The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics.
Author contributions
B.T.K.Y. performed the majority of the experiments reported: testes and sperm IHC-IF staining and analysis; sperm imaging and analysis; FACS sorting and analysis; western blotting and quantification; tubule and TUNEL staining, analysis and staging; microarray analysis; CENPA staining and quantification; qPCR; and data preparation and interpretation. K.M.B. performed TUNEL staining, qPCR, the majority of the western blotting, prepared RNA for microarrays, and conducted all mouse breeding and genotyping. B.L.B. performed ChIP-seq data analysis. R.C. prepared ChIP samples. P.S.K. conceived the project, supervised the studies, and along with B.T.K.Y., conducted experimental design and data interpretation. B.T.K.Y. wrote the manuscript. B.T.K.Y., P.S.K., K.M.B., B.L.B. and R.C. read, edited and approved the manuscript.
Funding
This work was supported by a National Institutes of Health grant [5K01CA114400-05] and a California Institute for Regenerative Medicine (CIRM) grant [RN2-00922-1], both to P.K.; and in part by grants made possible from the UC Davis Howard Hughes Medical Institute: Integrating Medicine into Basic Science Program [grant 56006769] and the CIRM Stem Cell Training Program [CIRM grant TG2-01163] to B.T.K.Y. Deposited in PMC for release after 12 months.
