Biology subjects

Serra, H.

Publications and source records attributed to Serra, H..

5 recordsLinked to original sources

bioRxiv2026

Role of small non-coding RNAs in DSB repair in Arabidopsis thaliana

DNA double-strand breaks (DSB) are highly toxic lesions that must be repaired to avoid major losses of genetic materials or genome rearrangements. A number of highly conserved molecular pathways are involved in DSB repair, each resulting in different repair outcomes for a given DSB. Over the past decade, accumulating evidence has revealed the emergence of an unexpected new actor in DSB repair: RNA. Notably, small RNAs have been observed at DSB-flanking sequences and hypothesized to be involved in their repair. However, these DSB-induced small RNAs (diRNAs) were so far observed in studies using reporter systems, and their natural occurrence as well as their role in DNA repair remains debated. To address this question, we investigated diRNA biogenesis in Arabidopsis thaliana using CRISPR/Cas12a to induce targeted DSBs at three actively transcribed loci and one untranscribed locus. We found that 21-nt diRNAs are generated at transcribed loci, in a Dicer-like 4 (DCL4) dependent manner, but not at the untranscribed locus, indicating that active transcription is required for diRNA production. Remarkably, long-read DNA sequencing analysis shows that these diRNAs do not detectably influence DSB repair via either end-joining or microhomology-mediated end-joining. Furthermore, endogenous diRNAs have a modest effect on homologous recombination-associated repair, as impairment of the diRNA pathway in ago2 mutants resulted in only a slight reduction in RAD51 focus formation following Cas12a-induced DSBs. Together, these findings indicate that diRNAs are not essential for DSB repair itself but may function in the post-transcriptional silencing of damaged genes, likely to prevent aberrant transcription until proper repair is completed.

genetics↗

bioRxiv2018

Interhomolog polymorphism shapes meiotic crossover within RAC1 and RPP13 disease resistance genes

During meiosis chromosomes undergo DNA double-strand breaks (DSBs), which can produce crossovers via interhomolog repair. Meiotic recombination frequency is variable along chromosomes and concentrates in narrow hotspots. We mapped crossovers within Arabidopsis thaliana hotspots located within the RAC1 and RPP13 disease resistance genes, using varying haplotypic combinations. We observed a negative non-linear relationship between interhomolog divergence and crossover frequency, consistent with polymorphism suppressing crossover repair of DSBs. Anti-recombinase mutants fancm, recq4a recq4b, figl1 and msh2, or lines with increased HEI10 dosage, are known to show increased crossovers. Surprisingly, RAC1 crossovers were either unchanged or decreased in these genetic backgrounds. We employed deep-sequencing of crossovers to examine recombination topology within RAC1, in wild type, fancm and recq4a recq4b mutant backgrounds. The RAC1 recombination landscape was broadly conserved in anti-recombinase mutants and showed a negative relationship with interhomolog divergence. However, crossovers at the RAC1 5-end were relatively suppressed in recq4a recq4b backgrounds, indicating that local context influences recombination outcomes. Our results demonstrate the importance of interhomolog divergence in shaping recombination within plant disease resistance genes and crossover hotspots.

genetics↗

bioRxiv2017

Nucleosomes and DNA methylation shape meiotic DSB frequency in Arabidopsis transposons and gene regulatory regions

Meiotic recombination initiates via DNA double strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Recombination frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced Arabidopsis SPO11-1-oligonucleotides. DSB hotspots occurred in gene promoters, terminators and introns, driven by AT-sequence richness, which excludes nucleosomes and allows SPO11-1 access. A strong positive relationship was observed between SPO11-1 DSBs and final crossover levels. Euchromatic marks promote recombination in fungi and mammals, and consistently we observe H3K4me3 enrichment in proximity to DSB hotspots at gene 5-ends. Repetitive transposons are thought to be recombination-silenced during meiosis, in order to prevent non-allelic interactions and genome instability. Unexpectedly, we found strong DSB hotspots in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. Hotspot transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination-enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the met1 DNA methyltransferase mutant to investigate the role of heterochromatin on the DSB landscape. Epigenetic activation of transposon meiotic DSBs occurred in met1 mutants, coincident with reduced nucleosome occupancy, gain of transcription and H3K4me3. Increased met1 SPO11-1 DSBs occurred most strongly within centromeres and Gypsy and CACTA/EnSpm coldspot transposons. Together, our work reveals complex interactions between chromatin and meiotic DSBs within genes and transposons, with significance for the diversity and evolution of plant genomes.

genomics↗

bioRxiv2017

Epigenetic activation of meiotic recombination in Arabidopsis centromeres via loss of H3K9me2 and non-CG DNA methylation

Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis centromeres are suppressed for crossovers, as recombination in these regions can cause chromosome mis-segregation. Plant centromeres are surrounded by repetitive, transposon-dense heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. Here we show that disruption of Arabidopsis H3K9me2 and non-CG DNA methylation pathways increases meiotic DNA double strand breaks (DSBs) within centromeres, whereas crossovers increase within pericentromeric heterochromatin. Increased pericentromeric crossovers in H3K9me2/non-CG mutants occurs in both inbred and hybrid backgrounds, and involves the interfering crossover repair pathway. Epigenetic activation of recombination may also account for the curious tendency of maize transposon Ds to disrupt CHROMOMETHYLASE3 when launched from proximal loci. Thus H3K9me2 and non-CG DNA methylation exert differential control of meiotic DSB and crossover formation in centromeric and pericentromeric heterochromatin.

genomics↗

bioRxiv2017

Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis

During meiosis homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double strand breaks, which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of [~]100-200 meiotic DSBs into [~]10 crossovers per meiosis, with the excess DSBs repaired as non-crossovers. In order to bias DSB repair towards crossovers, we simultaneously increased dosage of the pro-crossover E3 ligase gene HEI10 and introduced mutations in the anti-crossover helicase genes RECQ4A and RECQ4B. As HEI10 and recq4a recq4b increase interfering and non-interfering crossover pathways respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect of HEI10 on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases towards the sub-telomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover-suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

genetics↗