Sitbon D et al. (2020), Histone variant H3.3 residue S31 is essential f...

XB-ART-56795

Nat Commun 2020 Mar 09;111:1256. doi: 10.1038/s41467-020-15084-4.

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Histone variant H3.3 residue S31 is essential for Xenopus gastrulation regardless of the deposition pathway.

Sitbon D , Boyarchuk E , Dingli F , Loew D , Almouzni G .

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Vertebrates exhibit specific requirements for replicative H3 and non-replicative H3.3 variants during development. To disentangle whether this involves distinct modes of deposition or unique functions once incorporated into chromatin, we combined studies in Xenopus early development with chromatin assays. Here we investigate the extent to which H3.3 mutated at residues that differ from H3.2 rescue developmental defects caused by H3.3 depletion. Regardless of the deposition pathway, only variants at residue 31-a serine that can become phosphorylated-failed to rescue endogenous H3.3 depletion. Although an alanine substitution fails to rescue H3.3 depletion, a phospho-mimic aspartate residue at position 31 rescues H3.3 function. To explore mechanisms involving H3.3 S31 phosphorylation, we identified factors attracted or repulsed by the presence of aspartate at position 31, along with modifications on neighboring residues. We propose that serine 31-phosphorylated H3.3 acts as a signaling module that stimulates the acetylation of K27, providing a chromatin state permissive to the embryonic development program.

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Species referenced: Xenopus laevis
Genes referenced: abl1 arhgef5 daxx hira
GO keywords: chromatin
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	Fig. 1. H3.3 is essential for gastrulation of X. laevis.a Best-studied non-centromeric H3 histone variants in H. sapiens and X. laevis. The two well- characterized forms of non-centromeric H3 variants correspond to the replicative histones H3.1 and H3.2, and the non-replicative histone H3.3, depicted in purple and green, respectively. In humans, the replicative H3 variants differ by only one residue at position 96, a cysteine and a serine, respectively. The non-replicative form H3.3 shares more than 96% identity with the replicative forms, with five and four residue differences with H3.1 and H3.2, respectively. In addition, X. laevis embryos possess only one replicative histone variant, H3.2. Finally, histone sequences are conserved between H. sapiens and X. laevis. DSC: DNA-synthesis coupled, DSI: DNA-synthesis independent. b H3.3 depletion-associated defects and rescue. Morpholino and/or eH3.3 mRNA are injected at the two-cell stage and associated defects can be observed at the gastrulation stage if not rescued with eH3.3 WT. White arrowheads indicate the blastopore closure. Scale bar corresponds to 500 µm. See Supplementary Movie 1.
	Fig. 2. eH3.3 AIG mutants rescue depletion of H3.3 during X. laevis early development.a Highlights of the histone chaperone recognition motif residues of H3 variants. Dedicated histone chaperones recognize histone variants by the H3.2 SVM and H3.3 AIG motifs. Although both motifs are structurally similar, the main difference appears for the residue 90. Crystal structures adapted from PDB ID codes 5B0Z105 and 3 AV29. b Rescue assays with H3.3 AIG mutants. Injections are performed at two-cell stage. Effect on gastrulation is analyzed at stage 12. White arrowheads indicate the blastopore closure. Scale bar corresponds to 500 µm. Bar plot shows mean ± SD for three independent experiments with more than 30 embryos. Source data are provided as a Source Data file.
	Fig. 3. H3.3 AIG motif permutation changes histone chaperone interactions and histone modes of incorporation into chromatin in X. laevis.a Immunoprecipitation of eH3 AIG triple mutant and controls in interphase extracts. Recombinant proteins are produced in rabbit reticulocyte lysates and pulled down by their HA-tag after incubation. The binding of HIRA, DAXX, p60, and p150 is analyzed by western blotting with specific antibodies. α-HA antibody is used to detect eH3.3. H4 is used as an internal control. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. b Incorporation of eH3 AIG triple mutant and controls into sperm chromatin in interphase extracts. Purified nuclei remodeled in the interphase extracts supplemented with indicated eH3.3 in the presence or absence of aphidicolin are analyzed by western blotting with indicated antibodies. α-HA antibody is used to detect eH3.3. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. DSC: DNA-synthesis coupled, DSI: DNA-synthesis independent mode of incorporation. Source data are provided as a Source Data file.
	Fig. 4. In X. laevis, H3.3 S31 is critical for early development and is phosphorylated.a Rescue assays with eH3.3 S31A mutant. Injections are performed at two-cell stage. Effect on gastrulation is analyzed at stage 12. White arrowheads indicate the blastopore closure. Scale bar corresponds to 500 µm. Bar plot shows mean ± SD for three independent experiments with more than 30 embryos. b Representative immunofluorescence images showing 3D-distribution and timing of H3.3S31 and H3S10 phosphorylation in the Xenopus A6 cell line. Scale bar represents 10 µm. c H3.3 S31ph dynamics in Xenopus egg extracts. Nuclei remodeled in extracts cycled to interphase, mitosis, and then to the second interphase are purified at the indicated time. Kinetics (appearance and removal) of the H3 phosphorylation is analyzed by western blotting with indicated antibodies. H4 is used as a loading control. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. d Effects of kinase and phosphatase inhibitors on H3 phosphorylation in mitotic egg extracts. Purified nuclei remodeled in the CSF-arrested extracts supplemented with indicated inhibitors are analyzed by western blotting with indicated antibodies. H4 is used as a loading control. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. Source data are provided as a Source Data file.
	Fig. 5. H3.3 S31D phospho-mimic rescues H3.3 depletion.a Rescue assays with eH3.3 S31D mutant and controls. Injections are performed at two-cell stage. Effect on gastrulation is analyzed at stage 12. White arrowheads indicate the blastopore closure. Scale bar corresponds to 500 µm. Bar plot shows mean ± SD for three independent experiments with more than 30 embryos. b Immunoprecipitation of eH3.3 S31 mutants from interphase extracts. Recombinant proteins are produced in rabbit reticulocyte lysates and pulled down by their HA-tag after incubation. The binding of HIRA, DAXX, p60, and p150 is analyzed by western blotting with specific antibodies. α-HA antibody is used to detect eH3.3. H4 is used as an internal control. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. c Incorporation of eH3 S31 mutant forms and controls into sperm chromatin in interphase extracts. Purified nuclei remodeled in the interphase extracts supplemented with indicated eH3.3 proteins in the presence or absence of aphidicolin are analyzed by western blotting with indicated antibodies. α-HA antibody is used to detect eH3.3. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. DSC: DNA-synthesis coupled, DSI: DNA-synthesis independent mode of incorporation. Source data are provided as a Source Data file.
	Fig. 6. Mass spectrometry of H3.3 peptides in interphase and mitotic extracts reveals distinct protein associations.a Peptide pull-down strategy. Biotinylated peptides corresponding to H3 N-terminal tails are incubated in mitotic and interphase Xenopus egg extracts before pulldown, to identify interactors by proteomics mass spectrometry analysis performed from three replicates. b Venn diagrams of proteins identified by mass spectrometry analyses from H3 N-terminal biotinylated peptides in Xenopus egg extracts. Binding partners are selected with at least three peptides in “best analysis” either in interphase or mitotic extracts, specifically. c Summary of Gene Ontology analysis. Attraction refers to factors pulled down with the H3.3 S31D mutant. Repulsion refers to factors that are not pulled down with the H3.3 S31D mutant. Highlights from the top p-value ranked Gene Ontology molecular processes for attracted and repulsed factors are displayed with a selection of associated proteins. See Supplementary Fig. 6 for all top 5 p-value ranked molecular processes from Gene Ontology analysis for each condition obtained with the Enrichr tool106.
	Fig. 7. H3.3 S31D promotes H3K27ac and reduces H3K27me3 in-cis.a Scheme of potential crosstalk of H3.3 S31ph with neighboring PTMs. H3.3 S31 is located between H3.3K27 and H3.3K36, which can both be modified. b PTM crosstalk analyzed in H3.3-transfected Flp-In T-Rex 293 cell lines. Immunoprecipitations of H3.3 mutants enabled to enrich for the exogenous form. Presence of PTMs is analyzed by western blotting with indicated antibodies. α-HA antibody is used to detect eH3.3. H4 is used as an internal control. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. Exo: exogenous H3.3, endo: endogenous H3 and H3.3. c PTM crosstalk in Xenopus embryos. Immunoprecipitations of eH3.3 mutants and controls are performed at stage 12 after their injection at the 2-cell stage and are analyzed by western blotting with indicated antibodies. α-HA antibody is used to detect eH3.3. H4 is used as an internal control. Primary antibodies indicated on the right. Molecular weight (MW) markers in kDa indicated on the left. Exo: exogenous H3.3, endo: endogenous H3 and H3.3. Source data are provided as a Source Data file.
	Fig. 8. Model.Defects associated with H3.3 depletion can be rescued with H3 histone variants carrying a potential negatively charged residue, regardless of the mode of incorporation. MBT: midblastula transition.

References [+] :

Ahmad, Histone H3 variants specify modes of chromatin assembly. 2002, Pubmed