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. 2017 Jan 5;100(1):91-104.
doi: 10.1016/j.ajhg.2016.11.011. Epub 2016 Dec 8.

Mutations in the Chromatin Regulator Gene BRPF1 Cause Syndromic Intellectual Disability and Deficient Histone Acetylation

Kezhi Yan  1 , Justine Rousseau  2 , Rebecca Okashah Littlejohn  3 , Courtney Kiss  4 , Anna Lehman  5 , Jill A Rosenfeld  6 , Constance T R Stumpel  7 , Alexander P A Stegmann  7 , Laurie Robak  6 , Fernando Scaglia  6 , Thi Tuyet Mai Nguyen  2 , He Fu  2 , Norbert F Ajeawung  2 , Maria Vittoria Camurri  2 , Lin Li  1 , Alice Gardham  8 , Bianca Panis  9 , Mohammed Almannai  6 , Maria J Guillen Sacoto  10 , Berivan Baskin  10 , Claudia Ruivenkamp  11 , Fan Xia  6 , Weimin Bi  6 ; DDD Study  12 ; CAUSES Study  5 ; Megan T Cho  10 , Thomas P Potjer  11 , Gijs W E Santen  11 , Michael J Parker  13 , Natalie Canham  8 , Margaret McKinnon  5 , Lorraine Potocki  6 , Jennifer J MacKenzie  14 , Elizabeth R Roeder  15 , Philippe M Campeau  16 , Xiang-Jiao Yang  17
Affiliations

Mutations in the Chromatin Regulator Gene BRPF1 Cause Syndromic Intellectual Disability and Deficient Histone Acetylation

Kezhi Yan et al. Am J Hum Genet. .

Abstract

Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome.

Keywords: BRPF2; PHD finger; PWWP domain; PZP domain; bromodomain; developmental disorder; epigenetic regulator; histone acetylation; intellectual disability.

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Figures

Figure 1
Figure 1
Facial and Brain Characteristics of Individuals with BRPF1 Mutations (A) Photos for eight individuals. The photos are not in the same scale. Ages of the individuals when the photos were taken are as follows: P1, 6 years and 5 months; P2, 13 years and 3 months; P4, 10 years and 6 months; P5, 8 years and 3 months; P6, 4 years; P7, 12 years and 6 months; P9, 8 years; P10, 12 years. (B) Brain magnetic resonance imaging (MRI) images of individuals P2 and P9.
Figure 2
Figure 2
Domain Organization of BRPF1 and Its Variants from Individuals with Developmental Anomalies (A) Schematic representation of BRPF1 and nine variants as identified in ten individuals. See Table 1 for DNA sequence changes in the individuals. BRPF1 possesses multiple modules, including the PZP domain, bromodomain, and PWWP domain, for chromatin association. The PZP domain comprises two PHD fingers linked with a C2HC zinc finger. The first PHD finger recognizes the N terminus of histone H3., , The C2HC zinc knuckle and the second PHD finger form a non-specific DNA binding domain., The bromodomain has acetyllysine-binding ability, and the PWWP domain targets trimethylated histone H3., The EPC-like motif C-terminal to the PZP domain is essential for formation of a stable trimeric complex with ING5 and MEAF6., , Through the EPC-like motif N-terminal to the PZP domain and a conserved region further N-terminal to this motif, BRPF1 interacts with and activates KAT6A, KAT6B, and KAT7., , Unlike p.Pro370Ser, the other eight variants contain C-terminal truncations due to nonsense or reading-frameshift mutations. These mutations are not located within the last coding exon and might trigger NMD in vivo. Abbreviations are as follows: CH, C2H2 zinc finger; BN, conserved BRPF N-terminal domain; EPC, enhancer of polycomb-like motif; NLS, nuclear localization signal. (B) Sequence alignment of human BRPF1 with its paralogs (BRPF2 and BRPF3), as well as the orthologs from zebrafish (z), Xenopus (x), sea urchin (s), Drosophila (d), and C. elegans (Lin-49). There is one ortholog per organism from the worm to Xenopus, but there are three in zebrafish. Pro370 of BRPF1 and its corresponding residues in its paralogs and orthologs are boxed in red. (C) Pro370 is located within a loop connecting the C2HC knuckle to the second PHD finger. PHD1 recognizes the free N terminus of histone H3,, , whereas the C2HC zinc knuckle and PHD2 form a module for non-specific interaction with the DNA backbone of the nucleosome. The structural model was generated based on a published report, with kind help of Tatiana G. Kutateladze.
Figure 3
Figure 3
BRPF1 Loss Reduces Histone H3K23 Acetylation In Vivo (A and B) Histone H3 acetylation in thymus and spleen protein extracts from control and hematopoietic-specific Brpf1-knockout mice. (C) Histone H3 acetylation in dorsal cortical extracts from heterozygous and forebrain-specific Brpf1 knockouts. (D) Histone H3 acetylation in protein extracts from wild-type and epiblast-specific Brpf1-knockout embryos at embryonic day 10.5 (E10.5). (E) Quantification of H3K23 acetylation in proteins extracts from wild-type and epiblast-specific Brpf1 knockout embryos at E10.5. The blot was from the same experiment as shown in (D), but the exposure time was shorter than the bottom image in (D). The quantification was performed with NIH ImageJ. (F) Histone H3 acetylation in protein extracts from control and Brpf1-knockout mouse embryonic fibroblasts (MEFs). The fibroblasts were prepared from control and tamoxifen-inducible knockout embryos at E15.5. (G) Histone H3 acetylation in protein extracts from control and p.Pro370Ser lymphoblastoid cells. (H) Histone H3 acetylation in protein extracts from control and p.Arg455∗ fibroblasts.
Figure 4
Figure 4
Functional Characterization of BRPF1 Variants In Vitro (A) Interaction of BRPF1 and the variants with KAT6A, ING5, and MEAF6. KAT6A was produced in HEK293 cells as a FLAG-tagged fusion protein along with HA-tagged BRPF1 (or the p.Pro370Ser variant), ING5, and MEAF6 as indicated. Soluble protein extracts were prepared for affinity purification on anti-FLAG agarose, and bound proteins were eluted with the FLAG peptide for immunoblotting with anti-FLAG and -HA antibodies. (B) Histone acetylation assays. HeLa oligonucleosomes were used as substrates for acetylation by proteins affinity purified in (A). Acetylation of histone H3 was detected with antibodies recognizing histone H3 and its acetylated forms as indicated. (C and D) Same as (A) except that different BRPF1 variants were analyzed. Unexpectedly, the variant p.Trp315Leufs∗26 still showed strong interaction with MEAF6; whether this is due to the extra 26 residues introduced after the reading frameshift remains unclear. (E) Histone acetylation assays. Proteins affinity-purified in (D) were used for the enzymatic assays as in (B).
Figure 5
Figure 5
Distribution of Histone H3K23 Acetylation in the Mouse Brain Adult mouse brain sections were used for indirect immunofluorescence microscopy with the anti-H3K23ac antibody (upper), with nuclei counterstained with DAPI (middle). H3K23ac signals are enriched in the hippocampus, cerebellum, olfactory bulb (OB), and islands of Calleja (IC). Within the hippocampus, the fluorescence signals were high in Cornu Ammonis (CA) areas and the dentate gyrus (DG). Images of one representative parasagittal brain section are shown here. MB, mid-brain. Scale bar, 1 mm.
Figure 6
Figure 6
BRPF1 Governs H3K23 Acetylation through KAT6A and KAT6B (A) Competitive interaction of BRPF1 with KAT6A and KAT7. BRPF1 was produced in HEK293 cells as a FLAG-tagged fusion protein along with HA-tagged KAT6A (or KAT7), ING5, and MEAF6 as indicated. For KAT6A, only its MYST domain was produced (labeled as KAT6A∗). Soluble protein extracts were prepared for affinity purification on anti-FLAG agarose, and bound proteins were eluted with the FLAG peptide for immunoblotting with anti-FLAG and -HA antibodies. (B) Cartoon showing how BRPF1 mutations affect histone H3K23 acetylation and cause developmental abnormalities. In individuals without BRPF1 mutations (left), BRPF1 forms a trimeric complex with ING5 and EAF6 to control histone H3K23 acetylation by KAT6A and KAT6B, which in turn regulate developmental programs. In individuals with BRPF1 mutations (right), BRPF1 variants fail to exert proper control on histone H3K23 acetylation, thereby deregulating normal gene expression and developmental programs. For simplicity, the cartoon does not illustrate additional partners that BRPF1 might have in vivo.
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