RESUMO
Heritability and genome stability are shaped by meiotic recombination, which is initiated via hundreds of DNA double-strand breaks (DSBs). The distribution of DSBs throughout the genome is not random, but mechanisms molding this landscape remain poorly understood. Here, we exploit genome-wide maps of mouse DSBs at unprecedented nucleotide resolution to uncover previously invisible spatial features of recombination. At fine scale, we reveal a stereotyped hotspot structure-DSBs occur within narrow zones between methylated nucleosomes-and identify relationships between SPO11, chromatin, and the histone methyltransferase PRDM9. At large scale, DSB formation is suppressed on non-homologous portions of the sex chromosomes via the DSB-responsive kinase ATM, which also shapes the autosomal DSB landscape at multiple size scales. We also provide a genome-wide analysis of exonucleolytic DSB resection lengths and elucidate spatial relationships between DSBs and recombination products. Our results paint a comprehensive picture of features governing successive steps in mammalian meiotic recombination.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Instabilidade Genômica/genética , Recombinação Homóloga , Meiose/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nucleossomos/enzimologia , Nucleossomos/genética , Cromossomo X/genética , Cromossomo Y/genéticaRESUMO
Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Resection tracts averaged 1100 nt, but with substantial fine-scale heterogeneity at individual hot spots. Surprisingly, EXO1 is not the major 5' â 3' exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3' ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.
Assuntos
Reparo do DNA/fisiologia , Meiose , Animais , Cromatina/química , Cromatina/metabolismo , DNA/química , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Estrutura Molecular , Recombinação GenéticaRESUMO
The exchange of genetic information between parental chromosomes in meiosis is an integral process for the creation of gametes. To generate a crossover, hundreds of DNA double-strand breaks (DSBs) are introduced in the genome of each meiotic cell by the SPO11 protein. The nucleolytic resection of DSB-adjacent DNA is a key step in meiotic DSB repair, but this process has remained understudied. In this issue of Genes & Development, Yamada and colleagues (pp. 806-818) capture some of the first details of resection and DSB repair intermediates in mouse meiosis using a method that maps blunt-ended DNA after ssDNA digestion. This yields some of the first genome-wide insights into DSB resection and repair in a mammalian genome and offers a tantalizing glimpse of how to quantitatively dissect this difficult to study, yet integral, nuclear process.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Meiose , Animais , Cromatina/química , Cromatina/metabolismo , DNA/química , Meiose/genética , Estrutura Molecular , Recombinação GenéticaRESUMO
Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. They also have delayed and/or fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures-stochastic on autosomes, nearly absolute on X and Y-cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines a pleckstrin homology (PH) domain in REC114 and its direct intermolecular contacts with ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model in which ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, thereby regulating DSB formation.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Recombinação Homóloga/genética , Meiose/genética , Recombinases/química , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Pareamento Cromossômico , Segregação de Cromossomos/genética , Cromossomos , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Feminino , Masculino , Camundongos , Conformação Proteica , Recombinases/genética , Espermatócitos/química , Espermatócitos/metabolismoRESUMO
Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.
Assuntos
Proteínas de Transporte/genética , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga/genética , Meiose/genética , Animais , Proteínas de Transporte/química , Segregação de Cromossomos/genética , Masculino , Camundongos , Regiões Pseudoautossômicas/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo , Cromossomo X/genética , Cromossomo Y/genéticaRESUMO
In many mammals, recombination events are concentrated in hotspots directed by a sequence-specific DNA-binding protein named PRDM9. Intriguingly, PRDM9 has been lost several times in vertebrates, and notably among mammals, it has been pseudogenized in the ancestor of canids. In the absence of PRDM9, recombination hotspots tend to occur in promoter-like features such as CpG islands. It has thus been proposed that one role of PRDM9 could be to direct recombination away from PRDM9-independent hotspots. However, the ability of PRDM9 to direct recombination hotspots has been assessed in only a handful of species, and a clear picture of how much recombination occurs outside of PRDM9-directed hotspots in mammals is still lacking. In this study, we derived an estimator of past recombination activity based on signatures of GC-biased gene conversion in substitution patterns. We quantified recombination activity in PRDM9-independent hotspots in 52 species of boreoeutherian mammals. We observe a wide range of recombination rates at these loci: several species (such as mice, humans, some felids, or cetaceans) show a deficit of recombination, while a majority of mammals display a clear peak of recombination. Our results demonstrate that PRDM9-directed and PRDM9-independent hotspots can coexist in mammals and that their coexistence appears to be the rule rather than the exception. Additionally, we show that the location of PRDM9-independent hotspots is relatively more stable than that of PRDM9-directed hotspots, but that PRDM9-independent hotspots nevertheless evolve slowly in concert with DNA hypomethylation.
Assuntos
Histona-Lisina N-Metiltransferase , Recombinação Genética , Animais , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Mamíferos/genética , Ilhas de CpG/genética , Eutérios/genética , Camundongos , Feminino , Conversão Gênica , Evolução MolecularRESUMO
Canonical Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) act as major repressors of transposable elements (TEs) via the KRAB-mediated recruitment of the heterochromatin scaffold KRAB-associated protein (KAP)1. KZFP genes emerged some 420 million years ago in the last common ancestor of coelacanth, lungfish, and tetrapods, and dramatically expanded to give rise to lineage-specific repertoires in contemporary species paralleling their TE load and turnover. However, the KRAB domain displays sequence and function variations that reveal repeated diversions from a linear TE-KZFP trajectory. This Review summarizes current knowledge on the evolution of KZFPs and discusses how ancestral noncanonical KZFPs endowed with variant KRAB, SCAN or DUF3669 domains have been utilized to achieve KAP1-independent functions.
Assuntos
Proteínas Repressoras , Dedos de Zinco , Proteínas Repressoras/genética , Dedos de Zinco/genética , Fatores de Transcrição/genética , Elementos de DNA Transponíveis , HeterocromatinaRESUMO
The programmed formation of hundreds of DNA double-strand breaks (DSBs) is essential for proper meiosis and fertility. In mice and humans, the location of these breaks is determined by the meiosis-specific protein PRDM9, through the DNA-binding specificity of its zinc-finger domain. PRDM9 also has methyltransferase activity. Here, we show that this activity is required for H3K4me3 and H3K36me3 deposition and for DSB formation at PRDM9-binding sites. By analyzing mice that express two PRDM9 variants with distinct DNA-binding specificities, we show that each variant generates its own set of H3K4me3 marks independently from the other variant. Altogether, we reveal several basic principles of PRDM9-dependent DSB site determination, in which an excess of sites are designated through PRDM9 binding and subsequent histone methylation, from which a subset is selected for DSB formation.
Assuntos
Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Meiose/fisiologia , Animais , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Metilação , Camundongos , Camundongos Transgênicos , Domínios ProteicosRESUMO
Chromosomal fusions represent one of the most common types of chromosomal rearrangements found in nature. Yet, their role in shaping the genomic landscape of recombination and hence genome evolution remains largely unexplored. Here, we take advantage of wild mice populations with chromosomal fusions to evaluate the effect of this type of structural variant on genomic landscapes of recombination and divergence. To this aim, we combined cytological analysis of meiotic crossovers in primary spermatocytes with inferred analysis of recombination rates based on linkage disequilibrium using single nucleotide polymorphisms. Our results suggest the presence of a combined effect of Robertsonian fusions and Prdm9 allelic background, a gene involved in the formation of meiotic double strand breaks and postzygotic reproductive isolation, in reshaping genomic landscapes of recombination. We detected a chromosomal redistribution of meiotic recombination toward telomeric regions in metacentric chromosomes in mice with Robertsonian fusions when compared to nonfused mice. This repatterning was accompanied by increased levels of crossover interference and reduced levels of estimated recombination rates between populations, together with high levels of genomic divergence. Interestingly, we detected that Prdm9 allelic background was a major determinant of recombination rates at the population level, whereas Robertsonian fusions showed limited effects, restricted to centromeric regions of fused chromosomes. Altogether, our results provide new insights into the effect of Robertsonian fusions and Prdm9 background on meiotic recombination.
Assuntos
Cromossomos , Genômica , Masculino , Animais , Camundongos , AlelosRESUMO
In most mammals and likely throughout vertebrates, the gene PRDM9 specifies the locations of meiotic double strand breaks; in mice and humans at least, it also aids in their repair. For both roles, many of the molecular partners remain unknown. Here, we take a phylogenetic approach to identify genes that may be interacting with PRDM9 by leveraging the fact that PRDM9 arose before the origin of vertebrates but was lost many times, either partially or entirely-and with it, its role in recombination. As a first step, we characterize PRDM9 domain composition across 446 vertebrate species, inferring at least 13 independent losses. We then use the interdigitation of PRDM9 orthologs across vertebrates to test whether it coevolved with any of 241 candidate genes coexpressed with PRDM9 in mice or associated with recombination phenotypes in mammals. Accounting for the phylogenetic relationship among a subsample of 189 species, we find two genes whose presence and absence is unexpectedly coincident with that of PRDM9: ZCWPW1, which was recently shown to facilitate double strand break repair, and its paralog ZCWPW2, as well as, more tentatively, TEX15 and FBXO47ZCWPW2 is expected to be recruited to sites of PRDM9 binding; its tight coevolution with PRDM9 across vertebrates suggests that it is a key interactor within mammals and beyond, with a role either in recruiting the recombination machinery or in double strand break repair.
Assuntos
Proteínas de Ciclo Celular/genética , Deleção de Genes , Histona-Lisina N-Metiltransferase/genética , Animais , Evolução Molecular , Humanos , Camundongos , Filogenia , Recombinação Genética , Análise de Sequência de RNA/métodosRESUMO
Meiotic recombination is an important evolutionary force and an essential meiotic process. In many species, recombination events concentrate into hotspots defined by the site-specific binding of PRMD9. Rapid evolution of Prdm9's zinc finger DNA-binding array leads to remarkably abrupt shifts in the genomic distribution of hotspots between species, but the question of how Prdm9 allelic variation shapes the landscape of recombination between populations remains less well understood. Wild house mice (Mus musculus) harbor exceptional Prdm9 diversity, with >150 alleles identified to date, and pose a particularly powerful system for addressing this open question. We employed a coalescent-based approach to construct broad- and fine-scale sex-averaged recombination maps from contemporary patterns of linkage disequilibrium in nine geographically isolated wild house mouse populations, including multiple populations from each of three subspecies. Comparing maps between wild mouse populations and subspecies reveals several themes. First, we report weak fine- and broad-scale recombination map conservation across subspecies and populations, with genetic divergence offering no clear prediction for recombination map divergence. Second, most hotspots are unique to one population, an outcome consistent with minimal sharing of Prdm9 alleles between surveyed populations. Finally, by contrasting aggregate hotspot activity on the X versus autosomes, we uncover evidence for population-specific differences in the degree and direction of sex dimorphism for recombination. Overall, our findings illuminate the variability of both the broad- and fine-scale recombination landscape in M. musculus and underscore the functional impact of Prdm9 allelic variation in wild mouse populations.
Assuntos
Evolução Molecular , Variação Genética , Histona-Lisina N-Metiltransferase , Camundongos , Animais , Camundongos/genética , Cromossomos/genética , Genoma , Histona-Lisina N-Metiltransferase/genéticaRESUMO
Hybrid sterility (HS) is an early postzygotic reproductive isolation mechanism observed in all sexually reproducing species. Infertility of hybrids prevents gene flow between incipient species and leads to speciation. While Drosophila studies have focused almost exclusively on the genic control of HS, two other model species, Mus musculus and budding yeast, provided the first experimental evidence of hybrid sterility governed by the nongenic effects of DNA sequence divergence. Here, we propose that the nongenic effect of increasing DNA divergence between closely related species may impair mutual recognition of homologous chromosomes and disrupt their synapsis. Unsynapsed or mispaired homologs can induce early meiotic arrest, or their random segregation can cause aneuploidy of spermatids and sperm cells. Impaired recognition of homologs may thus act as a universal chromosomal checkpoint contributing to the complexity of genetic control of HS. Chromosomal HS controlled by the Prdm9 gene in mice and HS driven by the mismatch repair machinery in yeast are currently the most advanced examples of chromosomal homology search-based HS. More focus on the cellular and molecular phenotypes of meiosis will be needed to further validate the role of homolog recognition in hybrid sterility and speciation.
Assuntos
Infertilidade Masculina , Infertilidade , Masculino , Camundongos , Animais , Humanos , Hibridização Genética , Sementes , Infertilidade/genética , Cromossomos , Meiose , Saccharomyces cerevisiae/genética , Infertilidade Masculina/genética , Histona-Lisina N-Metiltransferase/genéticaRESUMO
Hybrid sterility is a critical step in the evolution of reproductive barriers between diverging taxa during the process of speciation. Recent studies of young subspecies of the house mouse revealed a multigenic nature and frequent polymorphism of hybrid sterility genes as well as the recurrent engagement of the meiosis-specific gene PR domain-containing 9 (Prdm9) and X-linked loci. Prdm9-controlled hybrid sterility is essentially chromosomal in nature, conditioned by the sequence divergence between subspecies. Depending on the Prdm9 interallelic interactions and the X-linked Hstx2 locus, the same homologs either regularly recombine and synapse, or show impaired DNA DSB repair, asynapsis, and early meiotic arrest. Thus, Prdm9-dependent hybrid sterility points to incompatibilities affecting meiotic recombination as a possible mechanism of reproductive isolation between (sub)species.
Assuntos
Infertilidade Masculina , Animais , Histona-Lisina N-Metiltransferase/genética , Recombinação Homóloga , Infertilidade Masculina/genética , Meiose/genética , Camundongos , Isolamento ReprodutivoRESUMO
The multidomain zinc finger (ZnF) protein PRDM9 (PRD1-BF1-RIZ1 homologous domain-containing 9) is thought to influence the locations of recombination hot spots during meiosis by sequence-specific DNA binding and trimethylation of histone H3 Lys4. The most common variant of human PRDM9, allele A (hPRDM9A), recognizes the consensus sequence 5'-NCCNCCNTNNCCNCN-3'. We cocrystallized ZnF8-12 of hPRDM9A with an oligonucleotide representing a known hot spot sequence and report the structure here. ZnF12 was not visible, but ZnF8-11, like other ZnF arrays, follows the right-handed twist of the DNA, with the α helices occupying the major groove. Each α helix makes hydrogen-bond (H-bond) contacts with up to four adjacent bases, most of which are purines of the complementary DNA strand. The consensus C:G base pairs H-bond with conserved His or Arg residues in ZnF8, ZnF9, and ZnF11, and the consensus T:A base pair H-bonds with an Asn that replaces His in ZnF10. Most of the variable base pairs (N) also engage in H bonds with the protein. These interactions appear to compensate to some extent for changes from the consensus sequence, implying an adaptability of PRDM9 to sequence variations. We investigated the binding of various alleles of hPRDM9 to different hot spot sequences. Allele C was found to bind a C-specific hot spot with higher affinity than allele A bound A-specific hot spots, perhaps explaining why the former is dominant in A/C heterozygotes. Allele L13 displayed higher affinity for several A-specific sequences, allele L9/L24 displayed lower affinity, and allele L20 displayed an altered sequence preference. These differences can be rationalized structurally and might contribute to the variation observed in the locations and activities of meiotic recombination hot spots.
Assuntos
Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Modelos Moleculares , Recombinação Genética , Alelos , Sequência de Bases , Cristalização , Histonas , Humanos , Meiose/genética , Metilação , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation.
Assuntos
Evolução Biológica , Especiação Genética , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos/classificação , Camundongos/genética , Recombinação Genética/genética , Alelos , Animais , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/genética , Hibridização Genética , Ligação ProteicaRESUMO
The PR domain-containing 9 or PRDM9 is a gene recognized for its fundamental role in meiosis, a process essential for forming reproductive cells. Recent findings have implicated alterations in the PRDM9, particularly its zinc finger motifs, in the onset and progression of cancer. This association is manifested through genomic instability and the misregulation of genes critical to cell growth, proliferation, and differentiation. In our comprehensive study, we harnessed advanced bioinformatic mining tools to delve deep into the intricate relationship between PRDM9F and cancer. We analyzed 136,752 breakpoints and found an undeniable association between specific PRDM9 motifs and the occurrence of double-strand breaks, a phenomenon evidenced in every cancer profile examined. Utilizing R statistical querying and the Regioner package, 55 unique sequence variations of PRDM9 were statistically correlated with cancer, from a pool of 1024 variations. A robust analysis using the Enrichr tool revealed prominent associations with various cancer types. Moreover, connections were noted with specific phenotypic conditions and molecular functions, underlining the pervasive influence of PRDM9 variations in the biological spectrum. The Reactome tool identified 25 significant pathways associated with cancer, offering insights into the mechanistic underpinnings linking PRDM9 to cancer progression. This detailed analysis not only confirms the pivotal role of PRDM9 in cancer development, but also unveils a complex network of biological processes influenced by its variations. The insights gained lay a solid foundation for future research aimed at deciphering the mechanistic pathways of PRDM9, offering prospects for targeted interventions and innovative therapeutic approaches in cancer management.
Assuntos
Instabilidade Genômica , Histona-Lisina N-Metiltransferase , Neoplasias , Humanos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga , Meiose , Neoplasias/genética , Neoplasias/metabolismoRESUMO
BACKGROUND: PRDM9 is a key regulator of meiotic recombination in most metazoans, responsible for reshuffling parental genomes. During meiosis, the PRDM9 protein recognizes and binds specific target motifs via its array of C2H2 zinc-fingers encoded by a rapidly evolving minisatellite. The gene coding for PRDM9 is the only speciation gene identified in vertebrates to date and shows high variation, particularly in the DNA-recognizing positions of the zinc-finger array, within and between species. Across all vertebrate genomes studied for PRDM9 evolution, only one genome lacks variability between repeat types - that of the North Pacific minke whale. This study aims to understand the evolution and diversity of Prdm9 in minke whales, which display the most unusual genome reference allele of Prdm9 so far discovered in mammals. RESULTS: Minke whales possess all the features characteristic of PRDM9-directed recombination, including complete KRAB, SSXRD and SET domains and a rapidly evolving array of C2H2-type-Zincfingers (ZnF) with evidence of rapid evolution, particularly at DNA-recognizing positions that evolve under positive diversifying selection. Seventeen novel PRDM9 variants were identified within the Antarctic minke whale species, plus a single distinct PRDM9 variant in Common minke whales - shared across North Atlantic and North Pacific minke whale subspecies boundaries. CONCLUSION: The PRDM9 ZnF array evolves rapidly, in minke whales, with at least one DNA-recognizing position under positive selection. Extensive PRDM9 diversity is observed, particularly in the Antarctic in minke whales. Common minke whales shared a specific Prdm9 allele across subspecies boundaries, suggesting incomplete speciation by the mechanisms associated with PRDM9 hybrid sterility.
Assuntos
Baleia Anã , Alelos , Animais , Histona-Lisina N-Metiltransferase/genética , Meiose , Baleia Anã/genética , Dedos de Zinco/genéticaRESUMO
BACKGROUND: Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mouse Prdm9 gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear. RESULTS: We introduced Prdm9 deletions into the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding to Prdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9 mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mouse Prdm9 mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants. CONCLUSIONS: We hypothesize that the relative increased fertility of rat versus mouse Prdm9 mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation-spermatid development and reproductive age-that may help to explain male-specific hybrid sterility.
Assuntos
Meiose , Animais , Cromatina , Quebras de DNA de Cadeia Dupla , Feminino , Fertilidade/genética , Histona-Lisina N-Metiltransferase/genética , Masculino , Meiose/genética , Camundongos , Ratos , Ratos Endogâmicos SHR , Espermatogênese/genéticaRESUMO
Krüppel-associated box (KRAB) zinc finger proteins are a large class of tetrapod transcription factors that usually exert transcriptional repression through recruitment of TRIM28/KAP1. The evolutionary root of modern KRAB domains (mKRAB) can be traced back to an ancestral motif (aKRAB) that occurs even in invertebrates. Here, we first stratified three subgroups of aKRAB sequences from the animal kingdom (PRDM9, SSX and coelacanth KZNF families) and defined ancestral subdomains for KRAB-A and KRAB-B. Using human ZNF10 mKRAB-AB as blueprints for function, we then identified the necessary amino acid changes that transform the inactive aKRAB-A of human PRDM9 into an mKRAB domain capable of mediating silencing and complexing TRIM28/KAP1 in human cells when employed as a hybrid with ZNF10-B. Full gain of function required replacement of residues KR by the conserved motif MLE (positionsA32-A34), which inserted an additional residue, and exchange of A9/S for F, A20/M for L, and A27/R for V. AlphaFold2 modelling documented an evolutionary conserved L-shaped body of two α-helices in all KRAB domains. It is transformed into a characteristic spatial arrangement typical for mKRAB-AB upon the amino acid replacements and in conjunction with a third helix supplied by mKRAB-B. Side-chains pointing outward from the core KRAB 3D structure may reveal a protein-protein interaction code enabling graded binding of TRIM28 to different KRAB domains. Our data provide basic insights into structure-function relationships and emulate transitions of KRAB during evolution.
Assuntos
Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Invertebrados/metabolismo , Fatores de Transcrição Kruppel-Like/química , Proteínas Repressoras/química , Proteína 28 com Motivo Tripartido/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Evolução Molecular , Mutação com Ganho de Função , Histona-Lisina N-Metiltransferase/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Proteínas Repressoras/genéticaRESUMO
Meiotic recombination in vertebrates is concentrated in hotspots throughout the genome. The location and stability of hotspots have been linked to the presence or absence of PRDM9, leading to two primary models for hotspot evolution derived from mammals and birds. Species with PRDM9-directed recombination have rapid turnover of hotspots concentrated in intergenic regions (i.e., mammals), whereas hotspots in species lacking PRDM9 are concentrated in functional regions and have greater stability over time (i.e., birds). Snakes possess PRDM9, yet virtually nothing is known about snake recombination. Here, we examine the recombination landscape and test hypotheses about the roles of PRDM9 in rattlesnakes. We find substantial variation in recombination rate within and among snake chromosomes, and positive correlations between recombination rate and gene density, GC content, and genetic diversity. Like mammals, snakes appear to have a functional and active PRDM9, but rather than being directed away from genes, snake hotspots are concentrated in promoters and functional regions-a pattern previously associated only with species that lack a functional PRDM9. Snakes therefore provide a unique example of recombination landscapes in which PRDM9 is functional, yet recombination hotspots are associated with functional genic regions-a combination of features that defy existing paradigms for recombination landscapes in vertebrates. Our findings also provide evidence that high recombination rates are a shared feature of vertebrate microchromosomes. Our results challenge previous assumptions about the adaptive role of PRDM9 and highlight the diversity of recombination landscape features among vertebrate lineages.