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.

Histone demethylase IBM1-mediated meiocyte gene expression ensures meiotic chromosome synapsis and recombination.

Histone methylation and demethylation play important roles in plant growth and development, but the involvement of histone demethylation during meiosis is poorly understood. Here we show that disruption of Arabidopsis thaliana INCREASE IN BONSAI METHYLATION 1 (IBM1) causes incomplete synapsis, chromosome entanglement and reduction of recombination during meiosis, leading to sterility. Interestingly, these ibm1 meiotic defects are rescued by mutations in either SUVH4/KYP or CMT3. Using transcriptomic analyses we show that mutation of IBM1 down-regulates thousands of genes expressed in meiocytes, and that expression of about 38% of these genes are restored to wild type levels in ibm1 cmt3 double mutants. Changes in the expression of 437 of these, including the ARABIDOPSIS MEI2-LIKE AML3-5 genes, are correlated with a significant reduction of gene body CHG methylation. Consistently, the aml3 aml4 aml5 triple have defects in synapsis and chromosome entanglement similar to ibm1. Genetic analysis shows that aml3 aml4 aml5 ibm1 quadruple mutants resembles the ibm1 single mutant. Strikingly, over expression of AML5 in ibm1 can partially rescue the ibm1 meiotic defects. Taken together, our results demonstrate that histone demethylase IBM1 is required for meiosis likely via coordinated regulation of meiocyte gene expression during meiosis.

DNA polymerase epsilon interacts with SUVH2/9 to repress the expression of genes associated with meiotic DSB hotspot in Arabidopsis.

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)-triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9. Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

DNA polymerase epsilon binds histone H3.1-H4 and recruits MORC1 to mediate meiotic heterochromatin condensation.

Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1's localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1. Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL ε beyond its classic role in DNA replication and appear to be conserved in animals and plants.

Regulation of interference-sensitive crossover distribution ensures crossover assurance in Arabidopsis.

During meiosis, crossovers (COs) are typically required to ensure faithful chromosomal segregation. Despite the requirement for at least one CO between each pair of chromosomes, closely spaced double COs are usually underrepresented due to a phenomenon called CO interference. Like Mus musculus and Saccharomyces cerevisiae, Arabidopsis thaliana has both interference-sensitive (Class I) and interference-insensitive (Class II) COs. However, the underlying mechanism controlling CO distribution remains largely elusive. Both AtMUS81 and AtFANCD2 promote the formation of Class II CO. Using both AtHEI10 and AtMLH1 immunostaining, two markers of Class I COs, we show that AtFANCD2 but not AtMUS81 is required for normal Class I CO distribution among chromosomes. Depleting AtFANCD2 leads to a CO distribution pattern that is intermediate between that of wild-type and a Poisson distribution. Moreover, in Atfancm, Atfigl1, and Atrmi1 mutants where increased Class II CO frequency has been reported previously, we observe Class I CO distribution patterns that are strikingly similar to Atfancd2. Surprisingly, we found that AtFANCD2 plays opposite roles in regulating CO frequency in Atfancm compared with either in Atfigl1 or Atrmi1. Together, these results reveal that although AtFANCD2, AtFANCM, AtFIGL1, and AtRMI1 regulate Class II CO frequency by distinct mechanisms, they have similar roles in controlling the distribution of Class I COs among chromosomes.

The cohesin loader SCC2 contains a PHD finger that is required for meiosis in land plants.

Cohesin, a multisubunit protein complex, is required for holding sister chromatids together during mitosis and meiosis. The recruitment of cohesin by the sister chromatid cohesion 2/4 (SCC2/4) complex has been extensively studied in Saccharomyces cerevisiae mitosis, but its role in mitosis and meiosis remains poorly understood in multicellular organisms, because complete loss-of-function of either gene causes embryonic lethality. Here, we identified a weak allele of Atscc2 (Atscc2-5) that has only minor defects in vegetative development but exhibits a significant reduction in fertility. Cytological analyses of Atscc2-5 reveal multiple meiotic phenotypes including defects in chromosomal axis formation, meiosis-specific cohesin loading, homolog pairing and synapsis, and AtSPO11-1-dependent double strand break repair. Surprisingly, even though AtSCC2 interacts with AtSCC4 in vitro and in vivo, meiosis-specific knockdown of AtSCC4 expression does not cause any meiotic defect, suggesting that the SCC2-SCC4 complex has divergent roles in mitosis and meiosis. SCC2 homologs from land plants have a unique plant homeodomain (PHD) motif not found in other species. We show that the AtSCC2 PHD domain can bind to the N terminus of histones and is required for meiosis but not mitosis. Taken together, our results provide evidence that unlike SCC2 in other organisms, SCC2 requires a functional PHD domain during meiosis in land plants.

Meiotic DNA Repair in the Nucleolus Employs a Nonhomologous End-Joining Mechanism.

Ribosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo nonallelic exchanges. During meiosis, DNA double-strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR), which subsequently ensures genetic exchange and chromosome disjunction. In Arabidopsis (Arabidopsis thaliana), we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications and meiotic cohesin and allows only very limited meiosis-specific DSB formation. DNA lesions within the rDNA arrays are repaired in an RAD51-independent but LIG4-dependent manner, establishing that nonhomologous end-joining maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats, we validate our findings and demonstrate that the rDNA constitutes an HR-refractory genome environment.

Meiocyte-Specific and AtSPO11-1-Dependent Small RNAs and Their Association with Meiotic Gene Expression and Recombination.

Meiotic recombination ensures accurate chromosome segregation and results in genetic diversity in sexually reproducing eukaryotes. Over the last few decades, the genetic regulation of meiotic recombination has been extensively studied in many organisms. However, the role of endogenous meiocyte-specific small RNAs (ms-sRNAs; 21-24 nucleotide [nt]) and their involvement in meiotic recombination are unclear. Here, we sequenced the total small RNA (sRNA) and messenger RNA populations from meiocytes and leaves of wild type Arabidopsis (Arabidopsis thaliana) and meiocytes of spo11-1, a mutant defective in double-strand break formation, and we discovered 2,409 ms-sRNA clusters, 1,660 of which areSPORULATION 11-1 (AtSPO11-1)-dependent. Unlike mitotic small interfering RNAs that are enriched in intergenic regions and associated with gene silencing, ms-sRNAs are significantly enriched in genic regions and exhibit a positive correlation with genes that are preferentially expressed in meiocytes (i.e. Arabidopsis SKP1-LIKE1 and RAD51), in a fashion unrelated to DNA methylation. We also found that AtSPO11-1-dependent sRNAs have distinct characteristics compared with ms-sRNAs and tend to be associated with two known types of meiotic recombination hotspot motifs (i.e. CTT-repeat and A-rich motifs). These results reveal different meiotic and mitotic sRNA landscapes and provide new insights into how sRNAs relate to gene expression in meiocytes and meiotic recombination.

DeepTetrad: high-throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.

Comparative transcriptomic analysis of thermally stressed Arabidopsis thaliana meiotic recombination mutants.

BACKGROUND: Meiosis is a specialized cell division that underpins sexual reproduction in most eukaryotes. During meiosis, interhomolog meiotic recombination facilitates accurate chromosome segregation and generates genetic diversity by shuffling parental alleles in the gametes. The frequency of meiotic recombination in Arabidopsis has a U-shaped curve in response to environmental temperature, and is dependent on the Type I, crossover (CO) interference-sensitive pathway. The mechanisms that modulate recombination frequency in response to temperature are not yet known. RESULTS: In this study, we compare the transcriptomes of thermally-stressed meiotic-stage anthers from msh4 and mus81 mutants that mediate the Type I and Type II meiotic recombination pathways, respectively. We show that heat stress reduces the number of expressed genes regardless of genotype. In addition, msh4 mutants have a distinct gene expression pattern compared to mus81 and wild type controls. Interestingly, ASY1, which encodes a HORMA domain protein that is a component of meiotic chromosome axes, is up-regulated in wild type and mus81 but not in msh4. In addition, SDS the meiosis-specific cyclin-like gene, DMC1 the meiosis-specific recombinase, SYN1/REC8 the meiosis-specific cohesion complex component, and SWI1 which functions in meiotic sister chromatid cohesion are up-regulated in all three genotypes. We also characterize 51 novel, previously unannotated transcripts, and show that their promoter regions are associated with A-rich meiotic recombination hotspot motifs. CONCLUSIONS: Our transcriptomic analysis of msh4 and mus81 mutants enhances our understanding of how the Type I and Type II meiotic CO pathway respond to environmental temperature stress and might provide a strategy to manipulate recombination levels in plants.

Conservation and Divergence in the Meiocyte sRNAomes of Arabidopsis, Soybean, and Cucumber.

Meiosis is a critical process for sexual reproduction. During meiosis, genetic information on homologous chromosomes is shuffled through meiotic recombination to produce gametes with novel allelic combinations. Meiosis and recombination are orchestrated by several mechanisms including regulation by small RNAs (sRNAs). Our previous work in Arabidopsis (Arabidopsis thaliana) meiocytes showed that meiocyte-specific sRNAs (ms-sRNAs) have distinct characteristics, including positive association with the coding region of genes that are transcriptionally upregulated during meiosis. Here, we characterized the ms-sRNAs in two important crops, soybean (Glycine max) and cucumber (Cucumis sativus). Ms-sRNAs in soybean have the same features as those in Arabidopsis, suggesting that they may play a conserved role in eudicots. We also investigated the profiles of microRNAs (miRNAs) and phased secondary small interfering RNAs in the meiocytes of all three species. Two conserved miRNAs, miR390 and miR167, are highly abundant in the meiocytes of all three species. In addition, we identified three novel cucumber miRNAs. Intriguingly, our data show that the previously identified phased secondary small interfering RNA pathway involving soybean-specific miR4392 is more abundant in meiocytes. These results showcase the conservation and divergence of ms-sRNAs in flowering plants, and broaden our understanding of sRNA function in crop species.

Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation.

Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer, and reduced fertility. So far, 19 genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homolog of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here, we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE81 (AtMUS81), known for its role in the formation of noninterfering COs. AtFANCD2 promotes noninterfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously.

Elevated temperature increases meiotic crossover frequency via the interfering (Type I) pathway in Arabidopsis thaliana.

For most eukaryotes, sexual reproduction is a fundamental process that requires meiosis. In turn, meiosis typically depends on a reciprocal exchange of DNA between each pair of homologous chromosomes, known as a crossover (CO), to ensure proper chromosome segregation. The frequency and distribution of COs are regulated by intrinsic and extrinsic environmental factors, but much more is known about the molecular mechanisms governing the former compared to the latter. Here we show that elevated temperature induces meiotic hyper-recombination in Arabidopsis thaliana and we use genetic analysis with mutants in different recombination pathways to demonstrate that the extra COs are derived from the major Type I interference sensitive pathway. We also show that heat-induced COs are not the result of an increase in DNA double-strand breaks and that the hyper-recombinant phenotype is likely specific to thermal stress rather than a more generalized stress response. Taken together, these findings provide initial mechanistic insight into how environmental cues modulate plant meiotic recombination and may also offer practical applications.

The Largest Subunit of DNA Polymerase Delta Is Required for Normal Formation of Meiotic Type I Crossovers.

Meiotic recombination contributes to the maintenance of the association between homologous chromosomes (homologs) and ensures the accurate segregation of homologs during anaphase I, thus facilitating the redistribution of alleles among progeny. Meiotic recombination is initiated by the programmed formation of DNA double strand breaks, the repair of which requires DNA synthesis, but the role of DNA synthesis proteins during meiosis is largely unknown. Here, we hypothesized that the lagging strand-specific DNA Polymerase δ (POL δ) might be required for meiotic recombination, based on a previous analysis of DNA Replication Factor1 that suggested a role for lagging strand synthesis in meiotic recombination. In Arabidopsis (Arabidopsis thaliana), complete mutation of the catalytic subunit of POL δ, encoded by AtPOLD1, leads to embryo lethality. Therefore, we used a meiocyte-specific knockdown strategy to test this hypothesis. Reduced expression of AtPOLD1 in meiocytes caused decreased fertility and meiotic defects, including incomplete synapsis, the formation of multivalents, chromosome fragmentation, and improper segregation. Analysis of meiotic crossover (CO) frequencies showed that AtPOLD1RNAi plants had significantly fewer interference-sensitive COs than the wild type, indicating that AtPOL δ participates in type I CO formation. AtPOLD1RNAi atpol2a double mutant meiocytes displayed more severe meiotic phenotypes than those of either single mutant, suggesting that the function of AtPOLD1 and AtPOL2A is not identical in meiotic recombination. Given that POL δ is highly conserved among eukaryotes, we hypothesize that the described role of POL δ here in meiotic recombination likely exists widely in eukaryotes.

Arabidopsis RAD51, RAD51C and XRCC3 proteins form a complex and facilitate RAD51 localization on chromosomes for meiotic recombination.

Meiotic recombination is required for proper homologous chromosome segregation in plants and other eukaryotes. The eukaryotic RAD51 gene family has seven ancient paralogs with important roles in mitotic and meiotic recombination. Mutations in mammalian RAD51 homologs RAD51C and XRCC3 lead to embryonic lethality. In the model plant Arabidopsis thaliana, RAD51C and XRCC3 homologs are not essential for vegetative development but are each required for somatic and meiotic recombination, but the mechanism of RAD51C and XRCC3 in meiotic recombination is unclear. The non-lethal Arabidopsis rad51c and xrcc3 null mutants provide an opportunity to study their meiotic functions. Here, we show that AtRAD51C and AtXRCC3 are components of the RAD51-dependent meiotic recombination pathway and required for normal AtRAD51 localization on meiotic chromosomes. In addition, AtRAD51C interacts with both AtRAD51 and AtXRCC3 in vitro and in vivo, suggesting that these proteins form a complex (es). Comparison of AtRAD51 foci in meiocytes from atrad51, atrad51c, and atxrcc3 single, double and triple heterozygous mutants further supports an interaction between AtRAD51C and AtXRCC3 that enhances AtRAD51 localization. Moreover, atrad51c-/+ atxrcc3-/+ double and atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutants have defects in meiotic recombination, suggesting the role of the AtRAD51C-AtXRCC3 complex in meiotic recombination is in part AtRAD51-dependent. Together, our results support a model in which direct interactions between the RAD51C-XRCC3 complex and RAD51 facilitate RAD51 localization on meiotic chromosomes and RAD51-dependent meiotic recombination. Finally, we hypothesize that maintenance of RAD51 function facilitated by the RAD51C-XRCC3 complex could be highly conserved in eukaryotes.

The Arabidopsis anaphase-promoting complex/cyclosome subunit 8 is required for male meiosis.

Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase-promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin-ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis. We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis. Meiocytes from apc8-1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8-1, including abnormal sister chromatid segregation and microtubule morphology. Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C-mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

Arabidopsis Cell Division Cycle 20.1 Is Required for Normal Meiotic Spindle Assembly and Chromosome Segregation.

Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At-Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1. Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation.