Systematic genetic and proteomic screens during gametogenesis identify H2BK34 methylation as an evolutionary conserved meiotic mark

Abstract

Background Gametes are highly differentiated cells specialized to carry and protect the parental genetic information. During male germ cell maturation, histone proteins undergo distinct changes that result in a highly compacted chromatin organization. Technical difficulties exclude comprehensive analysis of precise histone mutations during mammalian spermatogenesis. The model organism Saccharomyces cerevisiae possesses a differentiation pathway termed sporulation which exhibits striking similarities to mammalian spermatogenesis. This study took advantage of this yeast pathway to first perform systematic mutational and proteomics screens on histones, revealing amino acid residues which are essential for the formation of spores. Methods A systematic mutational screen has been performed on the histones H2A and H2B, generating ~ 250 mutants using two genetic backgrounds and assessing their ability to form spores. In addition, histones were purified at key stages of sporulation and post-translational modifications analyzed by mass spectrometry. Results The mutation of 75 H2A H2B residues affected sporulation, many of which were localized to the nucleosome lateral surface. The use of different genetic backgrounds confirmed the importance of many of the residues, as 48% of yeast histone mutants exhibited impaired formation of spores in both genetic backgrounds. Extensive proteomic analysis identified 67 unique post-translational modifications during sporulation, 27 of which were previously unreported in yeast. Furthermore, 33 modifications are located on residues that were found to be essential for efficient sporulation in our genetic mutation screens. The quantitative analysis of these modifications revealed a massive deacetylation of all core histones during the pre-meiotic phase and a close interplay between H4 acetylation and methylation during yeast sporulation. Methylation of H2BK37 was also identified as a new histone marker of meiosis and the mouse paralog, H2BK34, was also enriched for methylation during meiosis in the testes, establishing conservation during mammalian spermatogenesis. Conclusion Our results demonstrate that a combination of genetic and proteomic approaches applied to yeast sporulation can reveal new aspects of chromatin signaling pathways during mammalian spermatogenesis.

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

“…Consistent with this conclusion, the transcriptomic profile of the H2B-K123R mutant largely overlapped with those of the other mutants. We also observed an independent subset of transcripts specific to the H2B-K123R mutant, which might reflect loss of ubiquitylation-independent functions of K123 such as its role as a site for histone acetylation (57,58). However, we cannot rule out that some of the deviation between the transcriptomic profiles among the mutants might stem from slight variation imposed by tagged alleles and/or spike-in normalization (Fig.…”

The key role of a basic domain of histone H2B N‐terminal tail in the action of 5‐bromodeoxyuridine to induce cellular senescence

“…These findings support the view that nucleosome/chromatin organization is one of the determinants of the induction of cellular senescence, and also imply a possible implication of the HBR domain in the regulation of cellular senescence. However, we failed to observe the deletion of the HBR domain in human senescent cells induced by activated Ras , but it is still intriguing to speculate that the function of the HBR domain may be impaired in senescent cells by the loss of its positive charges through modifications, which have been reported in several studies [33,51].…”

Dynamic Histone H3 Modifications Regulate Meiosis Initiation via Respiration

“…During meiosis, chromatin structure is dynamically changed, and a series of events takes place to ensure genetic stability and diversity, such as the programmed DSBs formation, meiotic recombination, homologous chromosomes synapsis and crossover formation ( Zickler and Kleckner, 2015 ; Hillers et al, 2017 ). A number of mechanisms are used to ensure proper chromatin organization, including chromatin remodelers, non-coding regulatory RNAs (ncRNA), histone variants and histone modifications ( Crespo et al, 2020 ). Any defects in these events may cause genome instability, which is associated with the failure of haploid gamete production and infertility ( Wang et al, 2017 , 2019 ).…”