Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements withi...Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity ofNanog gene. In this report, we identified the role of two putative Spl binding sites located in the Nanog gene 5＇-flanking region in regulation ofmurine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Spl and Sp3. Furthermore, overexpression of dominant-negative Spl or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Spl and Sp3 are important for Murine Nanog gene expression.展开更多
GATA-1 is a hematopoietic transcription factor that is essential for the terminal maturation of proerythroblasts, megakaryocytic cells and mast cells. The erythroid-specific promoter of the human GATA-1 gene directs t...GATA-1 is a hematopoietic transcription factor that is essential for the terminal maturation of proerythroblasts, megakaryocytic cells and mast cells. The erythroid-specific promoter of the human GATA-1 gene directs the high expression of a reporter gene in K562 cells. Multiple putative transcription factor binding sites were identified in the promoter from the -860 to the -1 base pair （bp）. For a better understanding of the transcriptional control of human GATA-1 gene expression, we tested the transcriptional activity of a series of deletions from the 5′ end of the 860-bp promoter. A region between -221 and -128 bp retains most of the transcriptional activity of the full-length promoter. Deletion of the CGCCC box at-195 bp reduced reporter gene activity to 60.4%. Further deletion of the CACCC box at -173 bp nearly abolished reporter gene expression, indicating that the CACCC box is more critical. In vitro experiments of electrophoretic mobility shifts and in vivo studies using chromatin immuno-precipitation （CHIP） assays show that the Sp1/Sp3 proteins bind the CACCC site in the nuclei of K562 cells. Coincidently, hyperacetylation of histones in the GATA-1 erythroid promoter was also shown by ChIP assay. Co-transfection of Spl expression plasmids and plasmids with a wild-type promoter showed enhanced reporter gene activity in a dose-dependent manner. The combined data demonstrate that Sp1/Sp3, but not EKLF, is involved in the activation of the GATA-1 erythroid promoter, and that histones H3 and H4 are highly acetylated in this promoter region for an actively transcribed GATA-1 gene in K562 cells in which EKLF is barely detectable.展开更多
文摘Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity ofNanog gene. In this report, we identified the role of two putative Spl binding sites located in the Nanog gene 5＇-flanking region in regulation ofmurine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Spl and Sp3. Furthermore, overexpression of dominant-negative Spl or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Spl and Sp3 are important for Murine Nanog gene expression.
文摘GATA-1 is a hematopoietic transcription factor that is essential for the terminal maturation of proerythroblasts, megakaryocytic cells and mast cells. The erythroid-specific promoter of the human GATA-1 gene directs the high expression of a reporter gene in K562 cells. Multiple putative transcription factor binding sites were identified in the promoter from the -860 to the -1 base pair （bp）. For a better understanding of the transcriptional control of human GATA-1 gene expression, we tested the transcriptional activity of a series of deletions from the 5′ end of the 860-bp promoter. A region between -221 and -128 bp retains most of the transcriptional activity of the full-length promoter. Deletion of the CGCCC box at-195 bp reduced reporter gene activity to 60.4%. Further deletion of the CACCC box at -173 bp nearly abolished reporter gene expression, indicating that the CACCC box is more critical. In vitro experiments of electrophoretic mobility shifts and in vivo studies using chromatin immuno-precipitation （CHIP） assays show that the Sp1/Sp3 proteins bind the CACCC site in the nuclei of K562 cells. Coincidently, hyperacetylation of histones in the GATA-1 erythroid promoter was also shown by ChIP assay. Co-transfection of Spl expression plasmids and plasmids with a wild-type promoter showed enhanced reporter gene activity in a dose-dependent manner. The combined data demonstrate that Sp1/Sp3, but not EKLF, is involved in the activation of the GATA-1 erythroid promoter, and that histones H3 and H4 are highly acetylated in this promoter region for an actively transcribed GATA-1 gene in K562 cells in which EKLF is barely detectable.
