Meiotic recombination starts with the formation of DNA double-strand breaks (DSBs) at specific genomic locations that correspond to PRDM9 binding sites. The molecular steps occurring from PRDM9 binding to DSB formation are unknown. Using proteomic approaches to find PRDM9 partners, we identified HELLS, a member of the SNF2-like family of chromatin remodelers. Upon functional analyses during mouse male meiosis, we demonstrated that HELLS is required for PRDM9 binding and DSB activity at PRDM9 sites. However, HELLS is not required for DSB activity at PRDM9-independent sites. HELLS is also essential for 5-hydroxymethylcytosine (5hmC) enrichment at PRDM9 sites. Analyses of 5hmC in mice deficient for SPO11, which catalyzes DSB formation, and in PRDM9 methyltransferase deficient mice reveal that 5hmC is triggered at DSB-prone sites upon PRDM9 binding and histone modification, but independent of DSB activity. These findings highlight the complex regulation of the chromatin and epigenetic environments at PRDM9-specified hotspots.
PRIDE partner repository with the dataset identifier PXD017337. NGS data have been deposited at GEO under series record GSE145768. Source data have been provided for Figure 2C-D and Figure 3G
PRDM9 activity depends on HELLS and promotes local 5-hydroxymethylcytosine enrichmentNCBI Gene Expression Omnibus, GSE145768.
Identification of PRDM9 partnersPRIDE, PXD017337.
Extensive sex differences at the initiation of meiotic recombinationNCBI Gene Expression Omnibus, GSE99921.
Transient reduction of DNA methylation at the onset of meiosis in male miceNCBI Bioproject, PRJNA326117.
- Bernard de Massy
- Bernard de Massy
- Bernard de Massy
- Mathilde Biot
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All experiments were carried out according to the CNRS guidelines and were approved by the ethics committee on live animals (project CE-LR-0812 and 1295).
- Adèle L Marston, University of Edinburgh, United Kingdom
© 2020, Imai et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.
Imaging experiments reveal the complex and dynamic nature of the transcriptional hubs associated with Notch signaling.