PRDM9 points the zinc finger at meiotic recombination hotspots

Meiotic recombination events are spread nonrandomly across eukaryotic genomes in 'hotspots'. Recent work shows that a unique histone methyltransferase, PRDM9, determines their distribution.     

DNA binding specificities of the long zinc-finger recombination protein PRDM9

Background

Meiotic recombination ensures proper segregation of homologous chromosomes and creates genetic variation. In many organisms, recombination occurs at limited sites, termed ''hotspots'', whose positions in mammals are determined by PR domain member 9 (PRDM9), a long-array zinc-finger and chromatin-modifier protein. Determining the rules governing the DNA binding of PRDM9 is a major issue in understanding how it functions.

Results

Mouse PRDM9 protein variants bind to hotspot DNA sequences in a manner that is specific for both PRDM9 and DNA haplotypes, and that in vitro binding parallels its in vivo biological activity. Examining four hotspots, three activated by Prdm9^Cst and one activated by Prdm9^Dom2, we found that all binding sites required the full array of 11 or 12 contiguous fingers, depending on the allele, and that there was little sequence similarity between the binding sites of the three Prdm9^Cst activated hotspots. The binding specificity of each position in the Hlx1 binding site, activated by Prdm9^Cst, was tested by mutating each nucleotide to its three alternatives. The 31 positions along the binding site varied considerably in the ability of alternative bases to support binding, which also implicates a role for additional binding to the DNA phosphate backbone.

Conclusions

These results, which provide the first detailed mapping of PRDM9 binding to DNA and, to our knowledge, the most detailed analysis yet of DNA binding by a long zinc-finger array, make clear that the binding specificities of PRDM9, and possibly other long-array zinc-finger proteins, are unusually complex.     

Detecting sequence polymorphisms associated with meiotic recombination hotspots in the human genome

Background  

Meiotic recombination events tend to cluster into narrow spans of a few kilobases long, called recombination hotspots. Such hotspots are not conserved between human and chimpanzee and vary between different human ethnic groups. At the same time, recombination hotspots are heritable. Previous studies showed instances where differences in recombination rate could be associated with sequence polymorphisms.     

Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.     

Evidence of positive selection and concerted evolution in the rapidly evolving PRDM9 zinc finger domain in goats and sheep

Meiotic recombination contributes to augmentation of genetic diversity, exclusion of deleterious alleles and proper segregation of chromatids. PRDM9 has been identified as the gene responsible for specifying the location of recombination hotspots during meiosis and is also the only known vertebrate gene associated with reproductive isolation between species. PRDM9 encodes a protein with a highly variable zinc finger (ZF) domain that varies between as well as within species. In the present study, the ZF domain of PRDM9 on chromosome 1 was characterized for the first time in 15 goat breeds and 25 sheep breeds of India. A remarkable variation in the number and sequence of ZF domains was observed. The number of ZF repeats in the ZF array varied from eight to 12 yielding five homozygous and 10 heterozygous genotypes. The number of different ZF domains was 84 and 52 producing 36 and 26 unique alleles in goats and sheep respectively. The posterior mean of dN/dS or omega values were calculated using the codeml tool of pamlx to identify amino acids that are evolving positively in goats and sheep, as positions ?1, +3 and +6 in the ZF domain have been reported to experience strong positive selection across different lineages. Our study identified sites ?5, ?1, +3, +4 and +6 to be experiencing positive selection. Small ruminant zinc fingers were also found to be evolving under concerted evolution. Our results demonstrate the existence of a vast diversity of PRDM9 in goats and sheep, which is in concert with reports in many metazoans.     

A Highlights from MBoC Selection: EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis protein REC8

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 Lys-4 and Lys-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 model 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 through its component cohesin pREC8.     

Context dependence of meiotic recombination hotspots in yeast: the relationship between recombination activity of a reporter construct and base composition

Borde and colleagues reported that a reporter plasmid inserted at different genomic locations in Saccharomyces cerevisiae had different levels of meiotic recombination activity. We show that the level of recombination activity is very significantly correlated with the GC content of DNA sequences flanking the insertion.     

The synaptonemal complex and meiotic recombination in humans: new approaches to old questions

Meiotic prophase serves as an arena for the interplay of two important cellular activities, meiotic recombination and synapsis of homologous chromosomes. Synapsis is mediated by the synaptonemal complex (SC), originally characterized as a structure linked to pairing of meiotic chromosomes (Moses (1958) J Biophys Biochem Cytol 4:633–638). In 1975, the first electron micrographs of human pachytene stage SCs were presented (Moses et al. (1975) Science 187:363–365) and over the next 15 years the importance of the SC to normal meiotic progression in human males and females was established (Jhanwar and Chaganti (1980) Hum Genet 54:405–408; Pathak and Elder (1980) Hum Genet 54:171–175; Solari (1980) Chromosoma 81:315–337; Speed (1984) Hum Genet 66:176–180; Wallace and Hulten (1985) Ann Hum Genet 49(Pt 3):215–226). Further, these studies made it clear that abnormalities in the assembly or maintenance of the SC were an important contributor to human infertility (Chaganti et al. (1980) Am J Hum Genet 32:833–848; Vidal et al. (1982) Hum Genet 60:301–304; Bojko (1983) Carlsberg Res Commun 48:285–305; Bojko (1985) Carlsberg Res Commun 50:43–72; Templado et al. (1984) Hum Genet 67:162–165; Navarro et al. (1986) Hum Reprod 1:523–527; Garcia et al. (1989) Hum Genet 2:147–53). However, the utility of these early studies was limited by lack of information on the structural composition of the SC and the identity of other SC-associated proteins. Fortunately, studies of the past 15 years have gone a long way toward remedying this problem. In this minireview, we highlight the most important of these advances as they pertain to human meiosis, focusing on temporal aspects of SC assembly, the relationship between the SC and meiotic recombination, and the contribution of SC abnormalities to human infertility.The synaptonemal complex–50 years     

Effect of meiotic recombination on the production of aneuploid gametes in humans

Within the last decade, aberrant meiotic recombination has been confirmed as a molecular risk factor for chromosome nondisjunction in humans. Recombination tethers homologous chromosomes, linking and guiding them through proper segregation at meiosis I. In model organisms, mutations that disturb the recombination pathway increase the frequency of chromosome malsegregation and alterations in both the amount and placement of meiotic recombination are associated with nondisjunction. This association has been established for humans as well. Significant alterations in recombination have been found for all meiosis I-derived trisomies studied to date and a subset of so called "meiosis II" trisomy. Often exchange levels are reduced in a subset of cases where the nondisjoining chromosome fails to undergo recombination. For other trisomies, the placement of meiotic recombination has been altered. It appears that recombination too near the centromere or too far from the centromere imparts an increased risk for nondisjunction. Recent evidence from trisomy 21 also suggests an association may exist between recombination and maternal age, the most widely identified risk factor for aneuploidy. Among cases of maternal meiosis I-derived trisomy 21, increasing maternal age is associated with a decreasing frequency of recombination in the susceptible pericentromeric and telomeric regions. It is likely that multiple risk factors lead to nondisjunction, some age dependent and others age independent, some that act globally and others that are chromosome specific. Future studies are expected to shed new light on the timing and placement of recombination, providing additional clues to the link between altered recombination and chromosome nondisjunction.     

Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers

This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.     

The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3' long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP's activity, whereas disruption of the first and third fingers just slightly lowered its activity.     

The mammalian synaptonemal complex: protein components, assembly and role in meiotic recombination

The synaptonemal complex (SC) is a proteinaceous structure of chromosome bivalents whose assembly is indispensable for the successful progression of the first meiotic division of sexually reproducing organisms. In this mini-review we will focus on recent progress dealing with the composition and assembly of the mammalian SC. These advances mainly resulted from the systematic use of knockout mice for all known mammalian SC proteins as well as from protein polymerization studies performed in heterologous systems.     

Identification of four CCCH zinc finger proteins in Xenopus, including a novel vertebrate protein with four zinc fingers and severely restricted expression

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor alpha secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001-1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four Xenopus cDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx8Cx5Cx3H repeats, two of which are YKTEL Cx8Cx5Cx3H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.     

Genetic analysis of meiotic recombination in humans by use of sperm typing: reduced recombination within a heterozygous paracentric inversion of chromosome 9q32-q34.3.

To investigate patterns of genetic recombination within a heterozygous paracentric inversion of chromosome 9 (46XY inv[9] [q32q34.3]), we performed sperm typing using a series of polymorphic microsatellite markers spanning the inversion region. For comparison, two donors with cytogenetically normal chromosomes 9, one of whom was heterozygous for a pericentric chromosome 2 inversion (46XY inv[2] [p11q13]), were also tested. Linkage analysis was performed by use of the multilocus linkage-analysis program SPERM, and also CRI-MAP, which was adapted for sperm-typing data. Analysis of the controls generated a marker order in agreement with previously published data and revealed no significant interchromosomal effects of the inv(2) on recombination on chromosome 9. FISH employing cosmids containing appropriate chromosome 9 markers was used to localize the inversion breakpoint of inv(9). Analysis of inv(9) sperm was performed by use of a set of microsatellite markers that mapped centromeric to, telomeric to, and within the inversion breakpoints. Three distinct patterns of recombination across the region were observed. Proximal to the centromeric breakpoint, recombination was similar to normal levels. Distal to the telomeric breakpoint, there was an increase in recombination found in the inversion patient. Finally, within the inversion, recombination was dramatically reduced, but several apparent double recombinants were found. A putative model explaining these data is proposed.     

The ssDNA-binding protein MEIOB acts as a dosage-sensitive regulator of meiotic recombination

Meiotic recombination enables reciprocal exchange of genetic information between parental chromosomes and is essential for fertility. MEIOB, a meiosis-specific ssDNA-binding protein, regulates early meiotic recombination. Here we report that the human infertility-associated missense mutation (N64I) in MEIOB causes protein degradation and reduced crossover formation in mouse testes. Although the MEIOB N64I substitution is associated with human infertility, the point mutant mice are fertile despite meiotic defects. Meiob mutagenesis identifies serine 67 as a critical residue for MEIOB. Biochemically, these two mutations (N64I and S67 deletion) cause self-aggregation of MEIOB and sharply reduced protein half-life. Molecular genetic analyses of both point mutants reveal an important role for MEIOB in crossover formation in late meiotic recombination. Furthermore, we find that the MEIOB protein levels directly correlate with the severity of meiotic defects. Our results demonstrate that MEIOB regulates meiotic recombination in a dosage-dependent manner.     

Prdm9, a major determinant of meiotic recombination hotspots, is not functional in dogs and their wild relatives, wolves and coyotes

Meiotic recombination is a fundamental process needed for the correct segregation of chromosomes during meiosis in sexually reproducing organisms. In humans, 80% of crossovers are estimated to occur at specific areas of the genome called recombination hotspots. Recently, a protein called PRDM9 was identified as a major player in determining the location of genome-wide meiotic recombination hotspots in humans and mice. The origin of this protein seems to be ancient in evolutionary time, as reflected by its fairly conserved structure in lineages that diverged over 700 million years ago. Despite its important role, there are many animal groups in which Prdm9 is absent (e.g. birds, reptiles, amphibians, diptera) and it has been suggested to have disruptive mutations and thus to be a pseudogene in dogs. Because of the dog's history through domestication and artificial selection, we wanted to confirm the presence of a disrupted Prdm9 gene in dogs and determine whether this was exclusive of this species or whether it also occurred in its wild ancestor, the wolf, and in a close relative, the coyote. We sequenced the region in the dog genome that aligned to the last exon of the human Prdm9, containing the entire zinc finger domain, in 4 dogs, 17 wolves and 2 coyotes. Our results show that the three canid species possess mutations that likely make this gene non functional. Because these mutations are shared across the three species, they must have appeared prior to the split of the wolf and the coyote, millions of years ago, and are not related to domestication. In addition, our results suggest that in these three canid species recombination does not occur at hotspots or hotspot location is controlled through a mechanism yet to be determined.