Billings, T. | Search bioRxiv

bioRxiv2018

EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 lysine-4 and lysine-36 methyltransferase PRDM9 to ensure successful double-strand break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse models to deplete EWSR1 before the onset of meiosis, and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis. Author SummaryIn most mammals, including humans and mice, genetic recombination initiates when the meiosis-specific protein PRDM9 binds specific DNA sequences, known as hotspots, at the beginning of the extended prophase I of meiosis, and activates them by trimethylating histone 3 at lysine-4 and lysine-36 on nearby nucleosomes. Although this activation of hotspots is believed to occur on the chromatin loops, the subsequent double-strand break formation and repair occur on a proteinaceous structure known as the chromosome axis. We now show that Ewing sarcoma RNA binding protein 1 (EWSR1) is a key player in early recombination events, binding to PRDM9, promoting PRDM9-dependent histone methylation, and facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through the meiosis-specific cohesion REC8. As a result of these activities, EWSR1 assures sufficient numbers of properly positioned crossovers in each meiosis.

developmental biology↗

bioRxiv2018

CXXC1 is redundant for normal DNA double-strand break formation and meiotic recombination in mouse

In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break initiation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here we investigated whether CXXC1 has a similar function in mouse meiosis. We found that CXXC1 is co-expressed and interacts with PRDM9 in mouse spermatocytes. To investigate the meiotic function of CXXC1, we created a Cxxc1 conditional knockout mouse to deplete CXXC1 before the onset of meiosis. Surprisingly, knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on hotspot trimethylation activity, double-strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1.\n\nAuthor SummaryMeiotic recombination increases genetic diversity by ensuring novel combination of alleles passing onto the next generation correctly. In most mammals, the meiotic recombination sites are determined by histone methyltransferase PRDM9. These sites subsequently become associated with the chromosome axis with the participation of additional proteins and undergo double strand breaks, which are repaired by homologous recombination. In Saccharomyces cerevisiae, Spp1 (ortholog of CXXC1) binds to methylated H3K4 and connects these sites with chromosome axis promoting DSB formation. However, our data suggest that even though CXXC1 interacts with PRDM9 in male germ cells, it does not play a crucial role in mouse meiotic recombination. These results indicate that, unlike in S. cerevisiae, a recombination initiation pathway that includes CXXC1 could only serve as a non-essential pathway in mouse meiotic recombination.

cell biology↗