Hippo/YAP通路调控组蛋白H3K27修饰动态变化在炎症性肠病肠黏膜损伤修复中的作用研究

The pathogenesis of inflammatory bowel disease (IBD) is close related to the disorder of epithelial cell regeneration and intestinal mucosal barrier function and wound healing. Intestinal epithelial cell (IECs) is the critical component of mucosal barrier and the normal function of intestinal local immunity and homeostasis depend on the various effects of IECs. Previous studies reported that Hippo/YAP pathway could contribute to the intestinal mucosa wound healing. However, the underlying mechanism is not defined yet. In our preliminary experiments, we demonstrated that YAP activation upregulated the expression of histone demethylase JMJD3, and Yap, Jmjd3, Cyr61 expression and H3K27 acetylation level increased in the inflamed mucosa of DSS-induced chronic colitis in mouse and IBD patients. Our data suggested YAP might manipulate the CYR61 expression through JMJD3 regulating the H3K27 modification to involve the wound healing during the intestinal inflammation development. In this study, we will comprehensive analyze and determine the effect of YAP activation on the JMJD3, H3K27 modification and expression CYR61 through generation of Yap and Jmjd3 gene deletion cells by CRISPR/Cas9 gene editor technology and the intestinal epithelial cell conditional Yap- or Jmjd3-deficient mice. We will evaluate the effects of YAP activation on the JMJD3 expression, H3K27 modification, CYR61 expression and influence of mouse colitis as well (both in vivo and in vitro). Furthermore, to further clarify the underlying molecular mechanism and network system, we will apply the systemic biology approaches to comprehensive analyze the H3K27 epigenetic and gene transcription profile and dynamic change in IECs during the intestinal inflammation development by high throughput ChIP-Seq and RNA-Seq technology, combined with bioinformatics methods. The therapeutic efficacy of small inhibitor of JMJD3 or H3K27 on DSS-induced colitis will be systemically evaluated in mouse model. Successful completion of our proposed study will not only lead to the elucidation of the intestinal local immunity character and epigenetic mechanism of pathogenesis of IBD, but also provide a new experimental evidence for the translation from basic science to clinic medicine and therapeutic target for developing novel strategies of managing IBD through epigenetic-based gene-targeted technology.

炎症性肠病（IBD）的发病与肠黏膜屏障受损和再生修复能力失衡密切相关。Hippo/YAP通路可能通过靶基因CYR61参与肠黏膜损伤修复，但具体机制不明。我们前期发现YAP活化促进组蛋白去甲基化酶JMJD3上调，炎症肠黏膜Yap、Jmjd3、Cyr61转录水平和H3K27乙酰化富集增高，提示YAP可能通过JMJD3调控H3K27修饰影响CYR61表达。本课题利用CRISPR/Cas9敲除肠上皮细胞株Yap或Jmjd3，并构建肠上皮细胞Yap或Jmjd3条件性敲除小鼠，明确Yap活化对Jmjd3、Cyr61表达和H3K27修饰及小鼠肠炎的影响；高通量ChIP-Seq技术分析肠炎发展过程中组蛋白修饰的动态变化图谱，鉴定关键表观遗传事件并进一步阐明其分子机制，同时初步确定炎症状态下增强子和超级增强子编码模式，从而揭示炎症发展过程中Hippo/YAP通路的表观遗传学调控机制，为转化医学提供依据。

背景及目的：表观遗传学修饰是炎症性肠病发病的重要机制之一，然而迄今为止表观遗传调控如何参与IBD发病仍不明确。我们阐明了Hippo/YAP通路在肠道炎症发展中的作用及其潜在机制。此外，我们也探讨了 H3K27ac表观遗传事件以及组蛋白甲基化酶SETD8在炎症性肠病中的作用机制。.方法：（1）构建YAP肠上皮细胞特异性敲除小鼠，采用2.5%DSS诱导的小鼠肠炎模型，运用生物信息学方法筛选YAP下游靶基因JMJD3。（2）ChIP-seq分析基因组H3K27乙酰化分布，筛选出转录因子STAT1可能参与肠道炎症。（3）使用脂多糖（ lipopolysaccharide, LPS）刺激小鼠巨噬细胞系RAW264.7，建立炎症细胞模型。.结果：（1）YAP在体外、体内模型和IBD患者中的表达均明显增加。敲低YAP可显著提高JMJD3蛋白水平。YAP与JMJD3之间没有直接结合，而YAP和EZH2可相互结合形成复合物， EZH2沉默显著降低了JMJD3启动子H3K27me3富集。JMJD3药物抑制剂(GSK-J4)可缓解DSS诱导的YAP肠上皮特异性敲除小鼠结肠炎的病情进展。 .（2）STAT1/p-STAT1在细胞炎症模型、DSS诱导的慢性小鼠肠炎模型及IBD患者中表达增加；LCP2和TNFAIP2STAT1/p-STAT的靶基因； p-STAT1 可招募EP300形成复合物，通过促进LCP2和TNFAIP2基因增强子H3K27ac富集来调控这两个靶基因的表达；小鼠急性肠炎模型中，EP300抑制剂C646处理后，小鼠肠炎明显减轻。.（3）SETD8的表达在IBD患者及慢性肠炎小鼠的炎症肠黏膜中显著降低。SETD8敲低可上调p62蛋白水平，但不改变LC3表达，敲低SETD8后，p62基因启动子区域H4K20me1富集减少。腹腔注射UNC0379可加重DSS诱导的病理改变和临床症状。.结论：我们的研究阐明了YAP与 EZH2形成复合体共同调节JMJD3表达，抑制肠道炎症。p-STAT1招募EP300、介导LCP2和TNFAIP2基因增强子上H3K27ac富集，促进LCP2和TNFAIP2表达增加而促进肠炎的发病。SETD8通过表观遗传调控p62表达、抑制炎症反应参与IBD的发生。