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 fa ctor (PDGF), a potent mitogen for cultured cells of mesenchymal origin . PDGF is stored in the alpha-granules of blood platelets, which are d erived from bone marrow megakaryocytes and lack transcriptional machin ery. 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-s is 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 s ite. 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-hyp ersensitivity (DH) site. The enhancer at -9.9 kb increases c-sis promo ter activity by 5-10-fold in K562 cells and DH at that site accompanie s phorbol-ester-induced megakaryocytic differentiation. In phorbol-est er-treated K562 cells the two enhancers may be negatively influenced b y a silencer that comaps with DH at -10.7/-11.0 kb. Reporter gene anal ysis predicted that combined activity of the upstream enhancers and th e c-sis promoter may result in 100 - 1000-fold higher promoter activit y in phorbol-ester-treated K562 cells compared with untreated cells, w hich can fully explain the more than 200-fold increase in c-sis mRNA l evel. 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, respecti vely, undetectable, low and high levels of c-sis mRNA. Although the in dividual DH sites displayed 4-10-fold enhancer activity in all these c ells, they lost most of their biological activity when combined in a l arger 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, a t present, there is no indication that the upstream DH sites are invol ved in regulation of expression of the sur gene.