Cell Cycle regulation by hDREF

我们已经成功地制备了表达针对hDREF的shRNA的慢病毒。用病毒转导的细胞直接用于实验，例如FACS分析、细胞的免疫荧光和使用针对以细胞周期特异性方式表达的许多蛋白质的抗体的Western印迹。我们发现hDREF的减少特异性抑制G2-M进展。我们已经试图确定hDREF的控制下的基因，发现22 79人RP基因含有类似于人类DREF（DNA复制相关的元素结合因子; hDREF）结合序列（hDRE）在200 bp的区域上游的转录起始位点的序列。电泳凝胶迁移率变动分析和染色质免疫沉淀分析表明，hDREF结合hDRE样序列在RP基因在体外和体内。与hDREF类似，RP基因的表达在G1-S期晚期增加，并且使用shRNA介导的敲减的hDREF消耗降低正常人成纤维细胞中的RP基因表达和细胞增殖。RPS 6基因的敲低也导致细胞增殖受损。这些数据表明hDREF是细胞增殖的重要转录因子，其在许多RP基因的细胞周期依赖性调节中起作用

We have successfully made lentivirus expressing shRNA against hDREF. The cells transduced with virus were directly used for experiments such as FACS analysis, immunofluorescence of cells, and Western blotting using antibodies against a number of proteins expressing in cell cycle specific manner. We have found that decrease of hDREF specifically inhibits G2-M progression. We have tried to identify genes under control of hDREF and found that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel-mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. Like that of hDREF, expression of RP genes is increased during the late G1-S phases and depletion of hDREF using shRNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell-cycle dependent regulation of a number of RP genes