morc-1 regulates transgenerational heterochromatin maintenance at nuclear RNAi targets. Caenorhabditis elegans

The germline is an immortal cell lineage and therefore must accurately transmit genetic and epigenetic information over infinite cell divisions to ensure fertility and species survival. Endogenous siRNAs (endo-siRNAs), which are highly expressed in the parental germline and inherited in their progeny, represent one mechanism by which epigenetic information can be transmitted from one generation to the next. In C. elegans, this is accomplished by the nuclear RNAi pathway, which ensures robust inheritance of endo-siRNAs and of target gene silencing through deposition of H3K9me3 marks at target loci. How these marks are maintained in subsequent generations is unknown. Here, we show that the C. elegans micro-orchidia homolog, MORC-1, functions downstream of endo-siRNAs to mediate target silencing and maintenance of siRNA-dependent H3K9me3 marks. Like the established members of the nuclear RNAi pathway, morc-1 is essential for germline maintenance and thus morc-1(-) mutants exhibit a mortal germline phenotype. Furthermore, morc-1(-) mutants exhibit severe decondensation of germline chromatin. Through a forward genetic screen, we have identified mutations in the gene encoding MET-1, a H3K36 histone methyltransferase, that potently suppress morc-1(-) germline mortality. We show that morc-1(-) mutants exhibit progressive, met-1-dependent enrichment of H3K36me3 and that mutation of met-1 can restore germline chromatin organization in late generation morc-1(-) mutants. Our data suggest that MORC-1 is required to repress MET-1 activity at nuclear RNAi target genes and that in the absence of this repression, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality. Overall design: We performed replicate small RNA-seq, mRNA-seq, and ChIP-seq of two histone methylation marks (H3K9me3, H3K36me3) in morc-1 mutant animals with wild-type and hrde-1 mutant animals as controls. In addition, ChIP-seq was performed in the same strains in a met-1 mutant background.

生殖系（germline）是永生细胞谱系，因此必须在无限次细胞分裂中精准传递遗传与表观遗传信息，以保障繁殖能力与物种存续。内源性小干扰RNA（endogenous siRNAs, endo-siRNAs）在亲代生殖系中高表达并可传递至子代，是表观遗传信息跨代传递的重要机制之一。在秀丽隐杆线虫（C. elegans）中，这一过程由核RNA干扰通路（nuclear RNAi pathway）介导，该通路通过在靶位点沉积H3K9me3修饰，保障内源性小干扰RNA及靶基因沉默的稳定遗传。目前尚不清楚这些组蛋白修饰如何在后续世代中得以维持。本研究发现，秀丽隐杆线虫的微睾丸同源蛋白MORC-1可在内源性小干扰RNA下游发挥功能，介导靶基因沉默并维持依赖于小干扰RNA的H3K9me3修饰。与已报道的核RNA干扰通路成员一致，morc-1基因对生殖系维持至关重要，因此morc-1(-)突变体呈现生殖系致死表型。此外，morc-1(-)突变体还表现出生殖系染色质的严重解聚。通过正向遗传筛选，我们鉴定出编码组蛋白H3K36甲基转移酶的met-1基因的突变可强效抑制morc-1(-)的生殖系致死表型。我们发现，morc-1(-)突变体呈现渐进性、依赖于met-1的H3K36me3富集，而met-1的突变可恢复晚期世代morc-1(-)突变体的生殖系染色质组织状态。我们的研究结果表明，MORC-1可在核RNA干扰通路靶基因处抑制MET-1的活性；当缺乏该抑制作用时，MET-1介导的常染色质侵占会导致生殖系染色质发生有害解聚，最终引发生殖系致死。总体实验设计：我们对morc-1突变体动物开展了重复的小RNA测序（small RNA-seq）、信使RNA测序（mRNA-seq）及两种组蛋白甲基化修饰（H3K9me3、H3K36me3）的染色质免疫共沉淀测序（ChIP-seq），并以野生型及hrde-1突变体动物作为对照。此外，我们还在met-1突变背景下的上述菌株中开展了染色质免疫共沉淀测序实验。