Role of bHLH transcription factor DEC1 on chondrogenic differentiation

我们克隆了人和小鼠的DEC1基因，并测定了这些基因的完整核苷酸序列，包括3kb的上游区域。DEC1基因由5个外显子和4个内含子组成，上游含有3个E-box等多个序列元件。没有TATA盒，但在启动子区域发现了几个GC盒：我们构建了一系列不同长度的DEC1启动子与荧光素酶报告基因融合的缺失构建体，并将它们导入几种类型的细胞。结果表明，在转录起始点上游至少260个碱基对几个细胞具有显著的启动子活性。进一步，将多个编码CLOCK、BMAL1、MyoD和HlF1的表达载体共转染入细胞。Northern印迹和RT-PCR分析检测了DEC1基因的表达模式。几乎所有被检测的细胞和组织都表达DEC1mRNA，并且这种表达是由cAMP诱导的。DEC1基因在ATDC5细胞中的过表达可诱导其向软骨细胞分化。

We cloned human and mouse DEC1 genes and determined an entire nucleotide sequence of the genes including 3 kb of the upstream region. The DEC1 genes consisted of 5 exons and 4 introns, and there were several sequence elements including 3 E-boxes in the upstream region. There was no TATA box, but several GC boxes were identified in the promoter region :We constructed a series of deletion constructs of different lengths of the DEC1 promoter fused to the luciferase reporter gene and transfected them into several types of cells. The results showed that at least 260 bp region upstream from the transcription start site had significant promoter activity in the several cells. Further, when several expression vectors encoding CLOCK, BMAL1, MyoD and HlF1 were co-transfected into the cells,. the promoter activity of the DEC1 gene was increased.Expression pattern of DEC1 mRNA was also examined by Northern blot and RT-PCR analyses. DEC1 mRNA was expressed almost all of cells and tissues examined, and the expression was induced by the addition of cAMP to the cells. Overexpression of DEC1 gene in ATDC5 cells induced chondrogenic differentiation of the cell.