Search

The rat βB2-crystallin gene is active only during a specific stage of the differentiation of rat lens fibre cells directed by basic fibroblast growth factor. The regulatory elements that determine the transient activity of this gene are located in the -750/-123 region and in the first intron. Singly, these elements act as silencers, together they constitute an enhancer that is active only during the specific differentiation stage. An additional silencer is found between -123 and -77. The proximal promoter region contains a Pax-6 binding site at -65/-51 . In vitro, binding to this site could be detected but, according to in vivo footprinting experiments, this site is not occupied in the endogenous gene. Furthermore, co-expression of Pax-6 did not enhance promoter activity. Finally, mutation or deletion of this site did not affect promoter activity; the region -37/+10 sufficed for basal promoter activity. The cooperation between the -750/-123 region and the first intron of the βB2-crystanin gene not only determines the differentiation stage-specific activity of the gene, but also contributes to the highly increased expression in lens cells compared with non-lens cells. [ABSTRACT FROM AUTHOR]

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 α-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-estermediated megakaryocytic ddifferentiation 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 c0is 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. [ABSTRACT FROM AUTHOR]