Delineation of meiotic gene expression in male mice

Thesis

Meiosis is a specialised cell division producing the haploid gametes required for sexual reproduction. A key function of this process is to generate genetic diversity through recombination and independent assortment of homologous chromosomes. Delineation of gene expression during this process has been challenging due to the heterogeneity of testis tissue and the lack of faithful in vitro models.

Here we jointly analyse a single-cell resolution transcriptomic dataset of over 20,000 cells from both wild type and mutant mice. We use dimensionality reduction methods to infer latent components of variation that represent transcriptional programmes, mutant specific pathological processes, and technical effects. This approach simultaneously soft clusters cells, infers corresponding groups of co-expressed marker genes, and imputes sparse, noisy gene expression. We were also able to infer, de novo, transcription factor binding motifs for each component, revealing a general switch at the meiotic divisions. We facilitate access to this resource by providing an interactive website testisatlas.ml.

The high-resolution delineation of gene expression during the spermatogenic programme provides high-resolution clues to the function of understudied genes. One such gene, Zcwpw1, is highly co-expressed with Prdm9, and has domains capable of recognising the unique combination of histone marks that PRDM9 deposits (H3K4me3 and H3K36me3). By using a human in vitro system of Zcwpw1 co-transfection with either human or chimp Prdm9, we show that PRDM9 causes the recruitment of ZCWPW1 to its binding sites. This recruitment is stronger than for sites with H3K4me3 alone and is CpG dependent.

Male Zcwpw1^-/- mice have completely normal double strand break positioning, but severe repair and synapsis defects leading to complete testicular azoospermia. Although PRDM9’s effect of DSB positioning remains intact, PRDM9’s effect of aiding synapsis appears to be abolished, with persistent DMC1 signal - most dramatically at the most strongly PRDM9 bound hotspots.

Authors: Wells, DJ

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: epigenetics; single cell RNAseq; testis; meiosis; ZCWPW1; spermatogenesis
• Subjects: functional genomics; meiosis