Formation of destabilized nucleosome core particles by spermatogenesis-specific histone variant H2BFWT and its infertility-associated mutant H2BFWT-H100R: Insights from single-molecule optical tweezers and cryo-electron microscopy

Mammalian spermatogenesis relies on an ordered reorganization of male germ cell chromatin which involves several distinct testis-specific histone variants. H2BFWT is a primate testis-specific histone H2B variant of unclear biological functions in human spermatogenesis, despite the nonsense and missense mutations of the H2BFWT gene having been associated with varying degrees of male infertility in multiple populations. We found that H2BFWT is specifically expressed in differentiating and differentiated spermatogonia during early spermatogenesis. Our biochemical analyses indicated that H2BFWT generally destabilizes the nucleosome core particle, with SHL+3 & SHL+5 being most perturbed, and the infertility-associated mutant H2BFWT-H100R causes enhanced nucleosome destabilization. Cryo-EM structures of nucleosomes reconstituted using H2BFWT or H2BFWT-H100R with a 147 bp Widom 601 DNA template revealed a more relaxed structure with floppy DNA ends and reduced resolvable ranges in H2A and H3 for H2BFWT nucleosomes. In particular, we discovered that H2BFWT alters the conformations of H2B loop 1 (L1) and H2BFWT-H4 four-helix bundle interface. The H2BFWT-H100R cryo-EM structure suggested that the H100R substitution further disrupts interactions between H2BFWT amino acid position-100 and H4K91 or H4R92, leading to weaker H2BFWT-H100R-H4 interactions. Using single-molecule optical tweezers, we demonstrated that H2BFWT increases the nucleosome outer unwrapping rate by reducing the nucleosome outer rip energy barrier by 40% compared to canonical nucleosomes, whereas the more destabilizing H2BFWT-H100R further diminishes this energy barrier by 15%. Our findings suggest that H2BFWT aids the formation of destabilized nucleosomes in spermatogonia that may induce chromatin reorganization necessary for normal spermatogenesis progression, while the H2BFWT-H100R mutant's exacerbated nucleosome destabilization leads to male infertility.