Germline nuclear RNAi in C. elegans represses transgenerational inheritance of heat-induced gene transcriptional activation (sRNA profiling)

Environmental stress-induced transgenerational epigenetic effects have been observed in various model organisms and human. The capacity and mechanism of such phenomena, particularly in animals, are poorly understood. In C. elegans, siRNA mediates transgenerational gene silencing through the germline nuclear RNAi pathway. At the organismal level, this pathway plays a transgenerational role in maintaining the germline immortality when C. elegans is under a mild heat stress. However, the underlying molecular mechanism is unknown. In this study, we performed a 12-generation temperature-shift experiment (15˚C->23˚C->15˚C) using the wild type (N2) and a mutant strain that lacks the germline-specific nuclear AGO protein HRDE-1/WAGO-9. We found that the temperature-sensitive mortal germline (Mrt) phenotype of the hrde-1 mutant is reversible, indicating a transgenerational cumulative but also reversible nature of the underlying molecular cause. By taking the whole-genome RNA and chromatin profiling approaches, we revealed an epigenetic role of HRDE-1 in repressing heat stress-induced transcriptional activation of over 280 genes, predominantly in or near LTR retrotransposons. Strikingly, for some of these elements, the heat stress-induced transcription becomes progressively activated in the hrde-1 mutant over several generations under heat stress. Furthermore, the effect of heat stress-induced transcription activation is heritable for at least two generations after the heat stress. Interestingly, the siRNA expression of these genes tend to be heat-inducible in the wild type animals, but not in the hrde-1 mutant, suggesting a role of siRNAs in repressing heat-inducible elements. Our study revealed a novel phenomenon of transgenerational feed-forward transcriptional activation, which is normally repressed in the wild type C. elegans by the germline nuclear RNAi pathway. It also provides a new paradigm to study epigenetic circuitry that connects the environment and gene regulation in the germline. In this study, we performed a 12-generation temperature-shift experiment (15˚C->23˚C->15˚C) using the wild type and hrde-1 mutant. mRNA-seq, Pol II ChIP-seq, H3K9me3 ChIP-seq, and small RNA-seq analyses were performed for all or some of the generations. The effects of temperature change in whole-genome mRNA expression, siRNA expression, gene transcription, and H3K9me3 were investigated at the multigenerational time scale in both the WT and hrde-1 mutant animals.

环境应激诱导的跨代表观遗传效应已在多种模式生物及人类中被观测到。此类现象的调控能力与作用机制，尤其是在动物体内的相关机制，目前仍有待深入阐释。
在秀丽隐杆线虫（Caenorhabditis elegans, C. elegans）中，小干扰RNA（small interfering RNA, siRNA）可通过生殖系核RNA干扰（RNA interference, RNAi）通路介导跨代基因沉默。在生物体层面，当秀丽隐杆线虫处于轻度热应激环境时，该通路在维持生殖系永生性方面发挥跨代调控作用，但其背后的分子机制仍未明确。
本研究以野生型（N2）以及缺失生殖系特异性核AGO蛋白HRDE-1/WAGO-9的突变株为材料，开展了为期12代的温度转换实验（15℃→23℃→15℃）。研究发现hrde-1突变体的温度敏感型生殖系致死（temperature-sensitive mortal germline, Mrt）表型具有可逆性，这表明其潜在分子诱因具有跨代累积且可逆的特性。
通过全基因组RNA与染色质图谱分析手段，我们揭示了HRDE-1的表观遗传调控功能：它可抑制热应激诱导的280余个基因的转录激活，这些基因主要富集于长末端重复序列反转录转座子（LTR retrotransposons）内部或其邻近区域。值得注意的是，在部分此类元件中，hrde-1突变体在热应激环境下的数代培养过程中，热应激诱导的转录激活程度会逐渐升高。此外，热应激诱导的转录激活效应可在热应激结束后至少延续两代并传递给子代。
有趣的是，在野生型线虫中，这些基因的siRNA表达往往会受热应激诱导，但在hrde-1突变体中并未出现该现象，这提示siRNA在抑制热应激诱导元件方面发挥了作用。本研究揭示了一种全新的跨代前馈转录激活现象：在野生型秀丽隐杆线虫中，该现象通常会被生殖系核RNAi通路所抑制。本研究同时为研究连接环境与生殖系基因调控的表观遗传环路提供了全新的研究范式。
本研究同样以野生型线虫与hrde-1突变体为材料开展了12代温度转换实验（15℃→23℃→15℃）。对各代或部分代次样本进行了mRNA测序（mRNA sequencing, mRNA-seq）、RNA聚合酶II（RNA polymerase II, Pol II）染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）、组蛋白H3赖氨酸9三甲基化（histone H3 lysine 9 trimethylation, H3K9me3）染色质免疫共沉淀测序以及小RNA测序（small RNA sequencing, small RNA-seq）分析。本研究在多代时间尺度下，探究了温度变化对野生型与hrde-1突变体线虫全基因组mRNA表达、siRNA表达、基因转录以及H3K9me3修饰的影响。