MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis.

During meiosis, DNA double-strand breaks undergo interhomolog repair to yield crossovers between homologous chromosomes. To investigate how interhomolog sequence polymorphism affects crossovers, we sequenced multiple recombinant populations of the model plant Arabidopsis thaliana. Crossovers were elevated in the diverse pericentromeric regions, showing a local preference for polymorphic regions. We provide evidence that crossover association with elevated diversity is mediated via the Class I crossover formation pathway, although very high levels of diversity suppress crossovers. Interhomolog polymorphism causes mismatches in recombining molecules, which can be detected by MutS homolog (MSH) mismatch repair protein heterodimers. Therefore, we mapped crossovers in a msh2 mutant, defective in mismatch recognition, using multiple hybrid backgrounds. Although total crossover numbers were unchanged in msh2 mutants, recombination was remodelled from the diverse pericentromeres towards the less-polymorphic sub-telomeric regions. Juxtaposition of megabase heterozygous and homozygous regions causes crossover remodelling towards the heterozygous regions in wild type Arabidopsis, but not in msh2 mutants. Immunostaining showed that MSH2 protein accumulates on meiotic chromosomes during prophase I, consistent with MSH2 regulating meiotic recombination. Our results reveal a pro-crossover role for MSH2 in regions of higher sequence diversity in A. thaliana.

Natural variation identifies SNI1, the SMC5/6 component, as a modifier of meiotic crossover in Arabidopsis.

The frequency and distribution of meiotic crossovers are tightly controlled; however, variation in this process can be observed both within and between species. Using crosses of two natural Arabidopsis thaliana accessions, Col and Ler, we mapped a crossover modifier locus to semidominant polymorphisms in SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), which encodes a component of the SMC5/6 complex. The sni1 mutant exhibits a modified pattern of recombination across the genome with crossovers elevated in chromosome distal regions but reduced in pericentromeres. Mutations in SNI1 result in reduced crossover interference and can partially restore the fertility of a Class I crossover pathway mutant, which suggests that the protein affects noninterfering crossover repair. Therefore, we tested genetic interactions between SNI1 and both RECQ4 and FANCM DNA helicases, which showed that additional Class II crossovers observed in the sni1 mutant are FANCM independent. Furthermore, genetic analysis of other SMC5/6 mutants confirms the observations of crossover redistribution made for SNI1 The study reveals the importance of the SMC5/6 complex in ensuring the proper progress of meiotic recombination in plants.

AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1.

Expansion of an unstable CTG repeat in the 3'UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUGexp) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUGexp, we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUGexp from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUGexp. Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs' chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1.

The role of MutS complex and MSH2 protein in the crossover control in plants / Rola kompleksu MutS oraz bialka MSH2 w kontroli crossing-over u roslin.

Crossover is a reciprocal exchange of chromatid fragments between homologous chromosomes and takes place during second meiotic division. Many factors affect the distribution and frequency of crossovers ­ for instance, the activity of trans-acting modifiers, chromatin methylation level or the presence of polymorphisms between recombining chromosomes. MMR system, and specifically MSH2 protein, serves to recognize and repair mismatched DNA bases, and prevents recombination between divergent chromosomal regions during meiosis. Unexpectedly, MSH2 displays also a pro-recombination role in plants by detecting polymorphisms and directing crossover events into more diverged regions. In this review, we demonstrate how interhomolog polymorphism may affect crossover chromosomal distribution and, as a consequence, plant genomes evolution. It is especially important for self-fertilizing plants which naturally exhibit high level of homozygosity. If recombination were to occur only in homozygous regions, no new genotypes would be created in subsequent generations, slowing down the evolution of the organisms.

The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids.

In hybrid organisms, genetically divergent homologous chromosomes pair and recombine during meiosis; however, the effect of specific types of polymorphisms on crossover is poorly understood. Here, to analyze this in Arabidopsis, we develop the seed-typing method that enables the massively parallel fine-mapping of crossovers by sequencing. We show that structural variants, observed in one of the generated intervals, do not change crossover frequency unless they are located directly within crossover hotspots. Both natural and Cas9-induced deletions result in lower hotspot activity but are not compensated by increases in immediately adjacent hotspots. To examine the effect of single nucleotide polymorphisms on crossover formation, we analyze hotspot activity in mismatch detection-deficient msh2 mutants. Surprisingly, polymorphic hotspots show reduced activity in msh2. In lines where only the hotspot-containing interval is heterozygous, crossover numbers increase above those in the inbred (homozygous). We conclude that MSH2 shapes crossover distribution by stimulating hotspot activity at polymorphic regions.

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.

Quantifying Meiotic Crossover Recombination in Arabidopsis Lines Expressing Fluorescent Reporters in Seeds Using SeedScoring Pipeline for CellProfiler.

The number of crossovers during meiosis is relatively low, so multiple meioses need to be analyzed to accurately measure crossover frequency. In Arabidopsis, systems based on the segregation of fluorescent T-DNA reporters that are expressed in seeds (fluorescent-tagged lines, FTLs) allow for an accurate measurement of crossover frequency in specific chromosome regions. A major advantage of FTL-based experiments is the ability to analyze thousands of seeds for each biological replicate, which requires the use of automatic seed scoring. Here, we describe a protocol to computationally count the proportion of seeds that experienced a crossover event within the tested FTL interval and so measure the recombination frequency within that interval. We describe SeedScoring, a CellProfiler pipeline where the total time needed to measure crossover frequency in a single FTL line is approximately 5 min using a series of three images taken under a fluorescent stereomicroscope (3 min) and passing these images through the SeedScoring pipeline described in this protocol (2 min).

Where to Cross Over? Defining Crossover Sites in Plants.

It is believed that recombination in meiosis serves to reshuffle genetic material from both parents to increase genetic variation in the progeny. At the same time, the number of crossovers is usually kept at a very low level. As a consequence, many organisms need to make the best possible use from the one or two crossovers that occur per chromosome in meiosis. From this perspective, the decision of where to allocate rare crossover events becomes an important issue, especially in self-pollinating plant species, which experience limited variation due to inbreeding. However, the freedom in crossover allocation is significantly limited by other, genetic and non-genetic factors, including chromatin structure. Here we summarize recent progress in our understanding of those processes with a special emphasis on plant genomes. First, we focus on factors which influence the distribution of recombination initiation sites and discuss their effects at both, the single hotspot level and at the chromosome scale. We also briefly explain the aspects of hotspot evolution and their regulation. Next, we analyze how recombination initiation sites translate into the development of crossovers and their location. Moreover, we provide an overview of the sequence polymorphism impact on crossover formation and chromosomal distribution.