Unravelling the molecular mechanisms of histone code recognition by the BRD9 bromodomain

Dash, Pragyan (2021) Unravelling the molecular mechanisms of histone code recognition by the BRD9 bromodomain. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Pragyan Dash (16MS196)) 16MS196_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (7MB)

Abstract

Bromodomains (BRDs) are reader modules of epigenetic marks that facilitate gene transcription in our genome. The human genome encodes 46 diverse proteins that contain 61 BRDs. Structurebased classification has put them into eight families of human BRDs. The subfamily IV of BRDs is not extensively studied for binding to acetylated proteins, although their involvement in disease has been described. BRD-containing proteins can regulate gene expression by working as transcription factors or co-regulators, scaffolds and can be a part of a larger protein complex. BRD9, a subunit of SWI/SNF chromatin remodelling complexes, plays a critical role in tumor development. Its increased gene copy number and overexpression are associated with papillary thyroid carcinoma, cervical cancer, and it is a crucial regulator in Acute Myeloid Leukemia. The exact biological function of BRD9 is not discovered. To unravel the molecular mechanisms of action, we need to find the interacting histone partners of BRD9 and verify them experimentally. In this study, we used a combined approach of in silico and in vitro studies to determine the binding of BRD9 with modified histone peptides. First, molecular docking performs virtual screening of various acetylated histone peptides that shows a binding affinity for BRD9. Further, molecular dynamics simulations establish more reliable bromodomain-acetylated histone peptide complex interactions. Finally, fluorescence polarization and pull-down assays are performed from the available peptide library, that gave an idea about the affinity of the wild-type BRD9 to the histone peptides. Interestingly, we found that BRD9 can recognize H4K5ac, H4K8ac, H4K12ac, and H4Ktet-ac with higher affinity than the other histone modifications. This study provides us with novel insight that will help unravel the molecular mechanism of binding of BRD9 to chromatin, recruitment of transcriptional factors for gene expression, and unveil the exact biological function of BRD9 in humans. It will help open a scientific path for further research on the role of BRD9 in a disease-specific context.

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