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Germline mutations affecting the histone H4 core cause a developmental syndrome by altering DNA damage response and cell cycle control

Abstract

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure1,2. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders3,4,5,6 or has a role in tumorigenesis7,8. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis9,10. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

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Figure 1: Clinical and molecular defects in patients with HIST1H4C mutations.
Figure 2: Histone H4 variants (encoded in HIST1H4C) induce defective growth, microcephaly and double-strand break accumulation in zebrafish embryos.
Figure 3: Histone H4 (HIST1H4C) alterations at K91 perturb cell cycle progression.

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Acknowledgements

We thank the subjects and their parents for being willing to be part of this study, S. van der Elst for technical assistance for the FACS analysis, P. Nguyen for the zebrafish embryonic cell suspension protocol, D. Guardavaccaro for advice on western blotting and A. de Graaff.

We acknowledge support from the Netherlands Cardiovascular Research Initiative, Dutch Heart Foundation grant CVON2014-18 CONCOR-GENES and the 'Proteins at Work' program of the Netherlands Organization for Scientific Research (NWO; project 184.032.201).

The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant HICF-1009-003), a parallel funding partnership between the Wellcome Trust with the Department of Health and the Wellcome Trust Sanger Institute (grant WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of the Wellcome Trust or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South REC, and GEN/284/12, granted by the Republic of Ireland REC). The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network.

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Authors and Affiliations

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Contributions

K.L.I.v.G., J.C.G., J.A.H. and R.H.S. identified and matched the study subjects. F.T., J.B. and G.v.H. designed the study. K.L.I.v.G. contributed to the study design. F.T. carried out the zebrafish experiments. K.D. performed molecular cloning and carried out RNA sequencing on patient-derived fibroblasts. H.R.V. and R.M.v.E. carried out the mass spectrometry analysis. M.P.M. performed the bioinformatics analysis on the RNA sequencing data. F.T., M.P.M., J.B. and G.v.H. wrote the manuscript, and F.T., J.C.G., R.H.S., J.B. and G.v.H. reviewed the manuscript.

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Correspondence to Jeroen Bakkers or Gijs van Haaften.

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The authors declare no competing financial interests.

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A list of members and affiliations appears in the Supplementary Note.

Integrated supplementary information

Supplementary Figure 1 Sequencing traces of HIST1H4C DNA from patients.

For all patients, the causative substitutions are indicated (black arrowhead). Patient 1 carries the rs61735681 SNP (asterisk); Patients 2 and 3 carry the rs198852 SNP (hash).

Supplementary Figure 2 Sequence reads of the HIST1H4C gene indicate low-level mosaicism in the father of patients 2 and 3.

Integrated Genomics Viewer (IGV) screenshots from the mutated position of patient 2 (left), the mother (middle) and the father (right). The mother is homozygous for SNP rs198852. The mutation in the child only occurs on reference reads, indicating that the mutation occurred in the paternal germ line. The reads of the father (right) show 2 mutated reads and 47 reference reads. The two mutated reads are present on a duplicate read, indicating that these originated from the same molecule.

Supplementary Figure 3 Protein sequence alignment of HIST1H4C homologs.

The amino acid affected in the patients (K91) is indicated by a black arrow.

Supplementary Figure 4 Relative expression of histone H4 genes in adult human tissues.

Histograms were drawn using data retrieved from the EMBL-EBI expression atlas (https://www.ebi.ac.uk/gxa/home) and previously published in the Genotype-Tissue Expression (GTEx) project (Nat. Genet. 45, 580–585, 2013). Gene IDs for the reported genes are as follows: ENSG00000197061, HIST1H4C; ENSG00000158406, HIST1H4H; ENSG00000197238, HIST1H4J; ENSG00000197837, HIST4H4; ENSG00000270276, HIST2H4B; ENSG00000270882, HIST2H4A; ENSG00000273542, HIST1H4K; ENSG00000274618, HIST1H4F; ENSG00000275126, HIST1H4L; ENSG00000275663, HIST1H4G; ENSG00000276180, HIST1H4I; ENSG00000276966, HIST1H4E; ENSG00000277157, HIST1H4D; ENSG00000278637, HIST1H4A; ENSG00000278705, HIST1H4B.

Supplementary Figure 5 Histone H4 K91Q and K91R expression induces convergence-extension defects in zebrafish embryos.

(a) mRNA in situ hybridization for krox20 (*; hindbrain rhombomeres 3 and 5) and myoD (+; muscle cell precursors) at the ten-somite stage in embryos microinjected with mRNA encoding K91Q and K91R HIST1H4C. Note that, in comparison to the normal situation (class A), embryos microinjected with the K91Q or K91R mutant mRNA often display mildly broader and relatively shorter signals for both krox20 and myoD (class B) to stronger convergence-extension defects (class C). (b) Quantification of the classes defined in a per treatment. Scale bar, 100 μm.

Supplementary Figure 6 RNA sequencing data for HIST1H4C mutant carriers and control fibroblast cell lines.

RNA sequence traces as seen in IGV and pie charts showing the normalized relative amounts of sequence reads for HIST1H4 gene family members are shown for a control cell line and the two described HIST1H4C-mutant cell lines. (a) RNA sequencing results for the control fibroblast cell line. (b) RNA sequencing results for the HIST1H4C p.Lys91Gln variant; the numbers of reference and variant sequence reads are shown in parentheses. SNP rs61735681 is found in the variant haplotype. (c) RNA sequencing reads for the p.Lys91Arg variant; the numbers of reference and variant sequence reads are shown in parentheses. Just upstream of this variant, a very common SNP (rs198852) is present. As can be seen in the pie charts for all three variants, the relative amount of HIST1H4C sequence reads ranges from 14 to 17% of all HIST1H4 family member reads, of which approximately half correspond to the mutant allele.

Supplementary Figure 7 Mass spectrometry analysis shows the presence of mutant histones H4 in patient-derived fibroblasts.

(a) Fragmentation spectra from a single experiment on fibroblasts form patient 3 showing the existence of both WT (TVTAMDVVYALK) and mutant K91R (TVTAMDVVYALR) peptide species. (b) Also included are the data of C-terminal dibasic missed cleavage species of both peptides, again showing the existence of the mutant histone H4. (c) To estimate the amount of the K91R H4 mutant in vivo, we digested the chromatin fraction of patient fibroblasts (patients 2 and 3) with trypsin and used high-resolution LC–MS/MS to detect peptides derived exclusively from the mutant protein as well as wild-type H4. Using the precursor area detector node of Proteome Discoverer 2.1, we determined the summed intensity ratio between mutant (K91R) and wild-type peptides. Plotted are the ratios of three replicates. The black horizontal bar indicates the mean value for each condition.

Supplementary Figure 8 Apoptosis in K91Q- and K91R-expressing zebrafish larvae.

(a) Expression of the K91Q and K91R HIST1H4C variants induces increased apoptosis as shown by acridine orange (AcrOr) staining in live embryos at 28 h.p.f. Representative magnifications of the head and tail (corresponding to the whole-embryo pictures shown) show increased occurrence of cellular apoptosis in these regions. The experiment was replicated twice on embryos originating from group matings of adult zebrafish. For each condition and replicate, a sample consisted of a minimum of 25 embryos. (b) Increased numbers of apoptotic cells in the head and tail of zebrafish larvae are also shown by DAPI staining of fragmented nuclei visualized as bright fluorescent circular spots in K91Q- and K91R-expressing larvae at 28 h.p.f. Scale bars, 100 μm in a and 50 μm in b. Imaging was carried out on three different embryos per condition. One representative image is shown.

Supplementary Figure 9 Exogenous HAT4 and DTX3L retain their histone H4 acetyltransferase and ubiquitin ligase activity, respectively, in zebrafish embryos.

(a) H4K91 acetylation in control zebrafish embryos and zebrafish embryos injected with HAT4 mRNA as detected with anti-H4K91ac antibody. Detection with anti-H4 antibody was used as a loading control. Normalized ratios of H4K91ac and H4 are shown below the panels. HAT4 mRNA injection results in increased levels of H4K91ac. (b) Co-injection of DTX3L mRNA with H4-FLAG mRNA results in an increased fraction of monoubiquitinated H4. Detection of H4-FLAG and Ubi-H4-FLAG was carried out with anti-FLAG antibody. One representative immunoblot of a minimum of two fully independent experiments is shown.

Supplementary Figure 10 Coexpression of the histone acetyltransferase HAT4 does not rescue the occurrence of γ-H2AX signals in embryos expressing K91Q or K91R HIST1H4.

Expression of the K91Q and K91R HIST1H4C variants results in significant accumulation of DSBs. Coexpression of HAT4 does not reduce the occurrence of γ-H2AX-positive cells in K91Q- or K91R-expressing embryos. For all conditions, n = 20. For all graphs, significance was determined by two-tailed Mann–Whitney U test: ns, not significant; **P < 0.01; ***P < 0.001. The black horizontal bar indicates the mean value for each condition. Data were collected on one technical replicate. All embryos analyzed originated from group matings of adult zebrafish.

Supplementary Figure 11 Inactivated DTX3L-M2 does not rescue the occurrence of γ-H2AX signals in embryos expressing K91Q or K91R HIST1H4.

Expression of the K91Q and K91R HIST1H4C variants results in significant accumulation of DSBs. DTX3L was inactivated by introducing four mutations affecting its RING domain (Online Methods). The inactive form of DTX3L was named DTX3L-M2, abbreviated here as M2. Coexpression of M2 does not reduce the occurrence of γ-H2AX-positive cells in K91Q- or K91R-expressing embryos. For all graphs, significance was determined by two-tailed Mann–Whitney U test: ns, not significant; the black horizontal bar indicates the mean value for each condition. Data were collected on two technical replicates. All embryos analyzed originated from group matings of adult zebrafish.

Supplementary Figure 12 FACS analysis of zFUCCI embryos expressing H4 mutants.

The FACS profiles displayed show Hoechst 34580 staining (405-nm channel; x axis) in vitro of zebrafish FUCCI live cell suspensions either in G1 phase or S/G2/M phases as determined by their fluorescence (mKO2 for G1; mAG for S/G2/M). The ratio displayed is calculated based on the mKO2 (G1) and mAG (S/G2/M) fluorescence detected by the FACS analyzer.

Supplementary Figure 13 p53 levels are increased in embryos expressing H4 mutants.

Western blot analysis and quantification display upregulation of p53 in embryos expressing H4 mutants. One representative immunoblot of two biological and technical replicates is shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Table 1 and Supplementary Note. (PDF 2668 kb)

Reporting Life Sciences Summary (PDF 173 kb)

Supplementary Table 2

RNA sequencing: differentially expressed genes. (XLSX 20 kb)

Supplementary Table 3

RNA sequencing: GO term analysis. (XLSX 47 kb)

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Tessadori, F., Giltay, J., Hurst, J. et al. Germline mutations affecting the histone H4 core cause a developmental syndrome by altering DNA damage response and cell cycle control. Nat Genet 49, 1642–1646 (2017). https://doi.org/10.1038/ng.3956

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