Negative regulation of cyclin D3 expression by trans-cription factor c-Ets1 in umbilical cord hematopoietic cells
Abstract
Aim: To investigate the role of transcription factor c-Ets1 in cyclin D3 expression and its effects on the proliferation of umbilical cord hematopoietic cells.
Methods: Cyclin D3 promoter deletion constructs were generated and transfected into CD34+ cells. Dual luciferase reporter assays and TFSEARCH software were used to identify negative regulatory domains and to predict putative transcription factors involved in cyclin D3 downregulation. Expression of c-Ets1 in CD34+ cells was detected using electrophoretic mobility shift and super shift assays. Point mutants of c-Ets1 binding sites were constructed. The wild-type c-Ets1 and the mutant promoter constructs were co-transfected into CD34+ cells to determine the promoter activity. The impact of c-Ets1 expression on the proliferation of CD34+ cells was assessed using MTT assay.
Results: Nine cyclin D3 promoter deletion constructs were generated. A negative regulatory domain containing c-Ets1 binding sites was identified between −439 bp and −362 bp. Transfection of the promoter deletion constructs containing mutant c-Ets1 binding sites enhanced cyclin D3 promoter activity. However, the opposite results were observed when CD34+ cells were co-transfected with wildtype c-Ets1 and its promoter deletion constructs. The overexpression of c-Ets1 could suppress cyclin D3 mRNA and protein levels. In addition, it inhibits the proliferation of CD34+ cells.
Conclusion: c-Ets1 functions as a negative transcription factor, down-regulating the expression of cyclin D3, which leads to inhibition of CD34+ cell proliferation.
Keywords:
Methods: Cyclin D3 promoter deletion constructs were generated and transfected into CD34+ cells. Dual luciferase reporter assays and TFSEARCH software were used to identify negative regulatory domains and to predict putative transcription factors involved in cyclin D3 downregulation. Expression of c-Ets1 in CD34+ cells was detected using electrophoretic mobility shift and super shift assays. Point mutants of c-Ets1 binding sites were constructed. The wild-type c-Ets1 and the mutant promoter constructs were co-transfected into CD34+ cells to determine the promoter activity. The impact of c-Ets1 expression on the proliferation of CD34+ cells was assessed using MTT assay.
Results: Nine cyclin D3 promoter deletion constructs were generated. A negative regulatory domain containing c-Ets1 binding sites was identified between −439 bp and −362 bp. Transfection of the promoter deletion constructs containing mutant c-Ets1 binding sites enhanced cyclin D3 promoter activity. However, the opposite results were observed when CD34+ cells were co-transfected with wildtype c-Ets1 and its promoter deletion constructs. The overexpression of c-Ets1 could suppress cyclin D3 mRNA and protein levels. In addition, it inhibits the proliferation of CD34+ cells.
Conclusion: c-Ets1 functions as a negative transcription factor, down-regulating the expression of cyclin D3, which leads to inhibition of CD34+ cell proliferation.