组织特异性表达基因PLUNC调控元件的生物信息学分析

目的分析组织特异性表达基因PLUNC的调控元件。方法采用进化足迹法,结合生物信息学工具,预测PLUNC基因的顺式作用元件和反式作用因子。结果预测的PLUNC基因的转录起始位点位于-1～＋10bp区域间、-29bp存在TATA盒子,启动子集中在-490bp～＋89bp内,在人和小鼠PLUNC基因的启动子保守区内存在29个共同的转录因子结合部位及5个增强子。结论PLUNC基因调控元件的分析可为探讨上呼吸道特异性表达基因的调控机制提供模型。生物信息学技术结合进化足迹法,在基因表达调控机制的研究中具有重要的应用价值。     

C/EBPalpha and C/EBPdelta activate the clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites

The Clara cell secretory protein (CCSP) gene is a cell-specific differentiation marker for the bronchiolar Clara cell. Previous studies suggest that CCAAT/enhancer binding protein (C/EBP)alpha is involved in controlling differentiation-dependent gene expression in the distal lung. In this study, immunofluorescence studies demonstrated high level expression of C/EBPdelta in the bronchiolar epithelium as well as lower levels of C/EBPalpha. Cotransfection studies in the lung epithelial cell line A549 showed that both C/EBPalpha and C/EBPdelta activate the murine CCSP gene and that a C/EBP-response element resides in the proximal CCSP promoter. C/EBPdelta exhibits an approximately 2-fold higher transactivation potential than does C/EBPalpha. DNase I footprint analyses revealed a footprint region located at -100 to -62 bp, corresponding to two C/EBP-binding sites. Mutation of either site resulted in abolished or strikingly reduced transactivation of the CCSP promoter by C/EBPalpha and C/EBPdelta, as well as impaired binding of both factors, indicating that the two C/EBP-binding sites form a compound response element. In electrophoretic mobility shift assays, it was shown that C/EBPalpha and C/EBPdelta can bind to both C/EBP sites, whereas in DNase I footprint analyses, the interaction of C/EBPalpha with the proximal site was weak. Furthermore, electrophoretic mobility shift assays demonstrated that C/EBPalpha and C/EBPdelta preferentially form heterodimers at both binding sites. Cotransfections with C/EBPalpha and C/EBPdelta together resulted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBPalpha-C/EBPdelta heterodimers. Our findings demonstrate that C/EBPalpha and C/EBPdelta regulate the CCSP gene through a compound response element and suggest that these factors are important for the differentiation-dependent expression of CCSP.