Expression of the human PDGF-B gene is regulated by both positively and negatively acting cell type-specific regulatory elements located in the first intron.

Abstract

Potential cis‐acting regulatory elements of the human platelet derived growth factor‐B (PDGF‐B) gene were identified by DNase I hypersensitive site mapping. The transcription unit was examined for the presence of hypersensitive sites in chromatin DNA isolated from human term placental cytotrophoblasts, human placental fibroblasts, the JEG‐3 choriocarcinoma cell line and the U2‐OS osteosarcoma cell line. A number of cell type‐specific hypersensitive sites were identified, all within the 1st intron. Transient transfection of JEG‐3 cells with CAT constructs containing regions of the c‐sis 1st intron linked to the basal c‐sis promoter identified a cell type‐specific positive regulatory activity within the intron, composed of at least two distinct elements. One element appeared to be specific for JEG‐3 cells, while the other was also active in U2‐OS cells. The overall positive regulatory activity of the 1st intron region was specific for JEG‐3 cells, but did not function as a classically defined enhancer, as it was orientation‐dependent (unless stably integrated into chromatin DNA). In addition, the activator appears to require interaction with the c‐sis promoter, as little or no activation was seen when either the SV40 or human beta‐globin promoters were substituted for the c‐sis promoter. The 1st intron also contained a negative regulatory element, which was specific for U2‐OS cells and silenced an abnormally high basal c‐sis promoter activity in these cells. The complexity of the transcriptional control of the PDGF‐B gene is discussed.

Localization and functional analysis of DNase‐I‐hypersensitive sites in the human c‐sis/PDGF‐B gene transcription unit and its flanking regions

“…DH sites at + 1.9 kb and + 4.0/+ 4.4 kb are especially pronounced in the placental cell lines. Recently, Franklin et al (1991) showed that a genomic fragment harbouring DH + 1.9, when cloned in the sense orientation upstream of the c-sis promoter, activates transcription fourfold in JEG-3 cells, while it reduces promoter activity 2-3-fold in U2-0s cells. Moreover, a fragment that contains DH + 4.0 appeared to activate the c-sis promoter 4-8-fold in JEG-3 and U2-0s cells.…”

“…Reporter-gene studies showed that DNA elements within the first 100 bp upstream of the transcription-start site are important for phorbol-ester-mediated inducibility in vitro (Pech et al, 1989 b). Recently, DNase-Ihypersensitivity (DH) sites located in c-sis intron 1 of placental cytotrophoblasts were shown to enhance activity of the c-sis promoter in placental cells (Franklin et al, 1991).…”

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

“…In previous studies transcription regulatory elements have been found in the c-sis promoter, exon 1, intron 1 and downstream of the gene (Pech et al, 1989b;Franklin et al, 1991 ;Dirks et al, 1993). Transcription regulatory elements located upstream of the c-sis promoter have not been found earlier.…”

Hypervariable allelic expression patterns of the imprinted IGF2 gene in tumor cells

“…The JEG-3 choriocarcinoma cell line was maintained as has been described (Franklin et al, 1991). Subclones of the JEG-3 cell line were generated by transfecting a pSV2neo plasmid followed by neomycin selection as has been described (Franklin et al, 1991).…”

“…Subclones of the JEG-3 cell line were generated by transfecting a pSV2neo plasmid followed by neomycin selection as has been described (Franklin et al, 1991). Neomycin-resistant clones were isolated and expanded for 4 ± 5 passages before analysis.…”