFs) co-regulating the same genes, either independently or through direct or indirect interactions. Here, we explore the extent and diversity of combinatorial regulation in the Drosophila genome. We utilized the binding motifs of 322 TFs and chromatin accessibility data to produce computational TF-DNA interaction maps through different stages of embryonic development in fruit fly. We examined these binding maps to identify pairs of co-expressed TFs that either prefer to or avoid binding at common locations. We find that TF-TF aversion is as prevalent as co-binding, suggesting a less appreciated aspect of the combinatorial regulation. Several TFs had unusually many aversion partners including known chromatin remodeling TFs. We explored TF-TF co-binding and aversion partnerships in the context of nearly 100 gene expression domains and four stages of development, and found that the frequency of such partnerships varies greatly across expression domains. We then analyzed the common binding locations of TF-pairs for statistical patterns in terms of relative spacing and orientation between binding sites, using a newly designed statistical tool called interacting TF signatures (iTFs). We identified many instances of short distance biases between binding sites of TF-pairs including examples where such biases are stronger under certain relative orientations. To test if the genomic arrangement of these binding sites might reflect physical interactions between the corresponding TFs, we selected 28 TF-pairs whose binding sites exhibited short distance biases (<10bp) for further analysis. In vitro pull-down experiments revealed that ~65% of these pairs can directly interact with each other. For 5 of these pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. Overall, this study produces a comprehensive map of various types of sequence signatures of combinatorial TF action.