H3K9甲基化酶SETDB1调控ERVs对小鼠早期胚胎发育及干细胞命运决定机制研究

The H3K9 transmethylase SETDB1 plays an important role in the early embryonic development and pluripotency maintenance of mouse embryonic stem cells (mESCs). SETDB1-mediated H3K9me3 mainly silences endogenous retroviruses (ERVs) in ESCs. ERVs are specifically activated during early embryo development, suggesting that ERVs may be involved in the regulation of early embryonic development. Setdb1 conditional knockout mice and mESCs have been constructed in the early stage of this project. Next, single-cell RNA-seq will be applied to map the SETDB1-regulated ERVs during early embryonic development and to explore the cis-regulatory relationship between ERV activation and gene expression. Additionally, preliminary data exhibited that knockout of Setdb1 causes necroptosis of the ground state mESCs. The raised expression of Ripk3, a key necroptosis gene, is related to the regulation of IAPLTR2 transposon upon Setdb1-deficiency, but the mechanism of RIPK3 activation is still unknown. ERV activation can also stimulate type I interferon-induced immune response and necroptosis through pattern recognition receptors (PPR). This project will also explore the mechanism of ERV activation in cell fate determination through PPR sensing. In summary, this project will study the regulatory mechanism of Setdb1-mediated ERVs in early embryonic development and ESC cell fate determination through cis-acting elements and PPRs sensing.

H3K9甲基化酶Setdb1对早期胚胎发育及多能性维持有重要作用，且可介导mESC中逆转录病毒元件(ERV)沉默。ERV在早期胚胎发育过程被特异激活，表明ERV可能参与早期胚胎发育调控。本项目前期已构建Setdb1条件敲除小鼠，以期通过单细胞RNA-seq分析绘制出Setdb1于早期胚胎发育过程的ERV调控图谱以及探讨ERV激活与基因表达的顺式调控关系。前期研究还发现Setdb1敲除可引起ground state mESC发生程序性坏死(Necroptosis)，且Necroptosis关键基因Ripk3转录上升与Setdb1缺失激活IAPLTR2转座子调控相关，但RIPK3激活机制仍未知。ERV激活可通过模式识别受体(PRR)激活Ⅰ型干扰素免疫反应、Necroptosis。综上，本项目将研究Setdb1所调控ERV通过顺式作用元件和PPR感应方式对早期胚胎发育及干细胞命运决定的调控机制。

表观遗传调控与转座子激活状态在细胞命运决定过程中具有重要意义。生命体每个细胞都拥有相同的一套基因组序列，但每个细胞的命运及其基因调控水平却各不相同。表观遗传酶Setdb1在胚胎干细胞中协同Trim28抑制TEs激活，其抑制的TEs主要为逆转录病毒元件(Endogenous retroviruses, ERVs) 。同时，Setdb1是所以H3K9甲基化酶中，其敲除唯一可致小鼠早期胚胎致死(E3.5-5.5)，表明Setdb1在早期胚胎发育过程中至关重要。Setdb1催化的H3K9me3修饰绝大多数落在TEs位点沉默TEs表达，暗示Setdb1调控TEs的功能有可能参与到其对细胞命运维持与转变的调控作用。因此，本项目以Setdb1条件性敲除小鼠为模型，通过采用RNA-seq、ChIP-seq、ATAC-seq和RIP-seq等高通量测序及生物化学手段研究Setdb1所调控TEs在胚胎干细胞命运中的调控机制。.我们通过对Setdb1 KO前后的RNA-seq、H3K9me3 ChIP-seq以及ATAC-seq数据进行联合分析发现Setdb1调控的ERVs对基因的顺式调控作用，其中程序性坏死因子Ripk3上游IAPLTR2元件具有enhancer调控作用，并进一步通过荧光素酶报告系统实验、dCas9调控、CRISPR-Cas9敲除等实验进行验证。进一步的，我们发现TEs除可作为顺式调控元件调控基因表达，其转录出的RNA可模拟病毒入侵激活程序性坏死，且ZBP1为激活ERVs下游感应sensor。总的来说，我们发现SETDB1敲除激活的ERVs元件可同时通过顺式调控作用以及反式调控作用的方式影响胚胎干细胞发生程序性坏死命运。该工作已整理并于Cell death & disease中投稿并修回一审意见。.延伸的，我们参与YTHDC1通过SETDB1调控多能性-全能性的工作，阐明Dux位点的重要性，证明全能性干细胞在胚胎中参与胚外谱系的发育潜能，在Nature杂志发表题为The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity的研究论文。.同时，我们利用多年积累的干细胞编辑以及培养技术，参与人源化ACE2小鼠快速制备攻关项目，成功在35天内制备出hACE2小鼠。