Unraveling the Role of Bromodomain and PHD finger (BRPF) Proteins in Epigenetic Regulation of Hepatocellular Carcinoma

Barman, Soumen (2024) Unraveling the Role of Bromodomain and PHD finger (BRPF) Proteins in Epigenetic Regulation of Hepatocellular Carcinoma. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Soumen Barman (18RS093)) 18RS093.pdf - Submitted Version Restricted to Repository staff only Download (21MB)

Abstract

The bromodomain and PHD finger (BRPF) family proteins (BRPF1/2/3) are multivalent epigenetic regulators containing multiple chromatin reading modules, including three histone-binding modules and one nonspecific DNA-binding module. BRPF family proteins stimulate and modulate the enzymatic activity of histone acetyltransferases (HATs) and promote acetylation of histone lysine residues during transcription. In this thesis, we have explored the role of BRPF1/2 family proteins in regulating gene expression and disease development. The biological functions of acetylated histone recognition by BRPF1/3 bromodomains are well characterized; however, the function of BRPF2 regulation via histone acetylation is still unexplored. Herein, we utilized a series of computational, biophysical, and biochemical methods to explore acetyllysine recognition by the BRPF2 bromodomain. Our findings reveal novel interacting partners of the BRPF2 bromodomain that recognizes different acetyllysine residues on the N-terminal tails of histone H4, H3, and H2A and preferentially binds to H4K5ac, H4K8ac, and H4K12ac modifications. Further, the ChIP-seq data analysis revealed that the BRPF2 strongly co-localizes with different acetylated histone H4 marks near the transcription start sites in the genome. Similarly, to histones, transcription factors (TFs) can be functionally acetylated and recognized by bromodomains. We site-specifically introduced the unnatural photo-cross-linkable amino acid 4-azido-L-phenylalanine (AzF) into the bromodomain of BRPF1, which allows cross-linking of its interactome with spatiotemporal precision. We then characterized the novel interacting partners by proteomics. BRPF1 interaction with interleukin enhancer binding factor 3 (ILF3), one of these novel interacting partners, was further validated by isothermal titration calorimetry (ITC) and co-IP. We used publicly available ChIP-seq and RNA-seq datasets to understand the colocalization of BRPF1 and ILF3 in regulating gene expression in the context of hepatocellular carcinoma (HCC). In addition, to understand the role of BRPF1 in HCC, we checked its expression using TCGA. Our analysis identified BRPF1 as the most highly upregulated gene in HCC among the other 43 bromodomain-containing genes. Upregulation of BRPF1 was significantly associated with poorer patient survival. Therefore, targeting BRPF1 may be an approach for HCC treatment. We selected the top four compounds against the BRPF1 bromodomain using virtual screening, MDS, and QSAR modeling. This study discovered some promising compounds that have the potential to act as potent BRPF1 bromodomain inhibitors.

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