Molecular Insights into the Recognition of Acetylated Histone Modifications by the BRPF2 Bromodomain

Abstract

HBO1 (HAT bound to ORC), a member of the MYST family of histone acetyltransferases (HATs), was initially identified as a binding partner of the origin recognition complex (ORC) that acetylates free histone H3, H4, and nucleosomal H3. It functions as a quaternary complex with the BRPF (BRPF1/2/3) scaffolding protein and two accessory proteins, ING4/5 and Eaf6. BRPF2 interaction with HBO1 has been shown to be important for regulating H3K14 acetylation during embryonic development. However, how the BRPF2 directs the HBO1 HAT complex to chromatin to regulate its HAT activity towards nucleosomal substrates remains unclear. Our findings reveal novel interacting partners of the BRPF2 bromodomain that recognizes different acetyllysine residues on the N-terminus of histone H4, H3, and H2A and preferentially binds to H4K5ac, H4K8ac, and H4K5acK12ac modifications. Moreover, pull-down assays on the endogenous histones and mononucleosomes isolated from human cells confirmed that the BRPF2 bromodomain specifically showed a stronger affinity to H4K5ac marks. Further, mutational analysis of BRPF2 bromodomain coupled with ITC binding and pull-down assays on the histone substrates identified critical residues responsible for acetyllysine binding. Together, our study provides novel insights into how the histone binding function of the BRPF2 bromodomain directs the recruitment of the HBO1 HAT complex to chromatin to regulate gene expression.

Uncovering the Domain-Specific Interactome of the TAF1 Tandem Reader Using Site-Specific Azide-Acetyllysine Photochemistry

“…Molecular docking was performed by following the method for docking the bromodomain with the acetylated histone peptide as previously described. ,, Briefly, the crystal structure of the double-bromodomain module of TAF1 [Protein Data Bank (PDB) entry 1EQF] was obtained from the Protein Data Bank (RCSB). For docking studies, the receptor structure was prepared by adding the polar hydrogen atoms using PyMOL.…”

“…210519). Protein expression and purification were performed using methods described previously. ,,, The C-terminally His 6 -tagged TAF1 tandem-bromodomain plasmid was transformed into One Shot BL21 star (DE3) Escherichia coli competent cells (Invitrogen, catalog no. C601003) using the pNIC28-Bsa4 kanamycin-resistant vector.…”

“…The CD experiment was performed by following the method described previously. , Briefly, the CD spectra were recorded on a JASCO J-1500 CD spectropolarimeter at 20 °C using a quartz cell with a path length of 10 mm. Two scans were accumulated at a scan speed of 100 nm min –1 , with data being collected every nanometer from 195 to 260 nm.…”

“…The computational modeling and molecular dynamics (MD) simulation were performed as previously described with slight modifications. 20,29,30 Briefly, the crystal structure of the doublebromodomain module of TAF1 was obtained from PDB entry 1EQF to model the bromodomain with a photo-cross-linking amino acid AzF (1). The targeted amino acids of the bromodomain were replaced with AzF 1 using the PyMOL builder and the MMFF94 structural optimization algorithm for initial structure approximation.…”

Effect of chlorogenic acid plus donepezil on critical neurocortical enzyme activities, inflammatory markers, and synaptophysin immunoreactivity in scopolamine‐assaulted rats, supported by multiple ligand simultaneous docking

“…The protein structures were obtained from RCSB protein data bank and edited by adding non‐polar hydrogen atoms, while water molecules and hetero atoms were removed (Barman et al, 2022; Vasanthkumar et al, 2019). This was followed by Gasteiger charges calculation using Autodock tools (Vasanthkumar et al, 2019).…”