人类原钙粘蛋白基因簇调控元件的克隆及对其启动子活性的影响

Cloning and Functional Analysis of the Regulatory Elements in the Human Protocadherin Gene Cluster

人类原钙粘蛋白(Protocadherin,Pcdh)基因簇包含53个成串排列非常相似的基因,组成3个紧密相连的基因簇(α,β和γ)。原钙粘蛋白基因簇γ通过启动子选择性表达产生神经元细胞膜表面的分子多样性,但是,该多样性产生的分子机制还不清楚。调控元件HS7L和HS5-1aL作为候选的增强子可能具有调控Pcdhγ基因表达的作用。利用分子克隆的方法,将调控元件HS7L和HS5-1aL分别克隆至包含γa9、γa10、γb3、γb7和γc3启动子的荧光素酶报告基因的下游。通过荧光素酶报告基因试验检测其对该5种Pcdhγ启动子活性的影响,发现HS7L对5种启动子活性具有增强作用,HS5-1aL对γa10启动子活性具有增强作用。之后,通过基因沉默绝缘子CTCF,发现下调CTCF不仅降低γb1基因表达,而且能够显著降低γb1启动子报告基因活性。试验结果表明调控元件HS7L和HS5-1aL能够增强Pcdhγ启动子活性,推测可能通过CTCF介导的增强子-启动子相互作用调控Pcdhγ的细胞特异性基因表达。

Promoter choice of members of the protocadherinγ （Pcdhγ） gene cluster in single neurons may generate an enormous diversity on the cell surface of individual neurons in the brain, but the underlying molecular mechanisms are poorly understood. Here, candidate enhancer elements of the human PcdhT clus- ter, HS7L and HS5-1aL, were cloned into the downstream of the luciferase reporter gene which is under the control of the γa9, γa10, γb3, γb7, or γc3 promoter. Luciferase reporter gene assay, which was used to char- acterize the functions of these two elements, revealed that HS7L enhances the promoter activity of all of the five genes; by contrast, HS5-1aL increases the γa10 promoter activity. In addition, the insulator CTCF knockdown led to a decrease in the levels of γb 1 mRNAs and its promoter activity. These results suggested that HS7L and HS5-1aL play an important role in regulating the cell-specific PcdhT expression in the brain by CTCF-mediated long-distance enhancer-promoter DNA looping interactions.