Proper control of gene expression is important for coordinating biological processes. Transcription factors (TFs) play a critical role in maintaining proper gene expression by binding their specific binding sites at cis-regulatory elements (CREs) and recruiting cofactors (COFs). Given their important roles in mediating gene expression, there is a need for methods that can profile TF-COF binding to DNA in a high throughput (HT) manner. In this thesis, I present two novel, array-based, HT methods – CASCADE (Customizable Approach to Survey Complex Assembly at DNA Elements) and CoRec (Cofactor Recruitment) – to profile the indirect recruitment of any COF in any cell type or cell state in a HT manner. CASCADE is a technique to profile the indirect recruitment of COFs to DNA via TFs in a stimulus-dependent manner. We demonstrate how CASCADE can be used to characterize COF recruitment at single nucleotide resolution to CREs in LPS-stimulated macrophages and demonstrate how it can be applied to characterize COF recruitment to ~1700 non-coding single nucleotide polymorphisms (ncSNPs). To understand genetic alterations that drive cancer, we apply CASCADE to cancer-associated non-coding variants (NCVs). Using our newly developed genetic burden test, we predict 2,555 driver NCVs and find that 765 candidate drivers altered transcriptional activity, 510 led to differential binding of TF-COF complexes, and that they primarily impact the binding to ETS factors. Lastly, I introduce the CoRec technique for profiling COF recruitment to a broad set of TF families. I show how CoRec can be used to develop cell type-specific TF-COF interaction “fingerprints” and reveal distinct COF recruitment preferences at single-nucleotide resolution. I integrate the findings from CoRec with genomic accessibility and gene expression data and demonstrate that COF-recruitment motifs captured with CoRec are enriched at promoters and significantly related to highly expressed genes. Taken together, the CASCADE and CoRec approaches are powerful methods to investigate TF-COF complex binding and their use will add to our understanding of CRE regulation, uncover biophysical mechanisms of NCVs, and characterizations of cell state-specific TF-COF complexes.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/47996 |
| Date | 01 February 2024 |
| Creators | Hook, Heather |
| Contributors | Siggers, Trevor |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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