The role of the testis specific H3 variant H3t in mouse spermatogenesis

Spermatogenesis is an extremely efficient differentiation process, resulting in the generation of millions of male germ cells on a daily basis. In the past, a substantial number of testis specific histone variants have been reported and for some an essential function at distinct stages of male germ cell development has been demonstrated. This is particularly true for H1 variants, H2A and H2B variants, however, at the start of my PhD project, variation in the histone H3 family remained poorly characterized. A testis specific H3 variant termed H3t was initially reported in the 1980’s in rat, but its existence in mouse and especially its significance remained unclear.
During my PhD project, I identified the H3t gene in a wide variety of species (across mammals except monotremes) and could demonstrate that the genomic region shows extensive conserved synteny between these species. Experiments conducted in my host laboratory demonstrated high levels of H3t at most stages of spermatogenesis and an almost complete replacement of canonical histones by H3t prior to entry into meiosis. To study the function of H3t in vivo, I established a CRISPR/Cas9 approach, which allowed me to efficiently generate mutant mouse models. By deleting the H3t gene, I could demonstrate that the locus is essential for male germ cell development. H3t deficient males show a dramatic decrease in early meiotic cells and are completely devoid of post-meiotic cells, resulting in male sterility. In contrast, males heterozygous for H3t are fertile, but show a decrease in testis weight.
My main focus to determine the function of H3t has been on variations in its amino acid sequence relative to canonical H3.1 (V24A, H42R, S98A). I generated mouse models that have each of these positions substituted to a H3.1 state individually and in combination. Notably, I find a combinatorial effect of H3t specific amino acid variations on male germ cell development, which is particularly pronounced in a heterozygous H3t knock out context. Collectively, my data suggest that high levels and combined H3t specific amino acid variations promote efficient male germ cell development and thus, expression of H3t may provide a selective advantage in a population. Since H3t is prominently detectable in the genomes of all mammals investigated except monotremes, it is tempting to speculate that a wide variety of species relies on H3t for male germ cell development.