未折叠蛋白反应关键转录因子XBP1s与ATF4调控脂肪细胞分化的基因网络分析

Obesity has become the most common epidemic and a worldwide threat to the health of populations. Obesity is characterized by expansion of adipose tissue, which results from excess adipocyte differentiation. Adipocyte differentiation is orchestrated by many transcription factors, which forms network through lots of downstream effectors. Our previous study on the molecular mechanism of R17, a novel potential anti-obesity drug, revealed that two branches of UPR mediated by XBP1s and ATF4 (transcription factors of UPR), are activated during adipogenesis. Moreover, the inhibitory effect of R17 on adipogenesis is associated with the suppression of the two branches, indicating the proadipogenic role of UPR. However, how XBP1s and ATF4 regulate adipogenesis and whether they work synergistically remains unclear. On the basis of the background and previous study, we will study the role and molecular mechanism of XBP1s and ATF4, and their interrelationship during adipogenesis in 3T3-L1 cells by using CRISPR/Cas, ChIP-seq, microarray and luciferase reporter approches etc. Our findings will shed light on the role of UPR in adipogenesis , map the gene expression network of XBP1s and ATF4 in a genome-wide scale and also provide theoretical basis and novel targets for the treatment of obesity.

肥胖目前已成为威胁人类健康全球最大的流行病，主要特征为脂肪组织过度扩增。而脂肪细胞分化是导致脂肪组织过度扩增的重要因素。脂肪细胞分化受大量配合精密的转录因子及其介导的信号通路网络共同调控。我们在前期工作中发现，未折叠蛋白反应（UPR）的关键转录因子XBP1s和ATF4介导的信号通路在脂肪细胞分化过程中同时被激活，而一种潜在的减肥新药 R17抑制脂肪细胞分化的机制与下调这两条通路有关，提示UPR可能促进脂肪细胞分化。但XBP1s和ATF4是如何调节脂肪分化相关基因的表达，二者是否存在协同作用，还不清楚。本项目拟在3T3-L1细胞中，通过CRISPR/Cas、ChIP-seq、基因芯片等方法研究XBP1s和ATF4在脂肪细胞分化中的作用、调控机制及相互关系，并在全基因组范围初步绘制二者在分化中的基因调控网络，为进一步探讨脂肪细胞分化的分子机制提供更多证据，为肥胖的防治提供理论依据和新的治疗靶点

肥胖是目前全球最大的流行性疾病，而中国的肥胖人数居全球首位。脂肪细胞分化异常是肥胖发生发展过程中的重要环节。因此研究脂肪细胞分化的机制有助于揭示肥胖的发病机理。脂肪细胞分化的过程非常复杂，由众多转录因子及多条信号通路共同调控。近年来内质网应激（UPR）通路在脂肪细胞分化中的作用受到了关注，但是其调控脂肪细胞分化的机制尚未阐明。我们对UPR通路的关键转录因子ATF4，XBP1介导的两条UPR支路在脂肪细胞分化中的功能和机制进行深入研究，发现：1）敲低ATF4或XBP1能抑制脂肪细胞分化，并抑制UPR通路，下调脂肪细胞分化关键转录因子PPARg和C/EBPa的表达。2）过表达ATF4或XBP1能促进脂肪细胞分化，并激活UPR通路，促进PPARg和C/EBPa及下游脂肪分化蛋白的表达。3）敲低ATF4或者XBP1能抑制自噬。4）过表达ATF4或者XBP1能激活自噬。以上结果提示ATF4和XBP1介导的UPR通路可能通过自噬促进脂肪细胞分化。我们深入地挖掘ATF4调控脂肪细胞分化的机制，发现：5）ATF4能够调控3968个基因(DEGs)的转录，富集在脂肪酸代谢，细胞呼吸，能量代谢的进程。6）Chip-seq分析发现ATF4在全基因组的定位其中启动子占15.89%，涉及8913个基因。结合RNA-seq分析发现ATF4能够直接调控的差异表达基因有1955个，富集在代谢途径，脂肪细胞分化及脂肪组织扩增途径。且ATF4在PPARg及CTCF的启动子区有结合峰。7）Co-IP和Confocal分析发现ATF4与CTCF存在着直接的相互作用，而敲低CTCF能够抑制脂肪细胞分化，说明ATF4能够通过CTCF调控脂肪细胞的分化。8）对ATF4及CTCF的RNA-seq数据整合分析发现二者共同调控的差异表达基因富集在脂肪细胞分化等进程。综上，我们发现ATF4能够通过直接调控PPARg和CTCF的转录,并与CTCF相互作用，共同调控脂肪细胞的分化。