Frédéric Baudat

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.

Regulating double-stranded DNA break repair towards crossover or non-crossover during mammalian meiosis.

During meiosis the programmed induction of DNA double-stranded breaks (DSB) leads to crossover (CO) and non-crossover products (NCO). One key role of CO is to connect homologs before metaphase I and thus to ensure the proper reductional segregation. This role implies an accurate regulation of CO frequency with the establishment of at least one CO per chromosome arm. Current major challenges are to understand how CO and NCO formation are regulated and what is the role of NCO. We present here the current knowledge about CO and NCO and their regulation in mammals. CO density varies widely along chromosomes and their distribution is not random as they are subject to positive interference. As documented in the mouse and human, a significant excess of DSB are generated relative to the number of CO. In fact, evidence has been obtained for the formation of NCO products, for regulation of the choice of DSB repair towards CO or NCO and for a CO specific pathway. We discuss the roles of Msh4, Msh5 and Sycp1 which affect DSB repair and probably not only the CO pathway. We suggest that, in mammals, the regulation of NCO differs from that described in Saccharomyces cerevisiae.

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.

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.

Molecular basis for the regulation of the H3K4 methyltransferase activity of PRDM9.

PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2) and S-adenosylhomocysteine (AdoHcy), which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4) and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

Interallelic and Intergenic Incompatibilities of the Prdm9 (Hst1) Gene in Mouse Hybrid Sterility

The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect epistatic interactions. Although the mechanisms of speciation are of great interest, no incompatibility has been characterized at the gene level in mammals. The Hybrid sterility 1 gene (Hst1) participates in the arrest of meiosis in F1 males of certain strains from two Mus musculus subspecies, e.g., PWD from M. m. musculus and C57BL/6J (henceforth B6) from M. m. domesticus. Hst1 has been identified as a meiotic PR-domain gene (Prdm9) encoding histone 3 methyltransferase in the male offspring of PWD females and B6 males, (PWD×B6)F1. To characterize the incompatibilities underlying hybrid sterility, we phenotyped reproductive and meiotic markers in males with altered copy numbers of Prdm9. A partial rescue of fertility was observed upon removal of the B6 allele of Prdm9 from the azoospermic (PWD×B6)F1 hybrids, whereas removing one of the two Prdm9 copies in PWD or B6 background had no effect on male reproduction. Incompatibility(ies) not involving Prdm9B6 also acts in the (PWD×B6)F1 hybrids, since the correction of hybrid sterility by Prdm9B6 deletion was not complete. Additions and subtractions of Prdm9 copies, as well as allelic replacements, improved meiotic progression and fecundity also in the progeny-producing reciprocal (B6×PWD)F1 males. Moreover, an increased dosage of Prdm9 and reciprocal cross enhanced fertility of other sperm-carrying male hybrids, (PWD×B6-C3H.Prdm9)F1, harboring another Prdm9 allele of M. m. domesticus origin. The levels of Prdm9 mRNA isoforms were similar in the prepubertal testes of all types of F1 hybrids of PWD with B6 and B6-C3H.Prdm9 despite their different prospective fertility, but decreased to 53% after removal of Prdm9B6. Therefore, the Prdm9B6 allele probably takes part in posttranscriptional dominant-negative hybrid interaction(s) absent in the parental strains.

Dissecting the structure and mechanism of a complex duplication-triplication rearrangement in the DMD gene.

Pathogenic complex genomic rearrangements are being increasingly characterized at the nucleotide level, providing unprecedented opportunities to evaluate the complexities of mutational mechanisms. Here, we report the molecular characterization of a complex duplication–triplication rearrangement involving exons 45–60 of the DMD gene. Inverted repeats facilitated this complex rearrangement, which shares common genomic organization with the recently described duplication‐inverted triplication–duplication (DUP–TRP/INV‐DUP) events; specifically, a 690‐kb region comprising DMD exons from 45 to 60 was duplicated in tandem, and another 46‐kb segment containing exon 51 was inserted inversely in between them. Taking into consideration (1) the presence of a predicted PRDM9 binding site in the near vicinity of the junction involving two inverted L1 elements and (2) the inherent properties of X–Y chromosome recombination during male meiosis, we proposed an alternative two‐step model for the generation of this X‐linked DMD DUP–TRP/INV‐DUP event.

Parallel Detection of Crossovers and Noncrossovers in Mouse Germ Cells

The recombination between homologous chromosomes during the prophase of the first meiotic division plays an essential role in the formation of euploid gametes, as well as contributing to genetic diversity through the generation of new allele combinations. Two types of products are formed, crossovers (CO) and gene conversions not associated with a crossover, also called noncrossovers (NCO). They result from the repair through differentiated pathways of a common initiating event, DNA double-strand breaks, formation of which is programmed during early meiotic prophase. In contrast to CO, little is known on the frequency, distribution, and regulation of NCO in mammals, mostly due to the technical challenge represented by their detection. However, the development of batch sperm-typing methods allowed for the detection of meiotic NCO products at meiotic recombination hotspots in human and mice. Several recent studies based on this technique demonstrated that mammalian CO hotspots are sites of recombination initiation and that the formation of CO and NCO has different genetic requirements. Here, we describe a method for detecting and characterizing CO and NCO, which has been applied to the analysis of the mouse Psmb9 recombination hotspot.

Comment sont choisis les sites d’échanges entre chromosomes lors de la méiose ?

> Lors de la meiose, une seule replication du genome suivie de deux divisions permet a une cellule germinale diploide de produire des gametes haploides. La premiere division se distingue par le fait que les chromosomes homologues d’origines maternelle et paternelle segregent. Les echanges reciproques entre chromosomes homologues, ou crossingover, qui surviennent lors de la prophase de cette division jouent un role essentiel pour la formation de gametes fonctionnels : ils forment un lien physique entre les chromosomes homologues, necessaire a leur segregation. La recombinaison meiotique, a l’origine des crossingover, a egalement pour consequence de provoquer un reassortiment des alleles dans le genome, generant de la diversite genetique.

PRDM9, a driver of the genetic map

During meiosis, maternal and paternal chromosomes undergo exchanges by homologous recombination. This is essential for fertility and contributes to genome evolution. In many eukaryotes, sites of meiotic recombination, also called hotspots, are regions of accessible chromatin, but in many vertebrates, their location follows a distinct pattern and is specified by PR domain-containing protein 9 (PRDM9). The specification of meiotic recombination hotspots is achieved by the different activities of PRDM9: DNA binding, histone methyltransferase, and interaction with other proteins. Remarkably, PRDM9 activity leads to the erosion of its own binding sites and the rapid evolution of its DNA-binding domain. PRDM9 may also contribute to reproductive isolation, as it is involved in hybrid sterility potentially due to a reduction of its activity in specific heterozygous contexts.