During the post-meiotic phase of spermatogenesis, male germ cells called spermatids undergo profound transformations to differentiate into spermatozoa: acquisition of a flagellum and acrosome, elongation of the cell, elimination of the cytoplasm and compaction of the nucleus. These processes are essential for male fertility, and regulated by a genetic program that takes place at the end of meiosis and involves the expression of many genes. Nucleus compaction is a major event that occurs at the end of spermatogenesis, following the eviction of almost all histones (i.e. the proteins around which DNA is organized and which form the chromatin) and their replacement by more basic and smaller proteins, called protamines. This global and unique reorganization induces a high level of compaction necessary to protect the paternal genome and prepare its reprogramming in the zygote, if fertilization occurs.
In recent decades, histone variants and post-translational modifications have been shown to be required during the process of histones replacement by protamines. Hyperacetylation of histone H4 is, in particular, an essential modification for chromatin opening and histone removal. Several teams including ours have recently observed that lysine 79 of histone H3 is abundantly di- or trimethylated (H3K79me2 and me3) at the time of histone hyperacetylation. We also observed that the methyltransferase DOT1L - the only enzyme capable of catalyzing the methylation of H3K79 - is very strongly expressed in spermatids but its role during spermatogenesis remained unknown until now.
In order to understand the role of DOT1L during spermatogenesis, we generated a mouse model in which Dot1l gene is specifically knocked-out in male germ cells (Dot1l-KO). Phenotypic analyses show that fertility is impaired in KO males due to the production of non-functional spermatozoa, characterized by head and flagellum abnormalities, incomplete chromatin compaction as well as a reduction in their motility. Multiple defects in spermatogenesis are also observed, such as an increase in the number of elongated spermatids in apoptosis.
To identify the origin of the chromatin compaction defect, we quantified the main players of histone-to-protamine transition. By mass spectrometry, we demonstrated a deregulation of several histone modifications in Dot1l-KO cells, in particular a decrease in hyperacetylated forms of H4 in elongated spermatids. We also found abnormal retention of histones and transition proteins as well as accumulation of the immature form of protamine 2 in Dot1l-KO sperm. These results show the importance of DOT1L and H3K79 methylation in the process of sperm chromatin reorganization and compaction.
H3K79me2 being associated with expressed genes, we studied by RNA-seq (high-throughput sequencing of transcripts) the impact of DOT1L on gene expression at different stages of spermatogenesis. This analysis revealed that several hundred genes are deregulated in Dot1l-KO spermatids, in particular genes involved in flagellum function and energy production, chromatin organization and apoptosis. These results are correlated with the phenotype observed in Dot1l-KO males.
In addition to its role in chromatin reorganization, DOT1L therefore regulates the expression of many genes involved in spermatid differentiation into spermatozoa.
Our study shows that the histone methyltransferase DOT1L is essential for the differentiation of spermatids into functional spermatozoa. This enzyme – expressed ubiquitous – was known to be involved in multiple biological processes, including development. DOT1L has also been implicated in some forms of leukemia, and is a target of anti-cancer treatments.
Our work provides a better understanding of the mechanism by which DOT1L acts on chromatin organization (crosstalk with H4 hyperacetylation) and gene expression. In addition, our results identify Dot1l gene as an important player in male reproduction. They provide a better understanding of spermatogenesis that may be useful for future studies on infertility and male contraception.
This study has been funded by the Agence Nationale de la Recherche (ANR-17-CE12-0004-01 to J.C., ANR-21-CE44-0035 to D.P. and J.C.), the Fondation pour la Recherche Médicale (SPF201909009274 to C.G.), “Ministerio de Economía y competitividad” FI17/00224 to A.I., and “Ministerio de Ciencia e Innovación” PI20/00936 to R.O. and MV20/00026 to A.I.
Reference
Blanco M, El Khattabi L, Gobé C, Crespo M, Coulée M, de la Iglesia A, Ialy-Radio C, Lapoujade C, Givelet M, Delessard M, Seller-Corona I, Yamaguchi K, Vernet N, Van Leeuwen F, Lermine A, Okada Y, Daveau R, Oliva R, Fouchet P, Ziyyat A, Pflieger D, Cocquet J. DOT1L regulates chromatin reorganization and gene expression during sperm differentiation. EMBO Rep. 2023 Apr 26:e56316. doi: 10.15252/embr.202256316. Epub ahead of print. PMID: 37099396.
