Corinne Grey

PRDM9 is a Major Determinant of Meiotic Recombination Hotspots in humans and mice

Homing in on Hotspots The clustering of recombination in the genome, around locations known as hotspots, is associated with specific DNA motifs. Now, using a variety of techniques, three studies implicate a chromatin-modifying protein, the histone-methyltransferase PRDM9, as a major factor involved in human hotspots (see the Perspective by Cheung et al.). Parvanov et al. (p. 835, published online 31 December) mapped the locus in mice, and analyzed allelic variation in mice and humans, whereas Myers et al. (p. 876, published online 31 December) used a comparative analysis between human and chimpanzees to show that the recombination process leads to a self-destructive drive in which the very motifs that recruit hotspots are eliminated from our genome. Baudat et al. (p. 836, published online 31 December) took this analysis a step further to identify human allelic variants within Prdm9 that differed in the frequency at which they used hotspots. Furthermore, differential binding of this protein to different human alleles suggests that this protein interacts with specific DNA sequences. Thus, PDRM9 functions in the determination of recombination loci within the genome and may be a significant factor in the genomic differences between closely related species. A chromatin-modifying enzyme is implicated in the determination of recombination loci within the genome. Meiotic recombination events cluster into narrow segments of the genome, defined as hotspots. Here, we demonstrate that a major player for hotspot specification is the Prdm9 gene. First, two mouse strains that differ in hotspot usage are polymorphic for the zinc finger DNA binding array of PRDM9. Second, the human consensus PRDM9 allele is predicted to recognize the 13-mer motif enriched at human hotspots; this DNA binding specificity is verified by in vitro studies. Third, allelic variants of PRDM9 zinc fingers are significantly associated with variability in genome-wide hotspot usage among humans. Our results provide a molecular basis for the distribution of meiotic recombination in mammals, in which the binding of PRDM9 to specific DNA sequences targets the initiation of recombination at specific locations in the genome.

Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double‐strand breaks (DSBs) in Spo11−/− mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.

Mouse PRDM9 DNA-Binding Specificity Determines Sites of Histone H3 Lysine 4 Trimethylation for Initiation of Meiotic Recombination

The nature of the PRDM9 zinc finger domain determines the location of hotspots for meiotic recombination in the genome and promotes local histone H3K4 trimethylation.

Mutation of the Mouse Syce1 Gene Disrupts Synapsis and Suggests a Link between Synaptonemal Complex Structural Components and DNA Repair

In mammals, the synaptonemal complex is a structure required to complete crossover recombination. Although suggested by cytological work, in vivo links between the structural proteins of the synaptonemal complex and the proteins of the recombination process have not previously been made. The central element of the synaptonemal complex is traversed by DNA at sites of recombination and presents a logical place to look for interactions between these components. There are four known central element proteins, three of which have previously been mutated. Here, we complete the set by creating a null mutation in the Syce1 gene in mouse. The resulting disruption of synapsis in these animals has allowed us to demonstrate a biochemical interaction between the structural protein SYCE2 and the repair protein RAD51. In normal meiosis, this interaction may be responsible for promoting homologous synapsis from sites of recombination.

Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice. Principal Findings We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes. Conclusions/Significance Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.

The Molecular Chaperone Hsp90α Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.

MCM8- and MCM9-Deficient Mice Reveal Gametogenesis Defects and Genome Instability Due to Impaired Homologous Recombination

We generated knockout mice for MCM8 and MCM9 and show that deficiency for these genes impairs homologous recombination (HR)-mediated DNA repair during gametogenesis and somatic cells cycles. MCM8(-/-) mice are sterile because spermatocytes are blocked in meiotic prophase I, and females have only arrested primary follicles and frequently develop ovarian tumors. MCM9(-/-) females also are sterile as ovaries are completely devoid of oocytes. In contrast, MCM9(-/-) testes produce spermatozoa, albeit in much reduced quantity. Mcm8(-/-) and Mcm9(-/-) embryonic fibroblasts show growth defects and chromosomal damage and cannot overcome a transient inhibition of replication fork progression. In these cells, chromatin recruitment of HR factors like Rad51 and RPA is impaired and HR strongly reduced. We further demonstrate that MCM8 and MCM9 form a complex and that they coregulate their stability. Our work uncovers essential functions of MCM8 and MCM9 in HR-mediated DSB repair during gametogenesis, replication fork maintenance, and DNA repair.

Genome-wide control of the distribution of meiotic recombination.

Meiotic recombination events are not randomly distributed in the genome but occur in specific regions called recombination hotspots. Hotspots are predicted to be preferred sites for the initiation of meiotic recombination and their positions and activities are regulated by yet-unknown controls. The activity of the Psmb9 hotspot on mouse Chromosome 17 (Chr 17) varies according to genetic background. It is active in strains carrying a recombinant Chr 17 where the proximal third is derived from Mus musculus molossinus. We have identified the genetic locus required for Psmb9 activity, named Dsbc1 for Double-strand break control 1, and mapped this locus within a 6.7-Mb region on Chr 17. Based on cytological analysis of meiotic DNA double-strand breaks (DSB) and crossovers (COs), we show that Dsbc1 influences DSB and CO, not only at Psmb9, but in several other regions of Chr 17. We further show that CO distribution is also influenced by Dsbc1 on Chrs 15 and 18. Finally, we provide direct molecular evidence for the regulation in trans mediated by Dsbc1, by showing that it controls the CO activity at the Hlx1 hotspot on Chr 1. We thus propose that Dsbc1 encodes for a trans-acting factor involved in the specification of initiation sites of meiotic recombination genome wide in mice.

Mouse tetrad analysis provides insights into recombination mechanisms and hotspot evolutionary dynamics

The ability to examine all chromatids from a single meiosis in yeast tetrads has been indispensable for defining the mechanisms of homologous recombination initiated by DNA double-strand breaks (DSBs). Using a broadly applicable strategy for the analysis of chromatids from a single meiosis at two recombination hotspots in mouse oocytes and spermatocytes, we demonstrate here the unidirectional transfer of information—gene conversion—in both crossovers and noncrossovers. Whereas gene conversion in crossovers is associated with reciprocal exchange, the unbroken chromatid is not altered in noncrossover gene conversion events, providing strong evidence that noncrossovers arise from a distinct pathway. Gene conversion frequently spares the binding site of the hotspot-specifying protein PRDM9, with the result that erosion of the hotspot is slowed. Thus, mouse tetrad analysis demonstrates how unique aspects of mammalian recombination mechanisms shape hotspot evolutionary dynamics.

Interplay between the retinoblastoma protein and LEK1 specifies stem cells toward the cardiac lineage.

The molecular mechanisms governing early cardiogenesis are still largely unknown. Interestingly, the retinoblastoma protein (Rb), a regulator of cell cycle, has recently emerged as a new candidate regulating cell differentiation. Rb−/− mice die at midgestation and mice lacking E2f1/E2f3, downstream components of the Rb‐dependent transcriptional pathway, die of heart failure. To gain insight into the function of Rb pathway in early cardiogenesis, we used Rb−/− embryonic stem (ES) cells differentiating into cardiomyocytes. Rb−/− cells displayed a dramatic delay in expression of cardiac‐specific transcription factors and in turn in the whole process of cardiac differentiation. The phenotype of Rb−/− ES cell‐derived cardiomyocytes was rescued by reintroducing Rb in cardiac progenitors, by stimulating the BMP‐dependent cardiogenic pathway or by overexpression of Nkx2.5. ES cells deficient in the recently identified factor LEK1, a murine homolog of the cardiomyogenic factor 1, or specific disruption of Rb–LEK1 interaction into the nucleus of differentiating ES cells recapitulated the delay in cardiac differentiation of Rb−/− ES cells. Thus, we provide evidence for a novel Rb/LEK1‐dependent and BMP‐independent transcriptional program, which plays a pivotal role in priming ES cells toward a cardiac fate.