Abstract
We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser40 (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser40 and the other five have Ala40. 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 Ser40 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 Ala40 isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser40 isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser40 and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals.
Highlights
A wide functional and morphological diversity among the species is one of the features of the placenta[1], reflecting the evolutionary history of the organ
To investigate O-GlcNAcylation of histones, we raised a monoclonal antibody (20B2) that reacts with O-GlcNAcylated peptides and epitopes of YT(Gc)E or YS(Gc)E sequences (Supplementary data Fig. 1a)
Western blotting (WB) using 20B2 showed a single band (Supplementary data Fig. 1b,c) and its intensity was changed by O-GlcNAcase (OGA) or O-GlcNAc transferase (OGT) inhibitors in a dose-dependent manner (Fig. 1a)
Summary
A wide functional and morphological diversity among the species is one of the features of the placenta[1], reflecting the evolutionary history of the organ. Many placental genes are species-specific and are silenced in non-placental tissues[2,3]. The canonical histone isoforms have been assumed to encode functionally equivalent proteins because only a few differences exist in their amino acid sequence[11,13]. It is not clear whether such small but distinct sequence variations have any influence on the biochemical character of the histone isoforms. We report discovery of O-GlcNAcylated histone H2A at Ser[40] (H2AS40Gc) by the combination of production of monoclonal antibody and the immunoprecipitation using wildtype and mutated recombinant H2A isoforms and mass spectrometric analyses with RP-HPLC fractionation
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