Recent advances in mechanisms ensuring the pairing, synapsis and segregation of XY chromosomes in mice and humans.

Sex chromosome aneuploidies are among the most common variations in human whole chromosome copy numbers, with an estimated prevalence in the general population of 1:400 to 1:1400 live births. Unlike whole-chromosome aneuploidies of autosomes, those of sex chromosomes, such as the 47, XXY aneuploidy that causes Klinefelter Syndrome (KS), often originate from the paternal side, caused by a lack of crossover (CO) formation between the X and Y chromosomes. COs must form between all chromosome pairs to pass meiotic checkpoints and are the product of meiotic recombination that occurs between homologous sequences of parental chromosomes. Recombination between male sex chromosomes is more challenging compared to both autosomes and sex chromosomes in females, as it is restricted within a short region of homology between X and Y, called the pseudo-autosomal region (PAR). However, in normal individuals, CO formation occurs in PAR with a higher frequency than in any other region, indicating the presence of mechanisms that promote the initiation and processing of recombination in each meiotic division. In recent years, research has made great strides in identifying genes and mechanisms that facilitate CO formation in the PAR. Here, we outline the most recent and relevant findings in this field. XY chromosome aneuploidy in humans has broad-reaching effects, contributing significantly also to Turner syndrome, spontaneous abortions, oligospermia, and even infertility. Thus, in the years to come, the identification of genes and mechanisms beyond XY aneuploidy is expected to have an impact on the genetic counseling of a wide number of families and adults affected by these disorders.

RPA interacts with Rad52 to promote meiotic crossover and noncrossover recombination.

Meiotic recombination is initiated by programmed double-strand breaks (DSBs). Studies in Saccharomyces cerevisiae have shown that, following rapid resection to generate 3' single-stranded DNA (ssDNA) tails, one DSB end engages a homolog partner chromatid and is extended by DNA synthesis, whereas the other end remains associated with its sister. Then, after regulated differentiation into crossover- and noncrossover-fated types, the second DSB end participates in the reaction by strand annealing with the extended first end, along both pathways. This second-end capture is dependent on Rad52, presumably via its known capacity to anneal two ssDNAs. Here, using physical analysis of DNA recombination, we demonstrate that this process is dependent on direct interaction of Rad52 with the ssDNA binding protein, replication protein A (RPA). Furthermore, the absence of this Rad52-RPA joint activity results in a cytologically-prominent RPA spike, which emerges from the homolog axes at sites of crossovers during the pachytene stage of the meiotic prophase. Our findings suggest that this spike represents the DSB end of a broken chromatid caused by either the displaced leading DSB end or the second DSB end, which has been unable to engage with the partner homolog-associated ssDNA. These and other results imply a close correspondence between Rad52-RPA roles in meiotic recombination and mitotic DSB repair.

Control of meiotic crossover interference by a proteolytic chaperone network.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

COSA-1 mediated pro-crossover complex formation promotes meiotic crossing over in C. elegans.

Accurate chromosome segregation during meiosis requires the establishment of at least one crossover (CO) between each pair of homologous chromosomes. CO formation depends on a group of conserved pro-CO proteins, which colocalize at CO-designated sites during late meiotic prophase I. However, it remains unclear whether these pro-CO proteins form a functional complex and how they promote meiotic CO formation in vivo. Here, we show that COSA-1, a key component required for CO formation, interacts with other pro-CO factors, MSH-5 and ZHP-3, via its N-terminal disordered region. Point mutations that impair these interactions do not affect CO designation, but they strongly hinder the accumulation of COSA-1 at CO-designated sites and result in defective CO formation. These defects can be partially bypassed by artificially tethering an interaction-compromised COSA-1 derivate to ZHP-3. Furthermore, we revealed that the accumulation of COSA-1 into distinct foci is required to assemble functional 'recombination nodules'. These prevent early CO-designated recombination intermediates from being dismantled by the RTEL-1 helicase and protect late recombination intermediates, such as Holliday junctions, until they are resolved by CO-specific resolvases. Altogether, our findings provide insight into COSA-1 mediated pro-CO complex assembly and its contribution to CO formation.

A point mutation in the meiotic crossover formation gene HEI10/TFS2 leads to thermosensitive genic sterility in rice.

Thermosensitive genic female sterility (TGFS) is a promising property to be utilized for hybrid breeding. Here, we identified a rice TGFS line, tfs2, through an ethyl methyl sulfone (EMS) mutagenesis strategy. This line showed sterility under high temperature and became fertile under low temperature. Few seeds were produced when the tfs2 stigma was pollinated, indicating that tfs2 is female sterile. Gene cloning and genetic complementation showed that a point mutation from leucine to phenylalanine in HEI10 (HEI10tfs2), a crossover formation protein, caused the TGFS trait of tfs2. Under high temperature, abnormal univalents were formed, and the chromosomes were unequally segregated during meiosis, similar to the reported meiotic defects in oshei10. Under low temperature, the number of univalents was largely reduced, and the chromosomes segregated equally, suggesting that crossover formation was restored in tfs2. Yeast two-hybrid assays showed that HEI10 interacted with two putative protein degradation-related proteins, RPT4 and SRFP1. Through transient expression in tobacco leaves, HEI10 were found to spontaneously aggregate into dot-like foci in the nucleus under high temperature, but HEI10tfs2 failed to aggregate. In contrast, low temperature promoted HEI10tfs2 aggregation. This result suggests that protein aggregation at the crossover position contributes to the fertility restoration of tfs2 under low temperature. In addition, RPT4 and SRFP1 also aggregated into dot-like foci, and these aggregations depend on the presence of HEI10. These findings reveal a novel mechanism of fertility restoration and facilitate further understanding of HEI10 in meiotic crossover formation.

The genome-wide meiotic recombination landscape in ciliates and its implications for crossover regulation and genome evolution.

Meiotic recombination is essential for sexual reproduction and its regulation has been extensively studied in many taxa. However, genome-wide recombination landscape has not been reported in ciliates and it remains unknown how it is affected by the unique features of ciliates: the synaptonemal complex (SC)-independent meiosis and the nuclear dimorphism. Here, we show the recombination landscape in the model ciliate Tetrahymena thermophila by analyzing single-nucleotide polymorphism datasets from 38 hybrid progeny. We detect 1021 crossover (CO) events (35.8 per meiosis), corresponding to an overall CO rate of 9.9 cM/Mb. However, gene conversion by non-crossover is rare (1.03 per meiosis) and not biased towards G or C alleles. Consistent with the reported roles of SC in CO interference, we find no obvious sign of CO interference. CO tends to occur within germ-soma common genomic regions and many of the 44 identified CO hotspots localize at the centromeric or subtelomeric regions. Gene ontology analyses show that CO hotspots are strongly associated with genes responding to environmental changes. We discuss these results with respect to how nuclear dimorphism has potentially driven the formation of the observed recombination landscape to facilitate environmental adaptation and the sharing of machinery among meiotic and somatic recombination.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

Synthetically induced Arabidopsis thaliana autotetraploids provide insights into the analysis of meiotic mutants with altered crossover frequency.

Mutations affecting crossover (CO) frequency and distribution lead to the presence of univalents during meiosis, giving rise to aneuploid gametes and sterility. These mutations may have a different effect after chromosome doubling. The combination of altered ploidy and mutations could be potentially useful to gain new insights into the mechanisms and regulation of meiotic recombination; however, studies using autopolyploid meiotic mutants are scarce. Here, we have analyzed the cytogenetic consequences in colchicine-induced autotetraploids (colchiploids) from different Arabidopsis mutants with an altered CO frequency. We have found that there are three types of mutants: mutants in which chiasma frequency is doubled after chromosome duplication (zip4, mus81), as in the control; mutants in which polyploidy leads to a higher-than-expected increase in chiasma frequency (asy1, mer3, hei10, and mlh3); and mutants in which the rise in chiasma frequency produced by the presence of two extrachromosomal sets is less than doubled (msh5, fancm). In addition, the proportion of class I/class II COs varies after chromosome duplication in the control. The results obtained reveal the potential of colchiploid meiotic mutants for better understanding of the function of key proteins during plant meiosis. This is especially relevant considering that most crops are polyploids.

HEI10 is subject to phase separation and mediates RPA1a degradation during meiotic interference-sensitive crossover formation.

Reciprocal exchanges of DNA between homologous chromosomes during meiosis, or crossovers (COs), shuffle genetic information in gametes and progeny. In many eukaryotes, the majority of COs (class I COs) are sensitive to a phenomenon called interference, which influences the occurrence of closely spaced double COs. Class I COs depend on a group of factors called ZMM (Zip, Msh, Mer) proteins including HEI10 (Human Enhancer of Invasion-10). However, how these proteins are recruited to class I CO sites is unclear. Here, we show that HEI10 forms foci on chromatin via a liquid-liquid phase separation (LLPS) mechanism that relies on residue Ser70. A HEI10S70F allele results in LLPS failure and a defect in class I CO formation. We further used immunoprecipitation-mass spectrometry to identify RPA1a (Replication Protein A 1) as a HEI10 interacting protein. Surprisingly, we find that RPA1a also undergoes phase separation and its ubiquitination and degradation are directly regulated by HEI10. We also show that HEI10 is required for the condensation of other class I CO factors. Thus, our results provide mechanistic insight into how meiotic class I CO formation is controlled by HEI10 coupling LLPS and ubiquitination.

Meiosis: Dances Between Homologs.

The raison d'être of meiosis is shuffling of genetic information via Mendelian segregation and, within individual chromosomes, by DNA crossing-over. These outcomes are enabled by a complex cellular program in which interactions between homologous chromosomes play a central role. We first provide a background regarding the basic principles of this program. We then summarize the current understanding of the DNA events of recombination and of three processes that involve whole chromosomes: homolog pairing, crossover interference, and chiasma maturation. All of these processes are implemented by direct physical interaction of recombination complexes with underlying chromosome structures. Finally, we present convergent lines of evidence that the meiotic program may have evolved by coupling of this interaction to late-stage mitotic chromosome morphogenesis.

MSH2 stimulates interfering and inhibits non-interfering crossovers in response to genetic polymorphism.

Meiotic crossovers can be formed through the interfering pathway, in which one crossover prevents another from forming nearby, or by an independent non-interfering pathway. In Arabidopsis, local sequence polymorphism between homologs can stimulate interfering crossovers in a MSH2-dependent manner. To understand how MSH2 regulates crossovers formed by the two pathways, we combined Arabidopsis mutants that elevate non-interfering crossovers with msh2 mutants. We demonstrate that MSH2 blocks non-interfering crossovers at polymorphic loci, which is the opposite effect to interfering crossovers. We also observe MSH2-independent crossover inhibition at highly polymorphic sites. We measure recombination along the chromosome arms in lines differing in patterns of heterozygosity and observe a MSH2-dependent crossover increase at the boundaries between heterozygous and homozygous regions. Here, we show that MSH2 is a master regulator of meiotic DSB repair in Arabidopsis, with antagonistic effects on interfering and non-interfering crossovers, which shapes the crossover landscape in relation to interhomolog polymorphism.

FANCM interacts with the MHF1-MHF2 complex to limit crossover frequency during rice meiosis.

Crossovers (COs) are necessary for generating genetic diversity that breeders can select, but there are conserved mechanisms that regulate their frequency and distribution. Increasing CO frequency may raise the efficiency of selection by increasing the chance of integrating more desirable traits. In this study, we characterize rice FANCM and explore its functions in meiotic CO control. FANCM mutations do not affect fertility in rice, but they cause a great boost in the overall frequency of COs in both inbred and hybrid rice, according to genetic analysis of the complete set of fancm zmm (hei10, ptd, shoc1, mer3, zip4, msh4, msh5, and heip1) mutants. Although the early homologous recombination events proceed normally in fancm, the meiotic extra COs are not marked with HEI10 and require MUS81 resolvase for resolution. FANCM depends on PAIR1, COM1, DMC1, and HUS1 to perform its functions. Simultaneous disruption of FANCM and MEICA1 synergistically increases CO frequency, but it is accompanied by nonhomologous chromosome associations and fragmentations. FANCM interacts with the MHF complex, and ablation of rice MHF1 or MHF2 could restore the formation of 12 bivalents in the absence of the ZMM gene ZIP4. Our data indicate that unleashing meiotic COs by mutating any member of the FANCM-MHF complex could be an effective procedure to accelerate the efficiency of rice breeding.

NCOurd: modelling length distributions of NCO events and gene conversion tracts.

MOTIVATION: Meiotic recombination is the main driving force of human genetic diversity, along with mutations. Recombinations split into crossovers, separating large chromosomal regions originating from different homologous chromosomes, and non-crossovers (NCOs), where a small segment from one chromosome is embedded in a region originating from the homologous chromosome. NCOs are much less studied than mutations and crossovers as NCOs are short and can only be detected at markers heterozygous in the transmitting parent, leaving most of them undetectable. RESULTS: The detectable NCOs, known as gene conversions, hide information about NCOs, including their number and length, waiting to be unveiled. We introduce NCOurd, software, and algorithm, based on an expectation-maximization algorithm, to estimate the number of NCOs and their length distribution from gene conversion data. AVAILABILITY AND IMPLEMENTATION: https://github.com/DecodeGenetics/NCOurd.

Unequal crossing over between CYP11B2 and CYP11B1 causes 11 ß -hydroxylase deficiency in a consanguineous family.

Cytochrome P450 (CYP) family CYP11B2/CYP11B1 chimeric genes have been shown to arise from unequal crossing over of the genes encoding aldosterone synthase (CYP11B2) and 11ß-hydroxylase (CYP11B1) during meiosis. The activity deficiency or impaired activity of aldosterone synthase and 11ß-hydroxylase resulting from these chimeric genes are important reasons for 11ß-hydroxylase deficiency (11ß-OHD). Here，two patients with pseudoprecocious puberty and hypokalemia hypertension and three carriers in a consanguineous marriage family were studied. A single CYP11B2/CYP11B1 chimera consisting of the promoter and exons 1 through 5 of CYP11B2, exons 8 and 9 of CYP11B1, and a breakpoint consisting of part of exon 6 of CYP11B2 and part of exon 6, intron 6, and exon 7 of CYP11B1 were detected in the patients and carriers. At the breakpoint of the chimera, a c 0.1086 G > C ( p.Leu.362 =) synonymous mutation in exon 6 of CYP11B2, a c 0.1157 C＞G(p. A386V) missense mutation in exon 7 of CYP11B1, and an intronic mutation in intron 6 were detected. The allele model of the CYP11B2/CYP11B1 chimera demonstrated homozygosity and heterozygosity in the patients and the carriers, respectively. Molecular docking and enzymatic activity analyses indicated that the CYP11B2/CYP11B1 chimeric protein interacted with the catalytic substrate of aldosterone synthase and had similar enzymatic activity to aldosterone synthase. Our study indicated that deletion of CYP11B1 and CYP11B2 abolished the enzymatic activity of 11 ß-hydroxylase and aldosterone synthase; however, the compensation of the enzymatic activity of aldosterone synthase by the CYP11B2/CYP11B1 chimeric protein maintained normal aldosterone levels in vitro. All of the above findings explained the 11ß-OHD phenotypes of the proband and patients in the family.

Pathogenic bi-allelic variants of meiotic ZMM complex gene SPO16 in premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a heterogeneous disease affecting the physical and mental health of millions of women worldwide. The contribution of genetic factors in the pathogenesis of POI has increased, with quite a few of causative genes involved in meiosis. ZMM proteins are a group of conserved proteins participating in meiotic synapsis and crossover maturation. Here, by screening the variations of ZMM genes in our in-house WES database of 1030 idiopathic POI patients, one novel homozygous variation in SPO16 (c.160 + 8A > G) was firstly identified in one patient. The variation was verified to disturb mRNA splicing by minigene assay, produced a non-functional SPO16 protein, and was classified as pathogenetic according to American College of Medical Genetics guideline. During meiotic prophase I, SHOC1 binds to branched DNA and recruits SPO16 and other ZMM proteins to facilitate crossover formation. Together with our recent identified bi-allelic variations of SHOC1 in a published work, this study highlighted the essential roles of ZMM genes in the maintenance of ovarian function and expanded the POI gene spectrum.

Recombination and sterility in inversion homo- and heterokaryotypes under a general counting model of chiasma interference.

It has long been known that the chiasmata are not independently distributed in most organisms, a phenomenon known as chiasma interference. In this paper, I suggest a model of chiasma interference that generalizes the Poisson model, the counting model, the Poisson-skip model, and the two-pathway counting model into a single framework, and use it to derive infinite series expressions for the sterility and recombination pattern probabilities in inversion homo- and heterokaryotypes, and a closed-form expression for the special case of the two-pathway counting model in homokaryotypes. I then use these expressions to perform maximum likelihood parameter estimations for recombination and tetrad data from various species. The results imply that the simpler counting models perform well compared to more complex ones, that interference works in a similar way in homo- and heterokaryotypes, and that the model fits well with data for the latter as well as the former. I also find evidence that the interference signal is broken by the centromere in some species, but not others, suggestions of negative interference in Aspergillus nidulans, and no consistent support for the theory that a second noninterfering chiasma pathway exists only in organisms that require double-strand break for synapsis. I suggest that the latter finding is at least partly due to issues involved in analyzing aggregate data from different experiments and individuals.

The regulation of meiotic crossover distribution: a coarse solution to a century-old mystery?

Meiotic crossovers, which are exchanges of genetic material between homologous chromosomes, are more evenly and distantly spaced along chromosomes than expected by chance. This is because the occurrence of one crossover reduces the likelihood of nearby crossover events - a conserved and intriguing phenomenon called crossover interference. Although crossover interference was first described over a century ago, the mechanism allowing coordination of the fate of potential crossover sites half a chromosome away remains elusive. In this review, we discuss the recently published evidence supporting a new model for crossover patterning, coined the coarsening model, and point out the missing pieces that are still needed to complete this fascinating puzzle.

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.

Exo1 protects DNA nicks from ligation to promote crossover formation during meiosis.

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is essential to produce haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday junction (dHJ) intermediates. This dHJ resolution step involves the actions of Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. Here, we provide genetic evidence in baker's yeast that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 that interact with DNA, such as those required for the bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase reduced the crossover levels of exo1 DNA-binding mutants to levels that approached the exo1 null. In addition, our work identified a role for Exo1 in crossover interference. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution.

Heterozygous inversion breakpoints suppress meiotic crossovers by altering recombination repair outcomes.

Heterozygous chromosome inversions suppress meiotic crossover (CO) formation within an inversion, potentially because they lead to gross chromosome rearrangements that produce inviable gametes. Interestingly, COs are also severely reduced in regions nearby but outside of inversion breakpoints even though COs in these regions do not result in rearrangements. Our mechanistic understanding of why COs are suppressed outside of inversion breakpoints is limited by a lack of data on the frequency of noncrossover gene conversions (NCOGCs) in these regions. To address this critical gap, we mapped the location and frequency of rare CO and NCOGC events that occurred outside of the dl-49 chrX inversion in D. melanogaster. We created full-sibling wildtype and inversion stocks and recovered COs and NCOGCs in the syntenic regions of both stocks, allowing us to directly compare rates and distributions of recombination events. We show that COs outside of the proximal inversion breakpoint are distributed in a distance-dependent manner, with strongest suppression near the inversion breakpoint. We find that NCOGCs occur evenly throughout the chromosome and, importantly, are not suppressed near inversion breakpoints. We propose a model in which COs are suppressed by inversion breakpoints in a distance-dependent manner through mechanisms that influence DNA double-strand break repair outcome but not double-strand break formation. We suggest that subtle changes in the synaptonemal complex and chromosome pairing might lead to unstable interhomolog interactions during recombination that permits NCOGC formation but not CO formation.