Cell Cycle regulation by hDREF

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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