Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

Abstract

Lysine acetyltransferase 6A (KAT6A) and its paralog KAT6B form stoichiometric complexes with bromodomain- and PHD finger-containing protein 1 (BRPF1) for acetylation of histone H3 at lysine 23 (H3K23). We report that these complexes also catalyze H3K23 propionylation in vitro and in vivo. Immunofluorescence microscopy and ATAC-See revealed the association of this modification with active chromatin. Brpf1 deletion obliterates the acylation in mouse embryos and fibroblasts. Moreover, we identify BRPF1 variants in 12 previously unidentified cases of syndromic intellectual disability and demonstrate that these cases and known BRPF1 variants impair H3K23 propionylation. Cardiac anomalies are present in a subset of the cases. H3K23 acylation is also impaired by cancer-derived somatic BRPF1 mutations. Valproate, vorinostat, propionate and butyrate promote H3K23 acylation. These results reveal the dual functionality of BRPF1-KAT6 complexes, shed light on mechanisms underlying related developmental disorders and various cancers, and suggest mutation-based therapy for medical conditions with deficient histone acylation.

Fig. 1. BRPF1-KAT6 complexes catalyze H3K23 propionylation in vitro.

(A) Molecular architecture of the tetrameric complexes. BRPF1 has two EPC (enhancer of polycomb)–like motifs: EPC-I is required for association with the MYST domain of KAT6A or KAT6B, whereas EPC-II is necessary and sufficient for interaction with ING5 (or the paralog ING4) and MEAF6. The BRPF-specific N-terminal (BN) domain also contributes to the association with the MYST domain. BRPF1 contains the PZP domain, bromodomain, and PWWP domain for chromatin association. Unlike its paralogs BRPF2 and BRPF3, BRPF1 has an Sfp1-like C2H2 zinc finger (SZ). NLS, nuclear localization signal; H1-like, histone H1–like domain; PZP, PHD–zinc knuckle–PHD; bromo, bromodomain; PWWP, Pro-Trp-Trp-Pro containing domain; SM, serine/methionine-rich (8, 23). (B) BRPF1 promotes H3K23 propionylation. KAT6A was expressed in HEK293 cells as a FLAG-tagged fusion protein along with HA-tagged BRPF1, ING5, and MEAF6 as indicated. Affinity-purified proteins were used for acylation of HeLa oligonucleosomes in the presence of the respective acyl-CoA. Immunoblotting with antibodies recognizing histone H3 and its acylated forms was used to detect acylation states as indicated. See fig. S2A for immunoblotting analysis of the soluble extracts. Signals detected by the anti-H3K23cr and anti-H3K23bu antibodies need to be interpreted with caution due to cross-reactivity to H3K23ac and/or H3K23pr (see fig. S1B). (C) The H3K23 propionyltransferase activity is intrinsic to the MYST domain of KAT6A. Complex preparation and assays were performed as in (B) to compare KAT6A with its mutants. Recombinant mononucleosomes were used as substrate. See fig. S2B for immunoblotting analysis of the soluble extracts. Asterisks in (B) and (C) denote degraded products; the degradation varies from experiment to experiment. (D) Same as in (B) but ING4 and ING5 were compared. See fig. S2C for immunoblotting analysis of the soluble extracts. (E) Comparison of KAT6B fragments. Complex preparation and assays were performed as in (B) with the recombinant mononucleosome substrate, but KAT6B fragments were analyzed. Full-length KAT6B was difficult to express (21).

Fig. 2. Brpf1 inactivation impairs histone H3K23 acylation in mouse fibroblasts and embryos.

(A) Immunoblotting to detect histone H3 acylation in extracts from control and Brpf1^−/− MEFs. The fibroblasts were prepared from control and tamoxifen-inducible Brpf1 knockout embryos at E15.5 (39). (B) Immunoblotting to detect histone H3 acylation in extracts from wild-type and Brpf1^−/− embryos at E10.5. (C) Immunofluorescence microscopic analysis of histone H3 propionylation in control and Brpf1^−/− MEFs (E13.5). Scale bar, 20 μm. (D) Immunoblotting analysis to detect histone H3 acetylation and propionylation in extracts from control and Brpf1^−/− MEFs (E13.5) cultured in the MEF medium supplemented with or without 10 mM sodium propionate for 24 hours. (E) Histone H3 acylation in extracts from control and Kat6a^−/− MEFs. The fibroblasts were prepared from control and Kat6a^−/− embryos at E13.5. (F) Histone H3 acylation in extracts from wild-type and Kat6a^−/− embryos at E13.5. (G) Association of H3K23ac and H3K23pr with active chromatin. Soluble extracts from E13.5 wild-type (WT) and Brpf1^−/− MEFs (lanes 1 and 2) were used for immunoprecipitation (IP) with control immunoglobulin G (IgG) (lanes 3 and 4), anti-H3K23ac antibody (lanes 5 and 6), or anti-H3K23pr antibody (lane 7). Immunoblotting was carried out with the antibodies specific to the histone marks indicated at the right. (H and I) Active chromatin of E13.5 wild-type MEFs was labeled with ATAC-See before immunofluorescence microscopy with the anti-H3K23ac (H) or anti-H3K23pr (I) antibody. Scale bars, 20 μm.

Fig. 3. Clinical characteristics of newly identified individuals with BRPF1 variants.

(A) Schematic representation of variants identified in 12 newly identified individuals with syndromic intellectual disability. See Fig. 1A for domain nomenclature. (B) Facial features of seven individuals with heterozygous BRPF1 mutations. The ages when the photos were taken are indicated. See fig. S4C for additional photos. (C) Hand and foot photos of individuals P4 and P5. (D) Brain MRI images of individual P1. (E) Echocardiographic image of dilated ascending aorta in a previously reported case (12). Photo credit: The authors confirm that permissions were received to use all images, but to protect patient identity, photographer names remain anonymous.

Fig. 4. Functional impact of BRPF1 variants associated with neurodevelopmental disorders.

(A) Cartoon representation of variants previously identified in 29 individuals with syndromic intellectual disability (, –36) or autism (43). See Fig. 1A for domain nomenclature. (B) Nucleosomal acylation assays. HeLa oligonucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes. The complexes were prepared for assays as in Fig. 1B. See fig. S2D for immunoblotting analysis of the soluble extracts. (C) Nucleosomal histone acylation assays. Recombinant mononucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes. (D) Assays were carried out similarly as in (C), and the MYST domain of KAT6A was used. See fig. S2E for immunoblotting analysis of the soluble extracts. (E) Immunoblotting to detect histone H3 acylation in extracts from control and the Pro370Ser LCLs. (F) Immunoblotting to detect histone H3 acylation in protein extracts from the control and Arg455* LCLs. (G) Immunoblotting to detect histone H3 acylation in extracts from the control and Arg455* fibroblasts. (H) Assays were carried out similarly as in (C), but the Tyr406His variant was analyzed. See fig. S2F for immunoblotting analysis of the soluble extracts.

Fig. 5. Functional impact of somatic BRPF1 mutations from cancer.

(A) Cartoon representation of somatic mutants identified in cancer. While five mutations are missense and produce Pro20Leu, Glu253Gly, Leu298Pro, Trp348Arg, and Glu369Asp, the rest are nonsense or cause reading frameshift, resulting in C-terminal truncation. According to the published report on other truncation mutations of BRPF1 (12), these nonsense or frameshift mutations may not trigger nonsense-mediated mRNA decay. See Fig. 1A for domain nomenclature. (B) Sequence similarity among the C2H2 zinc fingers of BRPF1, the fly ortholog Br140, the yeast stress- and nutrient-sensing transcription factor Sfp1, the fly Sfp1-like protein CG12054, and the related human transcription factors JAZF1 and ZF609. The SZ of BRPF1 is similar to the first of two zinc fingers that Sfp1 has for DNA binding and homologous to the middle of three zinc fingers that JAZF1 uses to recognize DNA. The three key residues involved in known and potential DNA binding are highlighted in bold. (C) Nucleosomal acylation assays. Recombinant mononucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes as in Fig. 1C. Acylation of histone H3 was detected with antibodies recognizing histone H3 and its acylated forms as indicated. See fig. S2G (lanes 1 to 3) for immunoblotting analysis of the soluble extracts. (D) Subcellular localization of wild-type BRPF1 and its N-terminal mutants 1-51 and 1-71. Mutant 1-51 contains the SZ, and mutant 1-71 has this finger along with adjacent NLS1. Expression plasmids for BRPF1 or its mutants were transiently expressed in HEK293 cells as green fluorescent protein (GFP) fusion proteins with or without coexpression of FLAG- or HA-tagged KAT6A, ING5, and MEAF6 as indicated. Live green fluorescence images were taken. Scale bar, 50 μm. (E and F) Same as (C) except different variants were compared to wild-type BRPF1. See fig. S2G (lane 4) for immunoblotting analysis of extracts from HEK293 cells expressing Glu253Gly.

Fig. 6. Manipulation of histone H3K23 acylation with various compounds.

(A) Up-regulated histone H3K23 acylation in HEK293 cells treated with the indicated HDAC inhibitor valproate (VPA), vorinostat (SAHA), β-hydroxybutyrate (β-HB), TSA, or sodium butyrate (SB) for 24 hours. DMSO, dimethyl sulfoxide. (B) Rescue of histone H3K23 acylation deficiency in Brpf1^−/− MEFs by HDAC inhibitors. (C) Rescue of histone H3K23 acylation deficiency in patient-derived LCLs by HDAC inhibitors. For (A) to (C) and (E), results with the anti-H3K23cr and anti-H3K23bu antibodies need to be interpreted with caution due to cross-reactivity with H3K23ac and/or H3K23pr (fig. S1B). (D) Effect of an HDAC3-specific inhibitor. The indicated cells were treated with the inhibitor RGFP966 for 15 hours. (E) Rescue of histone H3K23 acylation deficiency in patient-derived LCLs by propionate. Control and patient-derived LCLs were cultured in a medium supplemented with or without 10 mM sodium propionate for 24 hours. For immunoblotting analysis with the anti-H3K23pr antibody, a long exposure (long e.) was also shown (panel 4 from the top). (F) Model explaining how BRPF1 acts through KAT6A and KAT6B for controlling histone H3K23 acylation and regulating developmental and pathological processes. HDAC inhibitors such as VPA, SAHA, TSA, and butyrate up-regulate acylation. These compounds offer therapeutic potential for the related diseases. Short-chain fatty acid (SCFAs) and other metabolites regulate acyl-CoA levels, thereby controlling H3K23 acylation. (G) Expanded version of the green box in (F) highlighting how various metabolites may differentially regulate acetylation and propionylation. For simplicity, only pyruvate and acetate are illustrated for events upstream from acetyl-CoA, a central player downstream from diverse metabolic pathways. On the basis of relative concentrations of acetyl-CoA and propionyl-CoA in vivo, we propose that acetylation plays a major role, whereas propionylation complements acetylation.