Zhou Hong, Li Le, Wang Qing, Hu Yan, Zhao Weiwei, Gautam Mayank, Li Lijia
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China.
Front Genet. 2020 Feb 6;11:43. doi: 10.3389/fgene.2020.00043. eCollection 2020.
The nucleolar structure and integrity are important for a range of cellular functions of the nucleoli. It has been shown that cells lacking histone H3 Lysine 9 (H3K9) methylation form fragmented nucleoli. However, the molecular mechanism involved remains poorly understood. Here, we present evidence suggesting that loss of H3K9 dimethylation (H3K9me2) triggers R-loop accumulation at the rDNA locus, which further leads to the multilobed nucleoli. We reveal that suppression of H3K9 methyltransferase G9a by the inhibitor BIX 01294 causes R-loop accumulation at the rDNA region as well as inducing formation of multiple nucleoli. SiRNA-mediated knockdown of RNase H1 which can hydrolyze the RNA chain in R-loops causes an increase in R-loop formation, which in turn results in multiple nucleoli in one nucleus, whereas H3K9me2 levels are not affected by R-loop accumulation. Inhibition of RNA polymerase I transcription elongation by small molecule inhibitors induces a substantial decrease in H3K9me2 levels, accumulation of R-loops at rDNA sites, and nucleolus fragmentation. These results provide a mechanistic insight into the role of H3K9me2 in the structural integrity and organization of nucleoli via regulating R-loop accumulation.
核仁结构与完整性对于核仁的一系列细胞功能至关重要。已有研究表明，缺乏组蛋白H3赖氨酸9（H3K9）甲基化的细胞会形成碎片化核仁。然而，其中涉及的分子机制仍知之甚少。在此，我们提供证据表明，H3K9二甲基化（H3K9me2）的缺失会触发rDNA位点处R环的积累，进而导致多叶核仁的形成。我们发现，抑制剂BIX 01294对H3K9甲基转移酶G9a的抑制会导致rDNA区域R环的积累以及多核仁的形成。RNA酶H1可水解R环中的RNA链，通过RNA干扰介导的RNA酶H1敲低会导致R环形成增加，进而导致一个细胞核中出现多核仁，而H3K9me2水平不受R环积累的影响。小分子抑制剂对RNA聚合酶I转录延伸的抑制会导致H3K9me2水平大幅下降、rDNA位点处R环的积累以及核仁碎片化。这些结果为H3K9me2通过调节R环积累在核仁结构完整性和组织中的作用提供了机制性见解。
