Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Abstract

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.

Fig. 1. H3F3A mutations (NM_002107.4) and H3F3B mutations (NM_005324.4).

N, 1, 2, and 3 refer to the N-Helix, Helix-1, Helix-2, and Helix-3 of H3.3, respectively. Upper mutations are encoded by H3F3B, and lower mutations are encoded by H3F3A. Red arrows indicate mutations found in two or more unrelated patients. A few variants, p.N108S, p.P121R, and p.Q125R, were found in the same position in both H3F3A and H3F3B. *p.S146X is only present in an H3F3B alternate transcript not shown here.

Fig. 2. Variant locations in the nucleosome.

At the top of the figure, we show the structure of the nucleosome with the H3.3 variants identified with spheres; the H3.3 monomer carrying them is colored in dark emerald green. The coloring of the variants reflects the predominating interactions at each location: DNA binding (magenta), intramonomer contacts (light orange), and contacts with other histones (dark blue). The same color code is used in the histogram below the structure, where we show the amount of the three interaction types at each location. Note that we use different y axis for these interactions: The y axis to the left corresponds to the H3.3-DNA binding contacts (magenta bars), and the y axis to the right corresponds to the intramonomer (light orange bars) and intermonomer contacts (dark blue bars).

Fig. 3. Variant locations in H3.3-epigenetic regulator complexes.

Variant locations could be mapped to the experimental structure of different complexes involving H3.3. In (A), we show the variants mapped and the gene names of the H3.3 partners in the corresponding complex. The same color is used for the lines originating in the same variant These complexes include CARM1 (Coactivator Associated Arginine Methyltransferase 1), ZMYND11, SETD2, NSD3 (Histone-lysine N-methyltransferase NSD3), MORC3 (MORC Family CW-Type Zinc Finger 3), MLLT3 (MLLT3 Super Elongation Complex Subunit), KDM1B (Lysine Demethylase 1B), and BRD4. In (B), we show the total amount of interatomic interactions at each location, for each H3.3-epigenetic regulator complex. To help interpretation, we give three examples where we can see the histone tail (blue spheres) interacting with its partner (continuous surface in light orange); the histone residue at the variant location is shown in magenta.

Fig. 4. qMS analysis of patient samples.

(A) Average profile of PTMs on canonical histones H3 and H4 across control lymphoblasts. Error bars represent SD (n = 9 donors; 3 biological replicates each). (B) Tukey boxplot depicting the coefficients of variation of 73 modified histone H3 and H4 peptides detected by nano–LC-MS/MS (biological variance: across all 14 donors; patient variance: across five patients; control variance: across nine controls). (C) Average histone H3.3 protein abundance (relative to total histone H3) in patient and control lymphoblasts. Error bars represent SD. (D) Volcano plot demonstrating significantly altered histone PTMs in patients versus controls. Dotted line represents P < 0.05 significance threshold. (E) K9 and K14 PTM abundances were compared between (i) protein transcribed from the mutant p.A15G allele from patient cells, (ii) protein transcript from the wild-type (WT) allele from the same patient cells, and (iii) protein transcribed from the WT alleles from a control. Note that the peptide from amino acids 9 to 17 is indistinguishable between canonical H3 and H3.3, so the WT peptide encompasses both. (F) A29P is the only mutation occurring on the same peptide that distinguishes H3.3 from H3. PTMs that fall on this peptide are compared across the mutant peptide, the WT peptide from the mutant sample, and the average profile of the peptide from control samples. *P < 0.05, **P < 0.01, and ***P < 0.001. This shows notable local deregulation of PTMs on the mutant peptide. qMS, quantitative mass spectrometry.

Fig. 5. Cellular dynamics of patient fibroblasts.

(A) Five H3F3A/B patient fibroblast lines (H3F3B: p.G34V; H3F3A: p.R17G; H3F3A: p.G90R; H3F3A: p.T45I; and H3F3B: p.V117V or p.S146X in alternate transcript) demonstrated increased proliferation over six matched controls. **P < 0.005 and ***P < 0.0005. Data represent means ± SEM of three biological replicates using three technical replicates each. (B) The same five H3F3A/B patient fibroblasts and six controls show no major differences in cell viability. The data represent the means ± SEM of four biological replicates using two technical replicates each. (C) Cell cycle analysis showed differences in the S (P = 0.0127) and G₂ (P = 0.0338) phase in the same five patient cell line compared to the six control fibroblast lines. Data represent the means ± SEM of four biological replicates using two technical replicates each.

Fig. 6. Requirement of H3.3A for neural crest–derived glia and pigment cells.

(A) Ventral whole-mount views of larval zebrafish heads at 5 dpf stained with Alcian Blue. Homozygous h3f3a^db1092 mutants display complete loss of neural crest–derived jaw cartilages (n = 10/10). (B to D) In situ hybridization of zebrafish embryos for markers of glia (foxd3; 24 hpf), melanocytes (dct; 27 hpf), and xanthophores (xdh; 27 hpf). Homozygous h3f3a^db1092 mutants injected at the one-cell stage with a control mCherry RNA show partial reductions in cranial glia (n = 5), melanocytes (n = 4), and xanthophores (n = 3), while those injected with dominant-negative H3f3a RNA to further reduced H3.3A function show complete loss of melanocytes (n = 5) and severe reductions of glia (n = 6) and xanthophores (n = 4) throughout cranial and trunk regions.