Mechanistic insights into DNA binding by BD1 of the TAF1 tandem bromodomain module.

Transcription initiation factor TFIID subunit 1 (TAF1) is a pivotal component of the TFIID complex, critical for RNA polymerase II-mediated transcription initiation. However, the molecular basis by which TAF1 recognizes and associates with chromatin remains incompletely understood. Here, we report that the tandem bromodomain module of TAF1 engages nucleosomal DNA through a distinct positively charged surface patch on the first bromodomain (BD1). Electrostatic potential mapping and molecular docking revealed a prominent basic region on BD1 that facilitates interaction with DNA, predominantly driven by hydrogen bonds and electrostatic forces, as supported by molecular dynamics simulations. Site-directed mutagenesis identified three key positively charged residues (R1435, K1436, and R1437) within the αA helix of BD1, constituting an "RKR" basic patch essential for DNA binding. Electrophoretic mobility shift assays demonstrated that the TAF1 tandem bromodomain binds DNA in a concentration-dependent manner with moderate preference for AT-rich sequences, attributed to this RKR motif. Importantly, DNA binding occurs independently of histone acetyllysine recognition by the bromodomains, as acetylated histone H4 peptides or mutations in the acetyllysine-binding pocket did not affect DNA interaction. Furthermore, nucleosome pulldown assays revealed that disruption of the BD1 RKR patch significantly reduces binding to acetylated nucleosomes, highlighting its role in facilitating chromatin engagement. Collectively, our findings establish the RKR basic patch on TAF1 BD1 as a critical determinant for DNA interaction, providing mechanistic insight into how TAF1 tandem bromodomains coordinate dual recognition of nucleosomal DNA and histone acetylation. These results offer a molecular basis for understanding how TAF1 may contribute to transcriptional regulation via chromatin engagement.