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.

As for pathological relevance, KAT6A was identified in 1996 as a gene rearranged in leukemia ( 8 , 23 ). KAT6B was then shown to be similarly rearranged in leukemia ( 8 , 23 ). In 2012 and 2015, they were reported to be mutated in individuals with intellectual disability and neurodevelopmental disorders ( 24 – 30 ). A recent study analyzed 76 individuals with KAT6A variants ( 31 ), and at least 210 persons with these variants have enrolled in the KAT6A Foundation. Known cases with KAT6B variants have also exceeded 60 ( 32 ). We and others have just identified BRPF1 variants in 28 individuals with syndromic intellectual disability ( 12 , 33 – 36 ). The variants cause deficient H3K23 acetylation ( 12 , 33 ). This site is also propionylated ( 3 ), but the enzymes are elusive. Here, we identify BRPF1-KAT6 complexes as the propionyltransferases in vitro and in vivo, demonstrate H3K23 propionylation deficiency resulting from germline BRPF1 variants in individuals with neurodevelopmental disorders or from somatic mutations in different types of cancer, and explore therapeutic strategies with histone deacetylase (HDAC) inhibitors and propionic acid.

( 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 ).

Lysine acetyltransferase 6A (KAT6A) and KAT6B are paralogous acetyltransferases of the MYST (Moz-, Ybf2/Sas3-, Sas2-, and Tip60-like domain) family, with similar molecular activities in vitro but distinct functions in vivo ( 8 ). The other three members are KAT5, KAT7, and KAT8 ( 8 ). Although they have conserved MYST domains for acetyltransferase activity ( 8 , 9 ), these five enzymes show site specificity in vivo. KAT6A and KAT6B govern acetylation of histone H3 at lysine 23 (H3K23) ( 10 – 12 ), whereas KAT7 is the major enzyme for H3K14 acetylation ( 13 – 15 ) and KAT8 is responsible for H4K16 acetylation ( 16 – 19 ). This specificity is conferred by multisubunit complexes. For example, KAT6A and KAT6B form tetrameric complexes with BRPF1 (bromodomain- and PHD finger–containing protein 1) and two other proteins ( Fig. 1A ) ( 20 , 21 ). BRPF1, in turn, activates KAT6A and KAT6B for H3K23 acetylation ( 20 – 22 ).

Histone modifications such as acetylation, phosphorylation, and methylation are critical for epigenetic regulation ( 1 ). Mass spectrometry has recently identified acetylation-like acylation, including propionylation, crotonylation, butyrylation, and succinylation ( 2 , 3 ). These modifications exhibit functional difference from acetylation in vitro and may serve as fine-tuning mechanisms in vivo. For example, both acetylation and crotonylation are recognized by the bromodomains, plant homeodomain-linked (PHD) fingers, and YEATS domains, albeit at different specificity and affinity ( 3 – 7 ). Like acetylation, the new acylations are reversible, but their responsible enzymes are largely unknown. Although some in vitro studies show that acetyltransferases and deacetylases also catalyze and reverse the acylations, respectively ( 2 , 3 ), the biological relevance remains unclear. Moreover, it is important to elucidate if and how the new acylations are linked to disease.

RESULTS

Deletion of mouse Brpf1 or Kat6a diminishes H3K23 propionylation in vivo We next investigated whether the BRPF1-KAT6 complexes are histone H3K23 propionyltransferases in vivo. Deletion of mouse Brpf1 markedly reduces H3K23 acetylation (11, 12). By analogy, we hypothesized that Brpf1 deletion has similar effects on H3K23 propionylation. To test this, we first carried out immunoblotting to detect histone H3 acylation in protein extracts from control and Brpf1−/− mouse embryonic fibroblasts (MEFs) (39). As previously reported (11, 12), H3K23 acetylation was abolished in Brpf1−/− MEFs (Fig. 2A). H3K23 propionylation was undetectable in these mutant cells (Fig. 2A). By contrast, acetylation or propionylation at H3K9 (or H3K14) was not affected (Fig. 2A). The H3K23 propionylation deficiency was also observed in Brpf1−/− embryos (Fig. 2B). Moreover, immunofluorescence microscopy detected marked reduction of H3K23 propionylation in Brpf1−/− MEFs (Fig. 2C). Notably, the H3K23 propionylation level was more robust in mouse embryos than various cultured cells (fig. S3A). Thus, BRPF1 is critical for H3K23 propionylation in MEFs and embryos, supporting its relevance in vivo. 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. Brpf1 deletion also abrogated signals detected by the anti-H3K23cr and anti-H3K23bu antibodies in MEFs and embryos (Fig. 2, A and B), but this may be, in part, due to cross-reactivity with H3K23ac and H3K23pr (fig. S1B). We also analyzed total histone H3 acylation using anti-propionyllysine and anti-crotonyllysine antibodies. Both detected H3 acylation in wild-type and Brpf1−/− MEF extracts (fig. S3, B and C). Coimmunoprecipitation revealed that the anti-propionyllysine antibody recognizes histone H3 (fig. S3D). Thus, BRPF1 is not critical for total H3 acylation in vivo. An interesting question is how H3K23 propionylation is regulated. One possibility is through propionyl-CoA. Because propionate CoA-transferase converts propionate and acetyl-CoA to propionyl-CoA, we treated MEFs with sodium propionate and analyzed the effect on H3K23 propionylation. As shown in Fig. 2D (lanes 1 and 2), this treatment enhanced H3K23 propionylation but not acetylation, highlighting their differential responses to different environmental conditions. The enhancement of propionylation in response to propionate treatment was abolished in Brpf1−/− MEFs (lanes 3 and 4), further supporting that Brpf1 is required for H3K23 propionylation in vivo. As BRPF1 activates KAT6A and KAT6B for H3K23 acylation in vitro (Fig. 1), we then examined histone H3 acylation in protein extracts from control and Kat6a−/− MEFs. As shown in Fig. 2E, H3K23 acetylation was reduced to about 30 to 50%, whereas H3K23 propionylation was barely detectable in Kat6a−/− MEFs (Fig. 2E). Moreover, in Kat6a−/− embryos, H3K23 acetylation was not affected, whereas H3K23 propionylation was reduced markedly (Fig. 2F). Residual H3K23 propionylation in mutant MEFs or embryos is likely due to Kat6b. These results support the view that KAT6A contributes substantially to H3K23 propionylation in vivo. To determine functional consequences of H3K23ac and H3K23pr, we investigated whether they are associated with active chromatin. For this, we performed coimmunoprecipitation using the anti-H3K23ac or anti-H3K23pr antibody for immunoblotting with antibodies recognizing active and repressive chromatin marks. The anti-H3K23ac and anti-H3K23pr antibodies coimmunoprecipitated histone H3K14ac (an active chromatin mark) from wild-type MEF extracts (Fig. 2G, top, lanes 1, 3, 5, and 7). The former antibody failed to do so with Brpf1−/− MEF extracts (lanes 2, 4, and 6), indicating that BRPF1-promoted H3K23 acetylation is required for coimmunoprecipitation of histone H3K14ac. In contrast, neither antibody coimmunoprecipitated histone H3K27me3 (a repressive chromatin mark) from either wild-type or mutant MEF extracts (Fig. 2G), supporting the association of H3K23ac and H3K23pr with active but not repressive chromatin. To substantiate the association with active chromatin, we also analyzed the subnuclear distribution of the modifications at the single-cell level. For this, active chromatin was labeled with ATAC-See, a recently developed technique to link oligonucleotide-coupled fluorophore covalently to active chromatin in situ (40). Then, H3K23 acylation was detected via fluorescence microscopy. H3K23ac- or H3K23pr-specific fluorescence signals were associated with chromatin labeled by ATAC-See in wild-type E13.5 (embryonic day 13.5) MEFs (Fig. 2, H and I). Moreover, similar results were obtained with sections prepared from wild-type mouse embryos (fig. S4, A and B). One noticeable difference between these two modifications was that propionylation, but not acetylation, was strong in the cerebrocortical neuroepithelium, highlighting the potential importance of H3K23 propionylation in cerebral development at the embryonic stage.

Developmental disorder-associated BRPF1 mutations diminish H3K23 acylation Because BRPF1 variants were found to cause H3K23 acetylation deficiency (12, 33), we asked whether H3K23 propionylation is also impaired (Fig. 3A). To assess this, we expressed and affinity-purified tetrameric KAT6A complexes containing wild-type or mutant BRPF1 proteins for histone acylation assays. As noticed previously (12), Gln629Hisfs*34, but not Arg455* or His563Profs*8, promoted expression of ING5 and MEAF6 (fig. S2D, lanes 1 to 5). This is because the latter two do not contain EPC-II (enhancer of polycomb II) essential for ING5/MEAF6 binding (Fig. 4A). ING5 and MEAF6 did not copurify with these two variants (Fig. 4B, bottom two panels, lanes 4 and 5, and fig. S2D). Unlike Arg455*, both His563Profs*8 and Gln629Hisfs*34 stimulated H3K23 acetylation or propionylation by KAT6A (Fig. 4B, top three panels). The observed activity of His563Profs*8 suggests that ING5/MEAF6 binding is dispensable for BRPF1 to stimulate acylation by KAT6A. As previously reported (12), the loss of the PWWP domain in Gln629Hisfs*34 (Fig. 4A) may affect histone methylation–based genome targeting in vivo. We also analyzed the four missense variants (Pro76Leu, Arg318His, Pro370Ser, and Cys389Arg). Among them, Pro370Ser is defective for stimulating H3K23 acetylation by KAT6A (12). Like this mutant, Cys389Arg was also inactive in stimulating H3K23 acetylation (Fig. 4C, top two panels). Neither variant stimulated H3K23 propionylation by KAT6A (Fig. 4C, middle). As shown in Fig. 4D (bottom two panels) and fig. S2E, Arg318His formed a tetrameric complex normally with the MYST domain of KAT6A, ING5, and MEAF6, but Thr434Profs*61 did not interact with ING5 and MEAF6. This is expected as this variant lacks EPC-II required for ING5/MEAF6 binding (Fig. 3A). Neither Thr434Profs*61 nor Arg318His stimulated H3K23 acetylation or propionylation by the MYST domain of KAT6A (Fig. 4D, top three panels). The Pro76Leu variant was normally expressed and promoted the expression of ING5 and MEAF6 as wild-type BRPF1 (fig. S7C). Moreover, similar to wild-type BRPF1, this variant stimulated H3K23 acylation by KAT6A (fig. S7D). As Pro76 is located at a serine-rich region (fig. S5), this variant may be affected in vivo in terms of regulation by phosphorylation. Clinical features such as macrocephaly also make this case stand out from the cohort (table S1). Further studies are needed to clarify this variant’s relationship to neurodevelopmental disorders and assess if it is affected differently from other variants in vivo. Thus, except for Pro76Leu, the BRPF1 variants are inactive in promoting H3K23 acylation by KAT6A. To establish the in vivo relevance, we analyzed H3K23 propionylation in cells from individuals with BRPF1 variants. As shown in Fig. 4E, this modification decreased in the lymphoblastoid cells (LCLs) prepared from the individual harboring the Pro370Ser variant. Moreover, a similar albeit less marked deficiency was also observed in the LCLs and fibroblasts from the individual with the Arg455* variant (Fig. 4, F and G). While BRPF1 variants identified so far are distributed across the entire protein (Figs. 3A and 4A) (12, 33), most of the KAT6A and KAT6B variants associated with syndromic intellectual disability delete the C-terminal part but leave the MYST domain intact (fig. S8, A and C) (24–31). Nonsense-mediated mRNA decay does not appear to affect transcripts with truncating mutations because most of them are located within the last large coding exon (24–30), so many KAT6A or KAT6B variants are not expected to alter H3K23 acetylation and propionylation. Neither was affected in cells with KAT6A or KAT6B variants (fig. S8, B and D). Thus, these variants do not impair H3K23 acylation. A BRPF1 Tyr406His variant was identified in an autistic individual (43), but the pathogenicity remains elusive. Tyr406 is highly conserved from Caenorhabditis elegans to humans (fig. S5), so we examined the activity of this variant. This variant was well expressed (fig. S2F), but it did not copurify well with KAT6A (Fig. 4H, bottom two panels). Moreover, the variant complex was also less active than the wild type in promoting H3K23 acetylation and propionylation by KAT6A (top three panels), supporting the pathogenicity of the corresponding variant. These results indicate that BRPF1 dysfunction also contributes to autism spectrum disorder (43), further attesting to phenotypic variation associated with BRPF1 variants and suggesting that they may be broadly associated with neurodevelopmental disorders.

Somatic cancer mutations perturb H3K23 acylation via different mechanisms In addition to germline mutations in individuals with neurodevelopmental disorders (Figs. 3A and 4A), somatic BRPF1 mutations have been identified in medulloblastoma, leukemia, and other types of cancer (Fig. 5A and figs. S5 and S9) (23, 44, 45). According to The Cancer Genome Atlas datasets, there are 211 cases with 236 BRPF1 mutations, corresponding to ~2% of the 10,240 cancer cases analyzed (fig. S9). Like those germline variants identified in individuals with neurodevelopmental disorders (Figs. 3A and 4A), these somatic mutations primarily comprise two groups: truncating and missense. According to published reports (12, 33) and the results in Fig. 4, the truncating somatic mutations are expected to inactivate the mutant alleles. 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. Among the missense mutants, Pro20Leu, Arg29Cys, and Ser36Ile alter an N-terminal region with no known function (Fig. 5A and fig. S5). This region is absent in the BRPF1 paralogs BRPF2 and BRPF3 but highly conserved in BRPF1 proteins from Drosophila to humans (fig. S5). BLAST (Basic Local Alignment Search Tool) search revealed high similarity to a C2H2 zinc finger shared by a group of proteins, including the yeast transcription factor Sfp1, so we referred to this unique N-terminal region as the Sfp1-like zinc finger (SZ) domain. Pro20 is highly conserved among this group of proteins (Fig. 5B) and invariant among BRPF1 proteins from Drosophila to humans (fig. S5), suggesting the importance of this residue. As shown in fig. S2G (lanes 1 to 3), this mutant was much more difficult to express than wild-type BRPF1 and also failed to promote expression of ING5 and MEAF6. Moreover, this mutant formed a suboptimal tetrameric complex with KAT6A, ING5, and MEAF6 (Fig. 5C, bottom two panels). The mutant barely stimulated H3K23 acetylation or propionylation by KAT6A (Fig. 5C, top three panels, compare lanes 2 and 4). Thus, the mutation affects expression, complex formation, and enzymatic activity. One hotspot mutation is recurrent in six cancer cases and encodes Arg66Cys (fig. S9). This residue is part of a putative nuclear localization signal, NLS1 (Fig. 1A and fig. S5), so the mutant may affect the function of this NLS. Moreover, three other cancer-associated mutants (Arg59His, Arg59Cys, and Gln67Pro) alter Arg59 and Gln67, both of which are part of NLS1. We thus analyzed the function of this NLS. As shown in Fig. 5D, a fragment (residues 1 to 57) containing the SZ domain of BRPF1 was localized to the nucleus and cytoplasm, with some enrichment in the former. Unlike the full-length BRPF1, coexpression of KAT6A, with or without ING5 and MEAF6, did not affect this subcellular distribution. Compared to the mutant 1-57, the fragment containing the N-terminal 71 residues was more enriched in the nucleus (Fig. 5D), indicating that NLS1 is a functional NLS. Thus, Arg66Cys, Arg59His, Arg59Cys, and Gln67Pro likely affect the function of NLS1. We then verified the activity of Glu253Gly, Leu298Pro, Trp348Arg, and Glu369Asp, all of which were identified in medulloblastoma (44). As illustrated in Fig. 5A, Glu253 and Leu298 are located at EPC-I required for KAT6A/B binding, whereas Trp348 and Glu369 are within the PZP domain important for nucleosome binding. Glu253Gly promoted expression of ING5 and MEAF6 (fig. S2G, compare lanes 1 and 2 with lane 4) but formed a suboptimal complex with KAT6A (Fig. 5E, bottom two panels, lanes 3 and 4). The interaction with ING5 and MEAF6 was also compromised (Fig. 5E, bottom, lanes 3 and 4, and fig. S2G, lanes 1, 2, and 4). This mutant was less active than wild-type BRPF1 in stimulating H3K23 acetylation and propionylation by KAT6A (Fig. 5E, top three panels, lanes 2 to 4). Unlike this mutant, Leu298Pro, Trp348Arg, and Glu369Asp all formed normal tetrameric complexes with KAT6A, ING5, and MEAF6 (Fig. 5E, bottom two panels, lanes 5 to 7). However, unlike Glu369Asp, neither Leu298Pro nor Trp348Arg stimulated H3K23 acetylation or propionylation by KAT6A (Fig. 5E, top three panels, lanes 5 to 7). Thus, Glu253Gly, Leu298Pro, and Trp348Arg, but not Glu369Asp, inactivated BRPF1. We also analyzed five other cancer-derived missense mutants that alter the PZP domain or its C-terminal region (Fig. 5F). Among them, Glu303Gln showed a modestly reduced activity in stimulating H3K23 acylation by KAT6A, whereas Asp344His, Arg347Leu, and the two other mutants were comparable to wild-type BRPF1 (Fig. 5F). Therefore, cancer-derived somatic BRPF1 mutations exert variable effects on H3K23 acylation, reiterating that the impact of each mutation needs to be verified experimentally.