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Novel O-GlcNAcylation on Ser(40) of canonical H2A isoforms specific to viviparity.
Hirosawa M
,
Hayakawa K
,
Yoneda C
,
Arai D
,
Shiota H
,
Suzuki T
,
Tanaka S
,
Dohmae N
,
Shiota K
.
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We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser(40) (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser(40) and the other five have Ala(40). The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser(40) isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala(40) isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser(40) isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser(40) and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals.
Figure 1. Characterization of monoclonal antibody 20B2 against O-GlcNAcylated Histone H2A.(a) WB by the monoclonal antibody (20B2) of mESC cell lysates treated with inhibitors of enzymes critical for O-GlcNAc modification. A pan-H2A antibody was used as a loading control. (b) IF of mESCs with 20B2 (green) and 4′, 6-diamidino-2-phenylindole (DAPI) (blue). Mouse IgG and 20B2 absorbed with O-GlcNAcylated peptides as negative controls. Scale bar = 20 μm. (c) Chromatogram, WB and Silver stain of separated mESC histones by RP-HPLC. Black line, protein retention; Thin blue line, acetonitrile gradient. Blotted with indicated antibodies. (d) IP of Flag-tagged H2A1A, H2A3, and H2A3-S40A mutants using anti-Flag antibody. Blotted with indicated antibodies.
Figure 2. Mass spectrometry of G37-K74 peptides on H2A.(a) The selected ion chromatograms of triply charged G37-K74 peptide ion modified with (lower panel m/z 1394.72 shown in red: G37-K74 with GlcNAc) and without GlcNAc (upper panel m/z 1327.02 G37-K74 without GlcNAc) in the LC-MS of API digested of the 20B2-positive fraction (Fig. 1c) is indicated. (b) LC-MS/MS spectrum of triply charged G37-K74 without GlcNAc ion (m/z 1327.02). (c) LC-MS/MS spectrum of triply charged G37-K74 with GlcNAc ion (m/z 1394.72). (d) L1 loop of canonical histone H2A of the mouse. Blue cylinders, a-helical regions; orange cylinder, the L1 loop. The numbers indicate amino acid position.
Figure 3. Species-dependent O-GlcNAcylation of H2ASer40.(a) WB by 20B2 in the crude histone fraction from various animals. (b) IF images of frozen testis sections from mouse and Xenopus with 20B2 (green) and DAPI (blue). (c–f) Chromatogram and WB of histones extracted from various animals. HeLa cells (human, (c)), CyEF cells (macaque, (d)), Calf thymus (cow, (e)), and Sf9 cells (silkworm, (f)). Black lines, protein retention; Thin blue lines, acetonitrile gradient. Each fraction was blotted with indicated antibodies and Silver stained. The fractions corresponding to H2A and H2AS40Gc were depicted. (g) Alignment of amino acid sequences around position 40 (boxed) of the canonical H2A of various animal species. Upper group has Ser40 or Ala40 and lower group has only Ala40.
Figure 4. Emergence of Ser40 isoform of canonical H2A in animal evolution.(a) The numbers of Ser40 isoforms and all isoforms of H2A for indicated species are shown by a phylogenetic tree. R1-3, rounds of whole-genome duplication15. (b) WB of serial dilutions of crude histone extract from CyEF (macaque) cells, PtK2 (rat kangaroo) cells and LMH (chicken) cells with 20B2. A pan-H2A antibody was used as a loading control. (c) Representative histone modifications functioning as a part of the epigenetic mechanisms in animal species. −, not found; +, considered present.
Figure 5. Genome-wide distribution of H2AS40Gc.(a) TS and dTS (day 6) were immunostained with 20B2 (green) and with DAPI (blue). Scale bar = 20 μm. (b) RP-HPLC chromatogram and WB of histones extracted from TS and dTS. (c–f) Summary of ChIP-seq analyses in TS and dTS. Distributions of H2AS40Gc peaks in TSS, GB and inter-genic regions (c), and in the distribution ratio exon and intron (d). (e) Venn diagrams showing the numbers of H2AS40Gc target genes in TS and dTS. (f) The box plots representing summary values of the expression of H2AS40Gc target genes in the TS and dTS relative to the ESCs.
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