CTCF蛋白调控造血干细胞发育的分子机理研究

Hematopoietic stem cell (HSC) is the origin of all mature blood cells. Its specification depends on the synergy of key signaling transduction pathways, transcription factors and epigenetic factors during embryonic development. Nevertheless, how the key chromatin topological structure regulates this process is still unknown. CCCTC-binding factor (CTCF) is a well-known protein involved in the establishment of chromatin three-dimension architecture, which plays the critical role in gene transcription and cell fate determination. Previously, we found that CTCF loss-of-function could result in apoptosis of hemogenic endothelium (HE) during endothelial to hematopoietic transition (EHT), and sequentially result in hematopoiesis failure, which was due to elevated level of p16. Further investigation revealed that CTCF, which was enriched in HE, could exert its enhancer blocking function by means of binding sites in both sides of p16 regulatory region, facilitating the negative regulation of p16 transcription by the synergy of Hoxb4a and repressive epigenetic factors. With the combination of biochemistry, molecular genetics and developmental biology approaches, we aim to illustrate the regulation mechanism of p16 by CTCF and Hoxb4a during HSC generation, to uncover the molecular mechanism regulating the specific expression of CTCF during EHT, and to analyze the development and function of HSC in the ctcf and p16 double knock-out animals. Our exploration will provide fundamental knowledge for optimizing HSC in vitro induction and for clinical diagnosis and therapy of relevant blood diseases.

造血干细胞（HSC）产生体内所有血细胞，已知HSC发育依赖信号转导途径、转录和表观遗传因子协同作用，而关键染色质结构如何参与此过程依然未知。CTCF蛋白是调控染色质三维结构形成并影响基因表达和细胞命运决定的关键因子。我们的前期工作发现斑马鱼ctcf基因失活可导致生血内皮细胞在向HSC分化过程中因p16水平上调而凋亡，血液系统发育失败；还发现CTCF在生血内皮中富集表达，并可能通过结合在p16调控区两侧形成增强子绝缘结构并介导Hoxb4a蛋白和抑制性表观遗传修饰协同负调控p16。本课题将综合运用生化、分子遗传和发育生物学手段，阐明HSC形成过程中CTCF及Hoxb4a对p16表达调控的作用机理；揭示CTCF在内皮-HSC转变过程中特异性表达的分子基础；展开ctcf和p16双缺失条件下HSC的发育与功能分析，以上研究结果将为HSC体外诱导技术优化及相关血液疾病诊治提供重要理论基础。

造血干细胞(HSC)是一类既具有自我更新能力又具有血细胞完全分化潜能的细胞，它们对维持机体造血系统稳态起到至关重要的作用。CTCF蛋白是调控染色质三维结构形成并影响基因表达和细胞命运决定的关键因子。ctcf突变造成斑马鱼在发育过程中AGM区造血干细胞形成缺陷，通过ChIP、CRISPR/Cas9等实验手段我们研究发现CTCF蛋白通过结合hoxb4a基因下游的特异DNA位点正向调控hoxb4a的表达。通过活体成像等我们检测了ctcf突变体AGM区的细胞凋亡和凋亡相关因子p16INK4a的表达，并通过RNASCope实验检测p16的表达，ctcf突变导致造血干细胞中p16表达升高，从而造成造血干细胞的凋亡。通过ChIP、CO-IP等实验进一步研究表明Hoxb4a蛋白直接结合p16启动子并招募PRC2复合体来维持p16启动子区的H3K27me3水平，从而抑制p16的表达。至此，我们发现 CTCF-Hoxb4a-p16调控轴在染色质高级结构的维持、基因表达调控和细胞命运监控三个环节影响造血干细胞的形成。该机制的发现不仅为造血干细胞的形成提供了染色质高级结构调控层面的科学理解，而且为体外诱导产生HSCs提供了新的理论依据，有望解决HSCs数量不足问题。在本项目支持下共发表具有国际影响力的研究论文2篇，核心成果在投稿中，另培养博士生6名。