Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity

Abstract

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

Keywords: BRPF1; MYST family; PHD fingers; acetyltransferase complexes; chromatin acetylation; histone tails.

Figure 1.

Characterization of the two PHD fingers in the PZP domain of BRPF1. (A) Subunit organization of human MYST acetyltransferase complexes used in this study. The core subunits have a scaffold protein (JADE, BRPF, or EPC), an ING tumor suppressor protein (ING3, ING4, or ING5), and a catalytic enzyme protein (Tip60, HBO1, or MOZ/MORF). (B) Schematic representation of the conserved protein domains found in the scaffold subunits of MYST–ING HAT complexes (yeast and human). (C) The PHD1 finger of the PZP domain of BRPF1 recognizes unmethylated H3 in vitro, while the PHD2 finger shows interaction with H3 peptides independently of methylation status. Peptide pull-down assays with different biotinylated peptides and recombinant PHD fingers fused to GST were analyzed by Western blotting with anti-GST antibody (Western blott: α-GST). (D) Superimposed ¹H,¹⁵N heteronuclear single quantum coherence (HSQC) spectra of BRPF1 PHD1, collected as unmodified H3 peptide, was titrated in. The spectrum is color-coded according to the protein–peptide ratio. (E) Binding affinities of the BRPF1 PHD1 finger for histone H3 peptides with different K4 methylation statuses were measured by tryptophan fluorescence. Numbers in parentheses represent the amino acid positions of histone H3 included in the peptides. (F) Superimposed ¹H,¹⁵N HSQC spectra of BRPF1 PHD2 (collected as indicated) peptides or unlabeled BRPF1 PHD1 were added stepwise. The spectra are color-coded according to the protein–peptide ratio. (G) The PZP domain of BRPF1 binds to unmethylated histone H3K4 in vitro. Peptide pull-down assays with different biotinylated peptides and recombinant PZP domain fused to GST were analyzed by Western blot with α-GST antibody. The PHD1 dictates the specificity of the entire domain toward unmethylated H3K4. (H) Binding affinities of the PZP domain for the indicated H3 peptides were measured by tryptophan fluorescence. Numbers in parentheses represent the amino acid positions included in the peptides. (NB) No binding was detected.

Figure 2.

BRPF1 PHD1 and PHD2 fingers are required for binding to chromatin and its acetylation. (A) BRPF1 associates with histone H3 in vivo, and this binding is lost when the PHD2 finger is deleted. 293T cells were cotransfected with HA-BRPF1 (wild-type [WT] or ΔPHD2) plasmids, and Flag-MOZ (MYST domain), Flag-ING5, and Flag-hEaf6 plasmids and whole-cell extracts (WCEs) were used for HA immunoprecipitation (IP). As the MYST domain of the MOZ catalytic subunit is sufficient for HAT activity and complex assembly (Ullah et al. 2008), it was used to avoid the high degradation sensitivity of the full-length protein (225 kDa). Mock control was cotransfected with both HA empty and Flag empty plasmids. Histone H3 binding was analyzed by Western blot α-H3. (B) PHD2 is required for proper in vitro chromatin acetylation. HAT assays with the same purified complexes as in A were performed on chromatin or free histones. Reactions were spotted on membranes and counted by liquid scintillation. Values are based on three independent experiments with standard error. (C) Western blot on purified wild-type, ΔPHD1, and ΔPHD2 complexes in 293T cells showing equal expression of the different subunits. WCE was used after cotransfection of HA-BRPF1 plasmids (wild type, ΔPHD1, or ΔPHD2), and Flag-MOZ, Flag-ING5, and Flag-hEaf6 plasmids were used for HA immunoprecipitation. HA beads were eluted with HA peptide, and fractions were loaded on gel. (D,E) PHD1 is also important for acetylation of chromatin in vitro. HAT assays on free histones (D) or chromatin (E) using the complexes purified in C were spotted on membranes for liquid scintillation counting. Values are based on three independent experiments with standard error.

Figure 3.

BRPF1 localizes to H3K4me3-enriched regions through its association with ING5. (A,B) ING5 directs the binding of the entire MORF complex toward H3K4me3 in vitro. Complexes ± ING5 were purified from SF9 cells and used in HAT assays on diverse modified histone peptides. Values are based on two independent experiments with standard error. (C–E) ING5, ING2, BRPF1/2, and HBO1 colocalize with H3K4me3 near the TSSs of genes. Heat maps of each protein ChIP-seq signal on ±5 kb surrounding the TSSs of genes are shown. Genes were sorted by gene expression level (see the Materials and Methods) from high (top) to low (bottom), and the signals were corrected over reads per million (RPM). (C) Enrichment of H3K4me3 signal compared with H3 signal. ING5 and ING2 (D) and BRPF1/2 and HBO1 (E) are bound at H3K4me3-enriched regions near the TSSs of genes. (F,G) BRPF1/2 and ING5 colocalize at the p21/CDKN1A gene and the HOXA cluster. The different signals for each protein are shown to illustrate their binding at H3K4me3-enriched region near the p21/CDKN1A TSS (F) and each TSS of the HOXA cluster (G).

Figure 4.

The BRPF1 scaffold subunit copurifies with both MOZ/MORF and HBO1 catalytic subunits. (A) A transduced stable HeLa cell line expressing 3xFlag-BRPF1 was used to purify associated proteins by anti-Flag immunoprecipitation/elution. Immunopurified proteins were analyzed on gel by silver staining. Nonspecific contaminants are shown by the asterisk. (B) The purified complex in A was analyzed by Western blot with the indicated antibodies. The two bands appearing with the MORF antibody are both MOZ and MORF, as the MORF antibody recognizes both proteins (Ullah et al. 2008). (C) Western blot analysis of a tandem affinity purification (TAP) from a transduced HeLa cell line stably expressing HA-HBO1-TAP (protein A and calmodulin-binding protein). (C) BRPF1 complexes acetylate histone H3 on chromatin in vitro. HAT assays were performed on free histones or native chromatin with the purified BRPF1 fraction in A. Acetylated histones were separated by SDS-PAGE and revealed by fluorography. (E–G) BRPF1 complexes acetylate histone H3 on chromatin in vivo. ChIP analysis of H3K14/K23 and H4 acetylation in transduced HeLa cells stably expressing BRPF1. Acetylation levels corrected for nucleosome occupancy (total H3 signal) were measured at the p21 TSS, 2 kb downstream, and at an intergenic locus. BRPF1 significantly increases H3K14ac and H3K23ac at the p21 TSS while showing no effect at the intergenic locus. (H) HBO1 occupancy on p21 gene does not change upon increased BRPF1 expression. ChIP analysis of HBO1 in BRPF1 and control transduced cell lines. All ChIP values are based on two independent experiments with standard error.

Figure 5.

Association of different scaffold subunits with the HBO1 HAT is responsible for switching its histone acetylation specificity between H4 and H3. (A) Purification of the MOZ–BRPF1, HBO1–BRPF1, and HBO1–JADE1L complexes. 293T cells were transfected with flagged scaffold subunit plasmids (BRPF1 and JADE1L), while other cotransfected subunits were HA-tagged. Mock control was cotransfected with Flag empty and HA empty plasmids. Complexes were purified from WCEs by Flag immunoprecipitation (IP) and eluted with 3xFlag peptides. (B) The scaffold subunit changes chromatin acetylation specificity. The purified complexes in A were used in HAT assays on both chromatin and free histones. Acetylated histones were separated by SDS-PAGE and revealed by fluorography. Coomassie-stained gels show equivalent amounts of histones between samples. (C) The HBO1–BRPF1 complex acetylates H3K14 and H3K23. HAT assays with the purified HBO1–BRPF1 complex were performed on chromatin followed by Western blot analysis of different histone marks. (D) Purification of various deletions of Brpf1. Cotransfected 293T cells with either wild-type (WT), ΔBromo, or ΔPWWP Flag-BRPF1 plasmids combined with HA-HBO1, HA-ING5, and HA-hEaf6 plasmids were used for Flag immunoprecipitation purification, and complexes were analyzed by Western blot with the indicated antibodies. (E) The PWWP domain and bromodomain of BRPF1 are not essential for acetylation specificity. The purified complexes in D were used in HAT assays on both chromatin and free histones. The amount of complex used for HAT assays was normalized to free histone activity. Acetylated histones were separated by SDS-PAGE and revealed by fluorography.

Figure 6.

A small N-terminal domain in the scaffold subunits of the MYST–ING complexes is responsible for directing specific histone tail acetylation. (A) Sequence alignment of scaffold subunits of the MYST–ING complexes with the N-terminal region of the EPcA domain found in the EPC proteins (EPC1/2 and Epl1). An arrow indicates the location of the N-terminal truncation in BRPF1 and JADE1 mutants. Domain I is the region of association with the MYST HAT. (B) Deletion of the N-terminal part of scaffold subunits BRPF1 and JADE1L alters chromatin acetylation specificity. 293T cells were cotransfected with the indicated expression plasmids, HA-tagged catalytic subunits, and Flag-tagged scaffold subunits. HA-ING5 and HA-hEAF6 expression plasmids were also cotransfected for each purification. Flag immunoprecipitations (IPs) were performed on WCE and were eluted with 3xFlag peptides. Purified complexes were used in HAT assays, and acetylated histones were separated by SDS-PAGE and revealed by fluorography. The complexes were normalized to the same HAT activity on free histones (by liquid assays). (C) Deletion of the N-terminal part of scaffold subunit EPC1 also modifies chromatin acetylation specificity. EPC1 wild-type and Δ1–12 complexes were purified and used for HAT assays by coexpressing Flag-EPC1, HA-ING3, HA-hEaf6, and HA-Tip60. (D) The N-terminal domain of the yeast homolog Epl1 acts similarly in directing histone specificity. Recombinant yeast piccolo NuA4 complexes with wild-type or Δ1–71 Epl1 were used for HAT assays on yeast chromatin, and acetylated histones were treated as in A.

Figure 7.

Model for MYST acetyltransferase assembly in alternate complexes, leading to different histone tail specificities. (A) Schematic representation of protein domains in BRPF paralogs and their demonstrated specific interactions/roles. (B) The HBO1 and MOZ/MORF catalytic subunits can be associated with different scaffold proteins, leading to a switch in histone tail specificity for acetylation of chromatin substrates. Thus, protein complexes associated with HAT proteins not only enable them to acetylate chromatin substrates, but also select which histone tail is targeted, a specificity previously thought to reside in the acetyltransferase itself. The arrow between MOZ/MORF and JADE1/2/3 is gray, since this interaction has only been reported in cotransfection experiments.