DNase-I-hypersensitive sites located far upstream of the human c-sis/PDGF-B gene comap with transcriptional enhancers and a silencer and are preceded by (part of) a new transcription unit.
The human c-sis gene encodes the B chain of platelet-derived growth factor (PDGF), a potent mitogen for cultured cells of mesenchymal origin. PDGF is stored in the alpha-granules of blood platelets, which are derived from bone marrow megakaryocytes and lack transcriptional machinery. Human myeloid leukemia cell line K562 can be used as a model for megakaryocytes. Phorbol-ester-mediated megakaryocytic differentiation of K562 cells is accompanied by more than 200-fold increase in the c-sis mRNA level. We have now localized transcriptional enhancers at -8.6 kb and -9.9 kb relative to the human c-sis gene transcription start site. The enhancer at -8.6 kb increases activity of the c-sis promoter by 40-60-fold specifically in K562 cells and comaps with a DNase-I-hypersensitivity (DH) site. The enhancer at -9.9 kb increases c-sis promoter activity by 5-10-fold in K562 cells and DH at that site accompanies phorbol-ester-induced megakaryocytic differentiation. In phorbol-ester-treated K562 cells the two enhancers may be negatively influenced by a silencer that comaps with DH at -10.7/-11.0 kb. Reporter gene analysis predicted that combined activity of the upstream enhancers and the c-sis promoter may result in 100-1000-fold higher promoter activity in phorbol-ester-treated K562 cells compared with untreated cells, which can fully explain the more than 200-fold increase in c-sis mRNA level. DH at -8.6 kb and -9.9 kb was also detected in human fibroblasts and in the carcinoma cell lines HeLa and PC3, which express, respectively, undetectable, low and high levels of c-sis mRNA. Although the individual DH sites displayed 4-10-fold enhancer activity in all these cells, they lost most of their biological activity when combined in a larger fragment. In addition we localized (part of) a new transcription unit at approximately 13 kb upstream of the c-sis transcription start site. The corresponding 0.45-kb sis upstream region (sur) transcript is constitutively expressed in all cell lines examined. The expression of the sur transcript is independent of the expression of c-sis mRNA and of the pattern of DH sites far upstream of the c-sis gene. Thus, at present, there is no indication that the upstream DH sites are involved in regulation of expression of the sur gene.
Dirks
R P
RP
Department of Biochemistry, University of Nijmegen, The Netherlands.
Jansen
H J
HJ
Onnekink
C
C
De Jonge
R J
RJ
Bloemers
H P
HP
D002460:Cell Line; D002874:Chromosome Mapping; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005091:Exons; D006801:Humans; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic
D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D015534:Trans-Activators; D011505:Protein-Tyrosine Kinases; D003850:Deoxyribonuclease I; D013755:Tetradecanoylphorbol Acetate