Decoding the Functional Roles of the TAF1 Tandem Bromodomain Module in Transcriptional Regulation

Yadav, Yogita (2025) Decoding the Functional Roles of the TAF1 Tandem Bromodomain Module in Transcriptional Regulation. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Yogita Yadav (18IP013)) 18IP013.pdf - Submitted Version Restricted to Repository staff only Download (26MB)

Abstract

Transcription initiation factor TFIID subunit 1 (TAF1) is a critical component of the TFIID complex and plays a central role in RNA polymerase II-mediated transcription initiation. Central to its function is the TAF1 tandem bromodomain module (BD1 and BD2), which recognizes acetylated lysine residues on histone tails. However, the mechanisms by which TAF1 engages chromatin, as well as its non-histone interaction landscape, remain incompletely understood. Here, we integrate structural, biochemical, and proteomic approaches to comprehensively define the DNA- and ligand-binding properties of the TAF1 tandem bromodomain. Electrostatic surface potential calculations and molecular docking revealed a positively charged region on BD1 that mediates electrostatic and hydrogen bonding interactions with nucleosomal DNA, particularly ATrich sequences. Mutational analysis identified key residues (R1435, K1436, R1437) essential for this DNA binding, while EMSAs and nucleosome pull-down assays confirmed that BD1-mediated DNA recognition is compatible with acetyl-lysine binding and contributes to nucleosome engagement. To dissect the ligand specificity and interactome of TAF1 bromodomains, we employed amber suppressor mutagenesis to incorporate the photoactivatable amino acid 4-azido- L-phenylalanine (AzF) at selected positions in BD1 (Y1403, L1466) and BD2 (W1526, Y1589). These engineered constructs retained structural integrity and ligand-binding capacity, as verified by circular dichroism, thermal shift assays, and isothermal titration calorimetry. Domain-specific photocrosslinking and mass spectrometry revealed that BD1 and BD2 independently bind distinct subsets of acetylated histone and non-histone proteins. Notably, BD2 selectively enriched the chromatin adaptor RBBP4, a component of the PRC2 and NuRD complexes, linking TAF1 to oncogenic transcriptional programs. ChIP-seq analysis demonstrated strong colocalization of TAF1 and RBBP4 at transcription start sites of cancer-related genes, and KEGG pathway analysis implicated the TAF1-RBBP4 axis in chromatin remodeling, neurodegeneration, and mitophagy. Collectively, our findings uncover the molecular basis for TAF1's dual recognition of acetylated histones and DNA, establish the independent functionality of its tandem bromodomains, and expand the TAF1 interactome to include non-histone substrates with key regulatory roles. These insights position TAF1 as a promising therapeutic target in cancers such as glioblastoma.

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