Histone H2A (H2A.X and H2A.Z) Variants in Molluscs: Molecular Characterization and Potential Implications For Chromatin Dynamics

Abstract

Histone variants are used by the cell to build specialized nucleosomes, replacing canonical histones and generating functionally specialized chromatin domains. Among many other processes, the specialization imparted by histone H2A (H2A.X and H2A.Z) variants to the nucleosome core particle constitutes the earliest response to DNA damage in the cell. Consequently, chromatin-based genotoxicity tests have been developed in those cases where enough information pertaining chromatin structure and dynamics is available (i.e., human and mouse). However, detailed chromatin knowledge is almost absent in most organisms, specially protostome animals. Molluscs (which represent sentinel organisms for the study of pollution) are not an exception to this lack of knowledge. In the present work we first identified the existence of functionally differentiated histone H2A.X and H2A.Z variants in the mussel Mytilus galloprovincialis (MgH2A.X and MgH2A.Z), a marine organism widely used in biomonitoring programs. Our results support the functional specialization of these variants based on: a) their active expression in different tissues, as revealed by the isolation of native MgH2A.X and MgH2A.Z proteins in gonad and hepatopancreas; b) the evolutionary conservation of different residues encompassing functional relevance; and c) their ability to confer specialization to nucleosomes, as revealed by nucleosome reconstitution experiments using recombinant MgH2A.X and MgH2A.Z histones. Given the seminal role of these variants in maintaining genomic integrity and regulating gene expression, their preliminary characterization opens up new potential applications for the future development of chromatin-based genotoxicity tests in pollution biomonitoring programs.

The characterization of macroH2A beyond vertebrates supports an ancestral origin and conserved role for histone variants in chromatin

“…38 Overall, these results seem indicative of an exclusion of macroH2A from the chromatin of terminally differentiated cells in non-vertebrates. 26 The low amount of Mg macroH2A observed in hemolymph is consistent with this notion, as recent studies suggest that progenitor cells differentiate into hemocytes in the gills 55 reaching the hemolymph in a highly differentiated state.…”

“…Accordingly, molecular phylogenetic analyses support a monophyletic origin for the macroH2A lineage 36,38 as early as before the diversification of Filozoans (including Filasterea, Choanoflagellata, and Metazoa) as suggested by the presence of macroH2A in Capsaspora (Fig. 2, Fig.…”

“…The tree was rooted with the human histone H2A.Z, as it constitutes a sister monophyletic group of macroH2A within the H2A phylogeny. 34,38 macroH2A encompasses an H2A domain (amino acids 1 to 120) displaying 58% identity with the homologous region in the canonical Mg H2A, followed by a basic linker region (amino acids 121 to 178) connecting the H2A domain with the macro domain (amino acids 179 to 369) (Fig. S1).…”

“…Its complete absence in mature sperm is noticeable, especially since the sperm of Mytilus, in addition to its characteristic protamine-like proteins, 40 retains approximately 20-25% of histones, among which variants are included. 38,41,42 Distribution and nucleosome organization of macroH2A in mussel chromatin…”

“…Fifteen per cent (w/v) polyacrylamide SDS gels 38 and native 4% (w/v) PAGE were prepared and run as described elsewhere. 38 The SDS-PAGE gels loaded with approximately 2 mg of histones were transferred onto nitrocellulose membranes at 100 V for 3 h at 4 C in 20 mM sodium phosphate (pH 6.8), 14.25% (v/v) ethanol and 0.1% (w/v) SDS transfer buffer.…”