The transcription of eukaryotic genes is regulated by both proximal promoters and distal enhancers. Some promoters also have enhancer activity. NOXA and BCL2 are pro-apoptotic and anti-apoptotic members of the BCL2 family of protein, respectively. Our previous study has found that the NOXA gene promoter and the BCL2 gene promoter interact at the level of three-dimensional chromatin structure. Moreover, the NOXA gene promoter region displays histone modifications characteristic of both promoters and enhancers. This study aimed to explore whether and when the NOXA promoter could act as an active enhancer to regulate BCL2 expression. Based on the apoptosis model of MCF-7 cells induced by camptothecin, we used chromosome conformation capture (3C), quantitative real-time PCR (qRT-PCR) and the luciferase reporter gene technology to demonstrate that the NOXA promoter could function as an active enhancer and physically interact with the BCL2 promoter through chromatin looping. The regulatory properties of the NOXA promoter were closely related to the strength of the apoptosis stimulation. Under weak apoptotic stimulation (1 μmol/L camptothecin treatment), the NOXA promoter mainly functioned as an enhancer; with the enhancement of apoptotic stimulation (10 μmol/L camptothecin treatment), the NOXA promoter activity increased and mainly regulated the expression of the gene itself to promote apoptosis. Chromatin immunoprecipitation (ChIP) confirmed that the dynamic changes of the promoter activity and enhancer activity in the NOXA promoter region are consistent with its histone modification marks. This study provides new clues for further exploring the mechanism underlying cooperative response of BCL2 family member to apoptosis stimuli.
真核生物基因的转录受到近端启动子和远端增强子的共同调控,部分基因的启动子可兼具有增强子的活性。NOXA与BCL2分别是BCL2蛋白家族促凋亡和抗凋亡成员。本课题组前期研究发现,NOXA基因启动子与BCL2基因启动子在染色质三维空间结构上存在相互作用,且NOXA基因启动子区兼有启动子和增强子特征性的组蛋白修饰标记。为进一步探究NOXA启动子是否具有增强子活性、能否在细胞凋亡过程中作为增强子调控BCL2基因表达,本研究利用染色质构象捕获(chromosome conformation capture, 3C)、实时荧光定量PCR (quantitative real-time PCR, qRT-PCR)和荧光素酶报告基因等检测技术在喜树碱诱导的MCF-7细胞凋亡模型中证实,NOXA启动子兼具增强子活性,并可通过形成染色质环结构远程调控BCL2基因表达。NOXA启动子的调控属性与凋亡信号强弱密切相关,在较弱凋亡信号刺激下(1 μmol/L喜树碱处理),NOXA启动子主要发挥增强子功能;随着凋亡刺激信号的加强(10 μmol/L喜树碱处理),NOXA启动子活性增强,主要调控其基因自身的表达,促进细胞凋亡。染色质免疫共沉淀(chromatin immunoprecipitation, ChIP)证实NOXA启动子区启动子活性和增强子活性的动态变化与其组蛋白修饰标志一致。本研究为进一步探讨BCL2家族成员对细胞凋亡刺激做出协同反应的机制提供了新的线索。.
Keywords: BCL2 gene; NOXA promoter; apoptosis; dual transcription regulation function; enhancer.