ABSTRACT
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 di-acetylated histone ligands bound 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 analysis, 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.
HIGHLIGHTS
Histone post-translational modifications recruit bromodomain-containing proteins such as BRPF1 to chromatin, but it is unknown how multiple modifications modulate acetyllysine binding.
The BRPF1 bromodomain preferentially binds to di-acetyllysine modifications on the histone H4 tail.
Di-acetyllysine recognition is coordinated by functional monomers of the BRPF1 bromodomain using amino acids near the canonical bromodomain binding pocket.
Adjacent phosphorylation and methylation modifications on the histone tail inhibit the BRPF1 bromodomain interaction with acetyllysine.
Cross-talk between multiple modifications on the histone tails is likely an important mechanism for regulating bromodomain interactions with chromatin.
ABBREVIATIONS
- AML
- acute myeloid leukemia
- AUC
- analytical ultracentrifugation
- BRPF1
- bromodomain-PHD finger protein 1
- CD
- circular dichroism
- DTT
- dithiothreitol
- HAT
- histone acetyltransferase
- MEAF6
- MYST/Esa1-associated factor 6
- ING5
- inhibitor of growth 5
- ITC
- isothermal titration calorimetry
- MOZ
- monocytic leukemic zinc-finger
- NMR
- nuclear magnetic resonance
- PCR
- polymerase chain reaction
- PHD
- plant homeodomain
- PTMs
- post-translational modifications
- PWWP
- proline-tryptophan-tryptophan-proline
- SV
- sedimentation velocity
- TCEP
- tris(2-carboxyethyl)phosphine).