HEIP1 is required for efficient meiotic crossover implementation and is conserved from plants to humans.

Crossovers (CO) shuffle genetic information and physically connect homologous chromosomal pairs, ensuring their balanced segregation during meiosis. COs arising from the major class I pathway require the activity of the well-conserved group of ZMM proteins, which, in conjunction with MLH1, facilitate the maturation of DNA recombination intermediates specifically into COs. The HEI10 Interacting Protein 1 (HEIP1) was identified in rice and proposed to be a new, plant-specific member of the ZMM group. Here, we establish and decipher the function of the Arabidopsis thaliana HEIP1 homolog in meiotic crossover formation and report its wide conservation in eukaryotes. We show that the loss of Arabidopsis HEIP1 elicits a marked reduction in meiotic COs and their redistribution toward chromosome ends. Epistasis analysis showed that AtHEIP1 acts specifically in the class I CO pathway. Further, we show that HEIP1 acts both prior to crossover designation, as the number of MLH1 foci is reduced in heip1, and at the maturation step of MLH1-marked sites into COs. Despite the HEIP1 protein being predicted to be primarily unstructured and very divergent at the sequence level, we identified homologs of HEIP1 in an extensive range of eukaryotes, including mammals.

A separation-of-function ZIP4 wheat mutant allows crossover between related chromosomes and is meiotically stable.

Many species, including most flowering plants, are polyploid, possessing multiple genomes. During polyploidisation, fertility is preserved via the evolution of mechanisms to control the behaviour of these multiple genomes during meiosis. On the polyploidisation of wheat, the major meiotic gene ZIP4 duplicated and diverged, with the resulting new gene TaZIP4-B2 being inserted into chromosome 5B. Previous studies showed that this TaZIP4-B2 promotes pairing and synapsis between wheat homologous chromosomes, whilst suppressing crossover between related (homoeologous) chromosomes. Moreover, in wheat, the presence of TaZIP4-B2 preserves up to 50% of grain number. The present study exploits a 'separation-of-function' wheat Tazip4-B2 mutant named zip4-ph1d, in which the Tazip4-B2 copy still promotes correct pairing and synapsis between homologues (resulting in the same pollen profile and fertility normally found in wild type wheat), but which also allows crossover between the related chromosomes in wheat haploids of this mutant. This suggests an improved utility for the new zip4-ph1d mutant line during wheat breeding, compared to the previously described CRISPR Tazip4-B2 and ph1 mutant lines. The results also reveal that loss of suppression of homoeologous crossover between wheat chromosomes does not in itself reduce wheat fertility when promotion of homologous pairing and synapsis by TaZIP4-B2 is preserved.

Deciphering complex genome rearrangements in C. elegans using short-read whole genome sequencing.

Genomic rearrangements cause congenital disorders, cancer, and complex diseases in human. Yet, they are still understudied in rare diseases because their detection is challenging, despite the advent of whole genome sequencing (WGS) technologies. Short-read (srWGS) and long-read WGS approaches are regularly compared, and the latter is commonly recommended in studies focusing on genomic rearrangements. However, srWGS is currently the most economical, accurate, and widely supported technology. In Caenorhabditis elegans (C. elegans), such variants, induced by various mutagenesis processes, have been used for decades to balance large genomic regions by preventing chromosomal crossover events and allowing the maintenance of lethal mutations. Interestingly, those chromosomal rearrangements have rarely been characterized on a molecular level. To evaluate the ability of srWGS to detect various types of complex genomic rearrangements, we sequenced three balancer strains using short-read Illumina technology. As we experimentally validated the breakpoints uncovered by srWGS, we showed that, by combining several types of analyses, srWGS enables the detection of a reciprocal translocation (eT1), a free duplication (sDp3), a large deletion (sC4), and chromoanagenesis events. Thus, applying srWGS to decipher real complex genomic rearrangements in model organisms may help designing efficient bioinformatics pipelines with systematic detection of complex rearrangements in human genomes.

Mechanistic Insight into Crossing over during Mouse Meiosis.

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.

Rad5 dysregulation drives hyperactive recombination at replication forks resulting in cisplatin sensitivity and genome instability.

The postreplication repair gene, HLTF, is often amplified and overexpressed in cancer. Here we model HLTF dysregulation through the functionally conserved Saccharomyces cerevisiae ortholog, RAD5. Genetic interaction profiling and landscape enrichment analysis of RAD5 overexpression (RAD5OE) reveals requirements for genes involved in recombination, crossover resolution, and DNA replication. While RAD5OE and rad5Δ both cause cisplatin sensitivity and share many genetic interactions, RAD5OE specifically requires crossover resolving genes and drives recombination in a region of repetitive DNA. Remarkably, RAD5OE induced recombination does not require other post-replication repair pathway members, or the PCNA modification sites involved in regulation of this pathway. Instead, the RAD5OE phenotype depends on a conserved domain necessary for binding 3' DNA ends. Analysis of DNA replication intermediates supports a model in which dysregulated Rad5 causes aberrant template switching at replication forks. The direct effect of Rad5 on replication forks in vivo, increased recombination, and cisplatin sensitivity predicts similar consequences for dysregulated HLTF in cancer.

Reducing MSH4 copy number prevents meiotic crossovers between non-homologous chromosomes in Brassica napus.

In allopolyploids, correct chromosome segregation requires suppression of non-homologous crossovers while levels of homologous crossovers are ensured. To date, no mechanism able to specifically inhibit non-homologous crossovers has been described in allopolyploids other than in bread wheat. Here, we show that reducing the number of functional copies of MSH4, an essential gene for the main crossover pathway, prevents non-homologous crossovers in allotetraploid Brassica napus. We show that non-homologous crossovers originate almost exclusively from the MSH4-dependent recombination pathway and that their numbers decrease when MSH4 returns to single copy in B. napus; by contrast, homologous crossovers remain unaffected by MSH4 duplicate loss. We also demonstrate that MSH4 systematically returns to single copy following numerous independent polyploidy events, a pattern that is probably not by chance. These results suggest that stabilization of allopolyploid meiosis can be enhanced by loss of a key meiotic recombination gene.

The impact of poly-A microsatellite heterologies in meiotic recombination.

Meiotic recombination has strong, but poorly understood effects on short tandem repeat (STR) instability. Here, we screened thousands of single recombinant products with sperm typing to characterize the role of polymorphic poly-A repeats at a human recombination hotspot in terms of hotspot activity and STR evolution. We show that the length asymmetry between heterozygous poly-A's strongly influences the recombination outcome: a heterology of 10 A's (9A/19A) reduces the number of crossovers and elevates the frequency of non-crossovers, complex recombination products, and long conversion tracts. Moreover, the length of the heterology also influences the STR transmission during meiotic repair with a strong and significant insertion bias for the short heterology (6A/7A) and a deletion bias for the long heterology (9A/19A). In spite of this opposing insertion-/deletion-biased gene conversion, we find that poly-A's are enriched at human recombination hotspots that could have important consequences in hotspot activation.

Meiotic MCM Proteins Promote and Inhibit Crossovers During Meiotic Recombination.

Crossover formation as a result of meiotic recombination is vital for the proper segregation of homologous chromosomes at the end of meiosis I. In many organisms, crossovers are generated through two crossover pathways: Class I and Class II. To ensure accurate crossover formation, meiosis-specific protein complexes regulate the degree to which each pathway is used. One such complex is the mei-mini-chromosome maintenance (MCM) complex, which contains MCM and MCM-like proteins REC (ortholog of Mcm8), MEI-217, and MEI-218. The mei-MCM complex genetically promotes Class I crossovers and inhibits Class II crossovers in Drosophila, but it is unclear how individual mei-MCM proteins contribute to crossover regulation. In this study, we perform genetic analyses to understand how specific regions and motifs of mei-MCM proteins contribute to Class I and II crossover formation, and distribution. Our analyses show that the long, disordered N-terminus of MEI-218 is dispensable for crossover formation, and that mutations that disrupt REC's Walker A and B motifs differentially affect Class I and Class II crossover formation. In rec Walker A mutants, Class I crossovers exhibit no change but Class II crossovers are increased. However, in rec Walker B mutants, Class I crossovers are severely impaired and Class II crossovers are increased. These results suggest that REC may form multiple complexes that exhibit differential REC-dependent ATP-binding and -hydrolyzing requirements. These results provide genetic insight into the mechanisms through which mei-MCM proteins promote Class I crossovers and inhibit Class II crossovers.

Mismatch recognition and subsequent processing have distinct effects on mitotic recombination intermediates and outcomes in yeast.

The post-replicative mismatch repair (MMR) system has anti-recombination activity that limits interactions between diverged sequences by recognizing mismatches in strand-exchange intermediates. In contrast to their equivalent roles during replication-error repair, mismatch recognition is more important for anti-recombination than subsequent mismatch processing. To obtain insight into this difference, ectopic substrates with 2% sequence divergence were used to examine mitotic recombination outcome (crossover or noncrossover; CO and NCO, respectively) and to infer molecular intermediates formed during double-strand break repair in Saccharomyces cerevisiae. Experiments were performed in an MMR-proficient strain, a strain with compromised mismatch-recognition activity (msh6Δ) and a strain that retained mismatch-recognition activity but was unable to process mismatches (mlh1Δ). While the loss of either mismatch binding or processing elevated the NCO frequency to a similar extent, CO events increased only when mismatch binding was compromised. The molecular features of NCOs, however, were altered in fundamentally different ways depending on whether mismatch binding or processing was eliminated. These data suggest a model in which mismatch recognition reverses strand-exchange intermediates prior to the initiation of end extension, while subsequent mismatch processing that is linked to end extension specifically destroys NCO intermediates that contain conflicting strand-discrimination signals for mismatch removal.

Male Meiotic Recombination in the Steppe Agama, Trapelus sanguinolentus (Agamidae, Iguania, Reptilia).

Meiotic recombination rates and patterns of crossover distributions along the chromosomes vary considerably even between closely related species. The adaptive significance of these differences is still unclear due to the paucity of empirical data. Most data on recombination come from mammalian species, while other vertebrate clades are poorly explored. Using immunolocalization of the protein of the lateral element of the synaptonemal complex (SYCP3) and the mismatch-repair protein MLH1, which marks mature recombination nodules, we analyzed recombination rates and crossover distribution in meiotic prophase chromosomes of the steppe agama (Trapelus sanguinolentus, Agamidae, Acrodonta, Iguania) and compared them with data obtained for the genus Anolis (Dactyloidae, Pleurodonta, Iguania). We found that, despite a smaller genome size, the total SC length and the MLH1 focus number per cell are much higher in the agama than in the anoles. The distributions of the MLH1 foci in the agama are multimodal in larger chromosomes and bimodal in smaller chromosomes without a significant centromere effect, resembling the patterns known for birds. A possible relationship between karyotype remodeling and the evolution of recombination in Iguania is discussed.

Discoveries and controversies in cutaneous mosaicism.

Genetic mosaicism is thought to be a common phenomenon in inherited skin disorders. It is the leading molecular mechanism explaining cutaneous hamartomas and nevoid disorders, skin manifestations of most X-linked genodermatoses and specific forms of clinical variability and topographic distribution in autosomal skin disorders. The developmental (in utero) origin and timing dependence are two major attributes for the current definition of cutaneous mosaicism. Chromosomal mosaicism, lyonization in X-linked genodermatoses, and various types of mosaicism (i.e. type 1, type 2 and revertant mosaicism) in autosomal skin disorders are mechanisms well defined at the molecular level. All these concepts have been fully included in the current medical terminology in dermatology and genetics. Mitotic crossing-over, paradominant inheritance, monoallelic expression of autosomal traits and mosaicism in acquired skin disorders remain without a formal molecular proof and still represent sources of debate in the scientific community. This review summarizes current concepts, discoveries and controversies in the field of cutaneous mosaicism for practitioners and clinical researchers to enhance their understanding of such a underestimated clinical phenomenon and its biological basis.

Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements.

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.

Mte1 interacts with Mph1 and promotes crossover recombination and telomere maintenance.

Mph1 is a member of the conserved FANCM family of DNA motor proteins that play key roles in genome maintenance processes underlying Fanconi anemia, a cancer predisposition syndrome in humans. Here, we identify Mte1 as a novel interactor of the Mph1 helicase in Saccharomyces cerevisiae. In vitro, Mte1 (Mph1-associated telomere maintenance protein 1) binds directly to DNA with a preference for branched molecules such as D loops and fork structures. In addition, Mte1 stimulates the helicase and fork regression activities of Mph1 while inhibiting the ability of Mph1 to dissociate recombination intermediates. Deletion of MTE1 reduces crossover recombination and suppresses the sensitivity of mph1Δ mutant cells to replication stress. Mph1 and Mte1 interdependently colocalize at DNA damage-induced foci and dysfunctional telomeres, and MTE1 deletion results in elongated telomeres. Taken together, our data indicate that Mte1 plays a role in regulation of crossover recombination, response to replication stress, and telomere maintenance.

Heterologous Synapsis and Crossover Suppression in Heterozygotes for a Pericentric Inversion in the Zebra Finch.

In the zebra finch, 2 alternative morphs regarding centromere position were described for chromosome 6. This polymorphism was interpreted to be the result of a pericentric inversion, but other causes of the centromere repositioning were not ruled out. We used immunofluorescence localization to examine the distribution of MLH1 foci on synaptonemal complexes to test the prediction that pericentric inversions cause synaptic irregularities and/or crossover suppression in heterozygotes. We found complete suppression of crossing over in the region involved in the rearrangement in male and female heterozygotes. In contrast, the same region showed high levels of crossing over in homozygotes for the acrocentric form of this chromosome. No inversion loops or synaptic irregularities were detected along bivalent 6 in heterozygotes suggesting that heterologous pairing is achieved during zygotene or early pachytene. Altogether these findings strongly indicate that the polymorphic chromosome 6 originated by a pericentric inversion. Since inversions are common rearrangements in karyotypic evolution in birds, it seems likely that early heterologous pairing could help to fix these rearrangements, preventing crossing overs in heterozygotes and their deleterious effects on fertility.

Mitotic crossover promotes leukemogenesis in children born with TEL-AML1 via the generation of loss of heterozygosity at 12p.

TEL-AML1 (ETV6-RUNX1) fusion gene which is formed prenatally in 1% of the newborns, is a common genetic abnormality in childhood Bcell precursor acute lymphoblastic leukemia. But only one child out of a hundred children born with this fusion gene develops leukemia (bottleneck phenomenon) later in its life, if contracts the second mutation. In other words, out of a hundred children born with TEL-AML1 only one child is at risk for leukemia development, which means that TEL-AML1 fusion gene is not sufficient for overt leukemia. There is a stringent requirement for a second genetic abnormality for leukemia development and this is the real or the ultimate cause of the leukemia bottleneck phenomenon. In most cases of TEL-AML1+ leukemia, the translocation t(12;21) is complemented with the loss of the normal TEL gene, not involved in the translocation, on the contralateral 12p. The loss of the normal TEL gene, i.e. loss of heterozygosity at 12p, occurs postnatally during the mitotic proliferation of TEL-AML1+ cell in the mitotic crossing over process. Mitotic crossing over is a very rare event with a frequency rate of 10-6 in a 10 kb region. The exploration and identification of the environmental exposure(s) that cause(s) proliferation of the TELAML1+ cell in which approximately 106 mitoses are generated to cause 12p loss of heterozygosity, i.e. TEL gene deletion, may contribute to the introduction of preventive measures for leukemia.

AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms.

Meiotic crossovers (COs) generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression.

Estrogenic exposure alters the spermatogonial stem cells in the developing testis, permanently reducing crossover levels in the adult.

Bisphenol A (BPA) and other endocrine disrupting chemicals have been reported to induce negative effects on a wide range of physiological processes, including reproduction. In the female, BPA exposure increases meiotic errors, resulting in the production of chromosomally abnormal eggs. Although numerous studies have reported that estrogenic exposures negatively impact spermatogenesis, a direct link between exposures and meiotic errors in males has not been evaluated. To test the effect of estrogenic chemicals on meiotic chromosome dynamics, we exposed male mice to either BPA or to the strong synthetic estrogen, ethinyl estradiol during neonatal development when the first cells initiate meiosis. Although chromosome pairing and synapsis were unperturbed, exposed outbred CD-1 and inbred C3H/HeJ males had significantly reduced levels of crossovers, or meiotic recombination (as defined by the number of MLH1 foci in pachytene cells) by comparison with placebo. Unexpectedly, the effect was not limited to cells exposed at the time of meiotic entry but was evident in all subsequent waves of meiosis. To determine if the meiotic effects induced by estrogen result from changes to the soma or germline of the testis, we transplanted spermatogonial stem cells from exposed males into the testes of unexposed males. Reduced recombination was evident in meiocytes derived from colonies of transplanted cells. Taken together, our results suggest that brief exogenous estrogenic exposure causes subtle changes to the stem cell pool that result in permanent alterations in spermatogenesis (i.e., reduced recombination in descendent meiocytes) in the adult male.

Brca2-Pds5 complexes mobilize persistent meiotic recombination sites to the nuclear envelope.

Homologous recombination is required for reciprocal exchange between homologous chromosome arms during meiosis. Only select meiotic recombination events become chromosomal crossovers; the majority of recombination outcomes are noncrossovers. Growing evidence suggests that crossovers are repaired after noncrossovers. Here, I report that persisting recombination sites are mobilized to the nuclear envelope of Drosophila pro-oocytes during mid-pachytene. Their number correlates with the average crossover rate per meiosis. Proteomic and interaction studies reveal that the recombination mediator Brca2 associates with lamin and the cohesion factor Pds5 to secure persistent recombination sites at the nuclear envelope. In Rad51(-/-) females, all persistent DNA breaks are directed to the nuclear envelope. By contrast, a reduction of Pds5 or Brca2 levels abolishes the movement and has a negative impact on crossover rates. The data suggest that persistent meiotic DNA double-strand breaks might correspond to crossovers, which are mobilized to the nuclear envelope for their repair. The identification of Brca2-Pds5 complexes as key mediators of this process provides a first mechanistic explanation for the contribution of lamins and cohesins to meiotic recombination.

Dual regulation of Dmc1-driven DNA strand exchange by Swi5-Sfr1 activation and Rad22 inhibition.

Both ubiquitously expressed Rad51 and meiosis-specific Dmc1 are required for crossover production during meiotic recombination. The budding yeast Rad52 and its fission yeast ortholog, Rad22, are "mediators;" i.e., they help load Rad51 onto ssDNA coated with replication protein A (RPA). Here we show that the Swi5-Sfr1 complex from fission yeast is both a mediator that loads Dmc1 onto ssDNA and a direct "activator" of DNA strand exchange by Dmc1. In stark contrast, Rad22 inhibits Dmc1 action by competing for its binding to RPA-coated ssDNA. Thus, Rad22 plays dual roles in regulating meiotic recombination: activating Rad51 and inhibiting Dmc1.