BRD4液相分离的体内功能及其调控超增强子的机制研究

细胞内“蛋白液相分离”是新发现的重要生物学现象，被认为是细胞高效管理多种生命活动（如超增强子调控的转录）的基础。最近，体外实验表明，超增强子关键蛋白BRD4通过LCD结构域发生液相分离，但体内功能及分子机制还不清楚。因此，我们建立了BRD4-LCD结构域缺陷小鼠（BRD4dLCD），破坏BRD4的液相分离。发现：70%的杂合型小鼠在出生1-3天内因肺发育不全死亡；从存活杂合型小鼠中分离的CD4+T细胞不能向Th9细胞分化；杂合型MEF细胞中，超增强子靶基因C-MYC等表达显著降低。因此，本课题提出BRD4通过液相分离调控超增强子的功能，从而参与肺发育和哮喘的发生。我们将系统研究BRD4液相分离1）在肺发育和哮喘中的作用，2）调控肺发育和哮喘的关键超增强子，3）调控超增强子的分子机制。本课题将从分子机制上阐明BRD4如何通过液相分离来组织超增强子调控靶基因，进而参与正常发育及疾病的发生。

Liquid-liquid phase separation (LLPS) is a recently observed cellular phenomenon that might be the key to understand how do multiple actors inside a cell get together at the right place and time to perform critical functions. BRD4 is a vital epigenetic reader with a unique C-terminal low complexity domain (LCD) that can drive liquid-liquid phase separation but with unknown physiological roles. Here, we established a mouse model (BRD4dLCD) to specifically destroy the C-terminal LCD but leave the N-terminus intact. BRD4dLCD heterozygotes display pre- and postnatal growth defects, and about 70% BRD4dLCD heterozygotes die within three weeks. BRD4dLCD nullizygous are embryonic lethal. The surviving BRD4dLC heterozygotes appear smaller than wild-type littermates. They also showed anatomical defects especially the shorter and bent nasal bone. Interestingly, BRD4dLCD/+ CD4+ T cells are failed to differentiate to Th9 cells in vitro. Furthermore, the expression of super-enhancer-dependent genes such as C-MYC is significantly reduced in BRD4dLCD/+ MEFs compared to wildtype MEFs. Our preliminary results suggest a critical role of BRD4-LLPS in the control of super-enhancers and the development of lung as well as asthma. Here, we will systematically study: (1) the function of BRD4-LLPS in the development of lung and its regulated super-enhancers; (2) the function of BRD4-LLPS in OVA-induced asthma and its regulated super-enhancers; (3) how does BRD4-LLPS regulate the organization and function of super-enhancers. The proposed study will not only provide new insights in the organization of super-enhancers but also provide novel data regarding the in vivo role of BRD4-LLPS.