Computational approaches to discover and characterize transcription regulatory complex binding from protein-binding microarray-based experiments
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Gene regulation is controlled by DNA-bound complexes of transcription factors (TFs) and indirectly recruited transcriptional cofactors (COFs). Understanding how and where these TF-COF complexes bind in the genome is fundamental to our understanding of the role of cis-regulatory elements (CREs) in gene regulation and our mechanistic interpretation of non-coding variants (NCVs) known to impact gene expression levels. In this thesis, I present three related array-based techniques for the high-throughput profiling of DNA-bound TFs and TF-COF complexes directly from cell nuclear extracts. First, I describe the nuclear extract protein-binding microarray (nextPBM) approach to profile TF-DNA binding using nuclear extracts to account for cell-specific post-translational modifications and cofactors. By analyzing cooperative binding of PU.1/SPI1 and IRF8 in monocytes, I demonstrate how nextPBM can be used to delineate DNA-sequence determinants of cell-specific cooperative TF complexes. Second, I present the CASCADE (Comprehensive ASsessment of Complex Assembly at DNA Elements) approach to simultaneously discover DNA-bound TF-COF complexes and quantify the impact of NCVs on their binding. To demonstrate applicability of CASCADE to screen NCVs, I profile differential TF-COF binding to ~1,700 single-nucleotide polymorphisms in human macrophages and discover a prevalence of perturbed ETS-related TF-COF complexes at these quantitative trait loci. Third, I present the human TF array (hTF array) as a general platform for surveying COF recruitment to a panel of 346 non-redundant consensus TF binding sites (TFBSs). Using the hTF array, one can examine the activity of a diverse panel of TFs by profiling TF-COF complexes in a cell state-specific manner. In addition to the hTF microarray design, I have developed analysis and visualization software that allows users to explore COF recruitment profiling results interactively. Collectively, nextPBM, CASCADE, and the hTF array represent a suite of new approaches to investigate TF-COF complex binding and their application will refine our understanding of CREs by linking NCVs with the biophysical complexes that mediate gene regulatory functions.
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