Bromodomains are evolutionarily conserved reader modules that recognize acetylated lysine residues on the histone tails to facilitate gene transcription. The bromodomain and PHD finger containing protein 3 (BRPF3) is a scaffolding protein that forms a tetrameric complex with HBO1 histone acetyltransferase (HAT) and two other subunits, which is known to regulate the HAT activity and substrate specificity. However, its molecular mechanism, histone ligands, and biological functions remain unknown. Herein, we identify mono‐ (H4K5ac) and di‐ (H4K5acK12ac) acetylated histone peptides as novel interacting partners of the BRPF3 bromodomain. Consistent with this, pull‐down assays on purified histones from human cells confirm the interaction of BRPF3 bromodomain with acetylated histone H4. Further, MD simulation studies highlight the binding mode of acetyllysine (Kac) and the stability of bromodomain‐histone peptide complexes. Collectively, our findings provide a key insight into how histone targets of the BRPF3 bromodomain direct the recruitment of HBO1 complex to chromatin for downstream transcriptional regulation.
The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...
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.
Combinatorial readout of histone
post-translational modifications
by tandem reader modules mediates crosstalk among different histone
modifications. To identify the domain-specific interactome of the
tandem reader, we engineered the dual bromodomain of TATA-binding
protein-associated factor-1 (TAF1) to carry a photoactivatable unnatural
amino acid, 4-azido-l-phenylalanine (AzF), via amber suppressor
mutagenesis. Using computational approaches, we modeled the targeted
residues of TAF1 with AzF to predict the cross-linking distance between
the reactive arylazide and its interacting partner. We developed three
photoactivatable TAF1 tandem-bromodomain analogues, viz., Y1403AzF
in bromodomain 1 (BD1), W1526AzF in bromodomain 2 (BD2), and Y1403AzF/W1526AzF in both BD1 and BD2. Circular dichroism and a thermal shift assay were used
to confirm the structural integrity of the engineered readers. Using
the TAF1 tandem-bromodomain analogues, we characterized their histone
ligand binding properties by isothermal titration calorimetry and
photo-cross-linking experiments. We found that the dual bromodomain
of TAF1 independently binds and cross-links to different acetylated
histone ligands. We further used the engineered BD1 and BD2 analogues of the TAF1 tandem readers to identify their
domain-specific interacting partners at the cellular level. Both BD1 and BD2 independently cross-link to a unique
interactome, and importantly, the dual cross-linker carrying TAF1
analogue could capture both BD1- and BD2-specific interactomes. Our work concludes that BD1 and BD2 of the TAF1 tandem reader independently recognize their
interacting partners to regulate downstream cellular functions.
To address the obstacle in insulin protein homeostasis leading to the formation of neurotoxic amyloid plaques associated with different diseases, herein we synthesized block copolymers using the reversible addition-fragmentation chain...
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