Abstract

Transcriptional dynamic and chromatin remodeling during spermatogenesis: role of theH3K79me2 mark and its methyltransferase DOT1LSpermatogenesis is the process by which male germ cells differentiate into spermatozoa. It is dividedinto three phases – spermatogonial proliferation, meiosis and spermiogenesis – associated with elevatedtranscriptional and chromatin dynamics. Transcriptomic and ChIP-Seq studies of these processes haverevealed an enrichment in dimethylation of histone H3 lysine 79 (an epigenetic mark known asH3K79me2) and strong expression of its sole methyltransferase DOT1L at meiotic and post-meioticstages. Our team has recently produced a Dot1l knock-out (KO) mouse model in male germ cells andshown that it leads to male hypofertility, characterized by sperm malformations and chromatincompaction defects.The aim of my thesis was to characterize, through bioinformatic analyses, the molecular role of DOT1Land to correlate the dynamics of H3K79me2 mark with transcriptional changes and chromatinremodeling during spermatogenesis. To this end, I analyzed ChIP-seq, RNA-seq and ATAC-seq dataproduced by the team and from the literature.To characterize the dynamics of H3K79me2 during spermatogenesis, I analyzed ChIP-seq data in 4stages of male germ cell development (from undifferentiated spermatogonia to post-meiotic, elongatedspermatids). A pipeline was developed using bowtie2 for alignment, MACS2 for mark enrichment andfollowed by characterization using ChIPseeker and ChromHMM. ChromHMM predicts male germ cellchromatin states from data derived from 6 different histone marks. This approach led me to develop anR package (ChromENVEE) to associate genes with their most likely distal regulatory region(enhancer). My analyses show that H3K79me2 is positively correlated with transcription, and enrichedat promoters and gene bodies, but also at enhancers. I also found that H3K79me2 is involved in theregulation of sex chromosomes-encoded genes during the transition from meiosis to spermatids.In parallel, I studied the impact of Dot1l-KO on transcription at different stages of spermatogenesisusing RNA-seq analyses. I developed a pipeline using STAR tool for alignment followed by differentialexpression analyses using DESeq2 and edgeR. These analyses were also carried out using externalnormalization by "spike-in", i.e. via exogenous sequences added to the samples in a knownconcentration. By integrating our RNA-seq and ChIP-seq data, we showed that Dot1l-KO leads to thederegulation of hundreds of genes. We found that the majority of downregulated genes in KO cellscorrespond to "direct" target genes, i.e. enriched in H3K79me2 at the gene body and/or enhancer.Counter-intuitively, we found many more genes upregulated than downregulated in KO cells. Thisphenomenon appears to be independent of H3K79me2, and these genes are mainly located inchromatin states characterized by bivalent and repressive marks, in particular the repressiveH3K27me3 mark.In conclusion, these results show that DOT1L and H3K79me2 are indispensable for gene regulationthroughout spermatogenesis. In addition to its activator role, these results also present a noveltranscription repressor role for DOT1L according to the chromatin environment.

Keywords : RNA-seq, ChIP-seq, spermatogenesis, H3K79me2, DOT1L, chromatin states