DNA polymerase epsilon interacts with SUVH2/9 to mediate meiotic DSB associated gene silencing beyond DNA methylation in Arabidopsis [RNA-seq]. DNA polymerase epsilon interacts with SUVH2/9 to mediate meiotic DSB associated gene silencing beyond DNA methylation in Arabidopsis [RNA-seq]

Meiotic recombination is initiated from 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 meiotic DSB sites appears to be detrimental for repair, and the mechanisms for gene silencing at DSB sites are largely undefined, especially in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3-9 homologs SUVH2 and SUVH9. N-SIM (Structured Illumination Microscopy) observation shows that the colocalization of SUVH2 with meiotic DSB marker γ- H2AX is dependent on POL2A. RNA-seq of male meiocytes identifies 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. Consequently, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9. Taken together, our results not only identify a new epigenetic regulatory mechanism for gene silencing in male meiocytes, but also reveal new roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis. Overall design: Examination of gene expression in meiocytes and seedlings of mutants, such as pol2a-1, suvh2 suvh9 etc, and wild type plants

减数分裂重组始于孢子形成11（SPORULATION 11, SPO11）介导的双链断裂（double strand breaks, DSBs）形成，这类断裂在多数真核生物中通常定位于具备活跃转录特征的开放染色质区域。然而，减数分裂DSB位点处的基因转录似乎对修复过程存在不利影响，而DSB位点的基因沉默机制在很大程度上仍未阐明，在植物中这一问题尤为突出。本研究证实，最大的DNA聚合酶ε亚基POL2A可与SU(VAR)3-9同源蛋白SUVH2及SUVH9发生相互作用。N-SIM（结构化照明显微镜，Structured Illumination Microscopy）观察结果显示，SUVH2与减数分裂DSB标记蛋白γ-H2AX的共定位现象依赖于POL2A的存在。对雄性减数细胞的RNA测序（RNA-seq）分析表明，POL2A与SUVH2可共同抑制865个基因的表达，这些基因具备与减数分裂DSB位点相关的多种已知特征。针对雄性减数细胞的亚硫酸氢盐测序（Bisulfite-seq）与小RNA测序（small RNA-seq）结果支持这一结论：POL2A与SUVH2/9对上述基因的沉默过程可能不依赖于CHH甲基化或24nt小干扰RNA（small interfering RNA, siRNA）的积累。进一步实验发现，pol2a suvh2 suvh9三突变体相较于单突变体pol2a或双突变体suvh2 suvh9，在减数分裂重组与育性方面呈现出更为严重的缺陷。综上，本研究不仅揭示了雄性减数细胞中基因沉默的新型表观遗传调控机制，还阐明了DNA聚合酶与SUVH2/9超越其经典有丝分裂功能的全新作用。整体实验设计：对pol2a-1、suvh2 suvh9等突变体及野生型植株的减数细胞与幼苗开展基因表达检测。