During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.chromosome segregation | meiosis | Schizosaccharomyces pombe | DSB formation | genetic separation of heterochromatin functions
Programmed DNA double-strand breaks (DSBs) in meiosis are formed by Spo11 (Rec12 in fission yeast), a topoisomerase II-like protein, which becomes covalently attached to DNA 5 ends. For DSB repair through homologous recombination, the protein must be removed from these DNA ends. We show here that Rec12 is endonucleolytically removed from DSB ends attached to a short oligonucleotide (Rec12-oligonucleotide complex), as is Spo11 in budding yeast. Fission yeast, however, has only one size class of Rec12-oligonucleotide complexes, whereas budding yeast has two size classes, suggesting different endonucleolytic regulatory mechanisms. Rec12-oligonucleotide generation strictly requires Ctp1 (Sae2 nuclease homolog), the Rad32 (Mre11) nuclease domain, and Rad50 of the MRN complex. Surprisingly, Nbs1 is not strictly required, indicating separable roles for the MRN subunits. On the basis of these and other data, we propose that Rad32 nuclease has the catalytic site for Rec12-oligonucleotide generation and is activated by Ctp1, which plays an additional role in meiotic recombination.The repair of DNA double-strand breaks (DSBs) is essential for living cells. Faithful repair requires processing of the DSB for creation of a singe-stranded DNA end that can invade an intact homologous DNA template for repair. In some cases, a protein is bound to the DSB end and must be removed for repair to proceed. One notable example occurs during meiosis, when programmed DSBs are made by Spo11 (or its homolog), which becomes covalently linked to the 5Ј DSB ends (17). Removal of the protein is essential for repair of the DSBs and subsequent formation of crossovers, which are important for the proper segregation of homologs at the first meiotic division as well as for the generation of genetic diversity. Removal of topoisomerases from DNA ends is also required for faithful repair when the topoisomerase reaction is aborted midway, as when cells are treated with topoisomerase inhibitors. Here, we address the mechanism of protein removal.Meiotic recombination in the fission yeast Schizosaccharomyces pombe is initiated by the formation of programmed DSBs by Rec12, its Spo11 homolog (4). To date, DSBs have been demonstrated by direct analysis of DNA only for S. pombe and the budding yeast Saccharomyces cerevisiae. This mechanism of recombination initiation (by DSB introduction) is thought to occur in most eukaryotes, since Spo11 homologs are widely conserved evolutionarily (17). After their formation, DSBs are repaired via homologous recombination. At least in S. cerevisiae, the DNA ends are subjected to 5Ј-to-3Ј exonucleolytic resection to create 3Ј OH single-stranded DNA (ssDNA) overhangs (29). The resected DNA ends are then coated with an ssDNA-binding protein and invade homologous duplex DNA by the action of Rad51, a RecA homolog, and its accessory proteins. Spo11 and its homologs, including Rec12, have sequence similarity to a type II topoisomerase from archaea, TopoVI (17). Consistent with this similarity, Spo11 and Rec12 introduce breaks in t...
In Schizosaccharomyces pombe, the RNAi pathway is required for the formation of pericentric heterochromatin, proper chromosome segregation, and repression of pericentric meiotic recombination. Here we demonstrate that, when the activity of the histone H3 Lys 14 (H3K14) acetyltransferase Mst2 is eliminated, the RNAi machinery is no longer required for pericentric heterochromatin functions. We further reveal that reducing RNA polymerase II recruitment to pericentric regions is essential for maintaining heterochromatin in the absence of RNAi.
PURPOSE. Affecting children by age 3, primary congenital glaucoma (PCG) can cause debilitating vision loss by the developmental impairment of aqueous drainage resulting in high intraocular pressure (IOP), globe enlargement, and optic neuropathy. TEK haploinsufficiency accounts for 5% of PCG in diverse populations, with low penetrance explained by variable dysgenesis of Schlemm's canal (SC) in mice. We report eight families with TEK-related PCG, and provide evidence for SVEP1 as a disease modifier in family 8 with a higher penetrance and severity. METHODS. Exome sequencing identified coding/splice site variants with an allele frequency less than 0.0001 (gnomAD). TEK variant effects were assayed in constructtransfected HEK293 cells via detection of autophosphorylated (active) TEK protein. An enucleated eye from an affected member of family 8 was examined via histology. SVEP1 expression in developing outflow tissues was detected by immunofluorescent staining of 7-day mouse anterior segments. SVEP1 stimulation of TEK expression in human umbilical vascular endothelial cells (HUVECs) was measured by TaqMan quantitative PCR. RESULTS. Heterozygous TEK loss-of-function alleles were identified in eight PCG families, with parent-child disease transmission observed in two pedigrees. Family 8 exhibited greater disease penetrance and severity, histology revealed absence of SC in one eye, and SVEP1:p.R997C was identified in four of the five affected individuals. During SC development, SVEP1 is secreted by surrounding tissues. SVEP1:p.R997C abrogates stimulation of TEK expression by HUVECs. CONCLUSIONS. We provide further evidence for PCG caused by TEK haploinsufficiency, affirm autosomal dominant inheritance in two pedigrees, and propose SVEP1 as a modifier of TEK expression during SC development, affecting disease penetrance and severity.
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