The BRPF1 bromodomain is a molecular reader of di-acetyllysine

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex, which is associated with chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of chromatin reader domains including two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) domain. BRPF1 is known to recruit the MOZ HAT complex to chromatin by recognizing acetylated lysine residues on the N-terminal histone tail region through its bromodomain. However, histone proteins can contain several acetylation modifications on their N-terminus, and it is unknown how additional marks influence bromodomain recruitment to chromatin. Here, we identify the BRPF1 bromodomain as a selective reader of di-acetyllysine modifications on histone H4. We used ITC assays to characterize the binding of di-acetylated histone ligands to the BRPF1 bromodomain and found that the domain binds preferentially to histone peptides H4K5acK8ac and H4K5acK12ac. Analytical ultracentrifugation (AUC) experiments revealed that the monomeric state of the BRPF1 bromodomain coordinates di-acetylated histone ligands. NMR chemical shift perturbation studies, along with binding and mutational analyses, revealed non-canonical regions of the bromodomain-binding pocket that are important for histone tail recognition. Together, our findings provide critical information on how the combinatorial action of post-translational modifications can modulate BRPF1 bromodomain binding and specificity.</jats:p>

Insights into the Molecular Mechanisms of Histone Code Recognition by the BRPF3 Bromodomain

“…Therefore, to get information on the protein surface properties, we calculated the electrostatic surface potential of BRPF1/2/3 bromodomains using APBS (Adaptive Poisson‐Boltzmann Solver) software [25] . We found that the surface area around the Kac‐binding site of the BRPF1 bromodomain is highly negatively charged compared to the BRPF2/3 bromodomains (Figure 1B and Figure S3), and these results are in accord with its histone ligands reported recently [21,26] . Notably, BRPF2/3 bromodomains show very similar electrostatic surface potentials, suggesting that the BRPF2/3 bromodomains possibly recognize a similar set of histone sequences.…”

“…More recent studies have shown that the BRPF1 bromodomain recognizes the histone H2AK5ac and H3K14ac modifications with moderate affinity [21,26] We also tested the binding affinity of BRPF3 bromodomain with H2AK5ac (1–12) and H3K14ac (9–19) peptides. Our ITC binding results revealed that the BRPF3 bromodomain binds to H2AK5ac (1–12) with a dissociation constant of (K D =85.4±9.2) (Figure S7A, Table 1), but failed to recognize the H3K14ac (9–19) modification (Table 1).…”

“…Our ITC binding studies revealed that the BRPF3 bromodomain demonstrated a very strong interaction with the acetylated histone peptides H4K5ac and H4K5acK12ac. More recently, it has been discovered that the family IV bromodomain‐containing proteins such as BRD9, BRPF1, BRPF2, and ATAD2B can bind to histone H4 peptides with di‐acetylation marks [21,23,33] . Specifically, the BRPF1 and ATAD2B bromodomains binds to H4K5acK8ac (1–10) peptide with a similar affinity (K D =27.7±5.2 μM versus 28.1±1.6 μM), but the ATAD2B binds to H4K5acK12ac (1–15) peptide with a higher affinity about 2.5‐fold stronger than the BRPF1 bromodomain (K D =18.7±0.9 μM versus 49.7±6.5 μM) [21,33] .…”

“…More recently, it has been discovered that the family IV bromodomain‐containing proteins such as BRD9, BRPF1, BRPF2, and ATAD2B can bind to histone H4 peptides with di‐acetylation marks [21,23,33] . Specifically, the BRPF1 and ATAD2B bromodomains binds to H4K5acK8ac (1–10) peptide with a similar affinity (K D =27.7±5.2 μM versus 28.1±1.6 μM), but the ATAD2B binds to H4K5acK12ac (1–15) peptide with a higher affinity about 2.5‐fold stronger than the BRPF1 bromodomain (K D =18.7±0.9 μM versus 49.7±6.5 μM) [21,33] . These results suggest that the family IV bromodomains prefer a similar set of histone modifications present on the N‐terminal tails of histone H4 with varying degrees of binding affinity.…”

“…Sequence similarity between the BRPF family bromodomains suggests that they may recognize a similar subset of acetylated histone modifications. Recent reports have shown that the bromodomain and PWWP domain of BRPF1 specifically bind to acetylated histone (H2A, H3, and H4) and methylated H3, respectively [21] . Two PHD fingers of BRPF1 and BRPF2 are connected by a zinc knuckle and form a bivalent domain that interacts with unmodified H3 and DNA [22] .…”