Deciphering the Chromatin Regulatory Functions of TRIM24 PHD-Bromodomain

Bardhan, Ishita (2025) Deciphering the Chromatin Regulatory Functions of TRIM24 PHD-Bromodomain. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Ishita Bardhan (19RS088)) 19RS088.pdf - Submitted Version Restricted to Repository staff only Download (20MB)

Abstract

TRIM24 is a multi-functional chromatin reader that plays a key role in transcription regulation and is often aberrantly expressed in various cancers. Initially identified as a nuclear receptor modulator, TRIM24’s expression correlates with poor prognosis in breast cancer, particularly through its association with the acetylation of histone H3 at lysine 23 (H3K23ac). However, the role of TRIM24 in recognizing acetylated histone H4 (H4ac) remains underexplored. Herein, we report novel H4ac binding partners (H4K5ac, H4K8ac, and H4K5acK8ac) of TRIM24 using an Isothermal titration calorimetry binding assay. Moreover, ChIP-seq analysis revealed that the H4K5ac and H4K8ac histone signatures strongly co-localize near the transcription start sites of different hub genes in breast cancer. In addition, the KEGG pathway analysis demonstrates that the TRIM24 and its H4ac targets are associated with several important biological pathways. Our findings describe that the H4ac recognition by TRIM24 PHD-Bromodomain enables access to the chromatin for specific transcriptional regulation. Next, to explore the broader interactome of TRIM24, we employed photo-crosslinking-based interactome profiling (PBIP), a chemoproteomic approach capable of identifying transient and dynamic acetylated non-histone proteins. Using amber suppressor mutagenesis, we successfully incorporated photo-crosslinkable unnatural amino acids, 4-azido-L-phenylalanine (AzF) and 4- benzoyl-L-phenylalanine (BzF) into the TRIM24 bromodomain. The TRIM24-AzF mutant exhibited enhanced crosslinking efficiency, surpassing the TRIM24-BzF variant in its ability to capture the cellular proteome. We found several novel nonhistone interactomes of TRIM24, including MAP4, NPM1, CDK9, RAN, and SND1 proteins involved in various cellular processes beyond transcriptional regulation. Finally, using high-throughput virtual screening, we identified potential small molecule inhibitors targeting the TRIM24 bromodomain. These findings open new avenues for targeting TRIM24 in the treatment of human malignancies.

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