Identification of a Cell Type-specific Enhancer in the Distal 5′-Region of the Platelet-derived Growth Factor A-chain Gene

Abstract

Transient transfection analysis of DNA subfragments from the distal 5-flanking region of the human plateletderived growth factor A-chain gene (؊18.3 to ؊1.8 kilobase pairs (kb)) revealed enhancer and silencer elements that contribute significantly to transcriptional regulation. Two adjacent regions (؊8.2 to ؊7.5 kb and ؊7.5 to ؊7.0 kb) enhanced transcription of both A-chain and heterologous thymidine kinase promoters, whereas repression was observed in two other nearby regions (؊9.9 to ؊8.2 kb and ؊7.0 to ؊5.9 kb). The ؊7.5 to ؊7.0-kb fragment, or J, was the strongest enhancer, and its activity was localized to a 66-base pair element (A-chain cell type-specific enhancer (ACE 66)). ACE66 activity was highly cell type-specific, with greatest activity seen in choriocarcinoma cell lines (4 -10-fold enhancement). Progressive 5-and 3-deletions of the ACE66 revealed distribution of activity across the element, with nucleotides 1-33 being critical for function. Electrophoretic mobility shift assays revealed cell type-specific patterns of high affinity protein binding to the element. Ethylation interference footprinting of JEG-3 extract localized guanine contacts on nucleotides 1-18 of both strands of the ACE element, whereas more extensive contacts were made with the phosphate backbone (nucleotides 1-32). The ACE66 element is a potent transcriptional regulator in placental cells and represents a valuable model of long distance regulation in a growth factor gene. Platelet-derived growth factor (PDGF)1 is a potent mitogen and chemoattractant for mesenchymal cells that was first purified from human platelet extracts but later shown to be expressed in a variety of normal and transformed cell types (1, 2). PDGF is a family of dimeric glycoproteins that arise from different combinations of two polypeptide chains, termed A and B, that are encoded by separate genes located on different chromosomes. The human A-chain gene contains 7 exons and is located on chromosome 7 (7pter-7q22), spanning approximately 24 kb (3, 4). Transcription of the A-chain gene is activated rapidly by a wide range of growth factors, cytokines and other mitogens (5-7), whereas complex cell type-specific patterns of A-chain transcription appear to underlie the regulation of cell proliferation during embryogenesis (8) and cellular differentiation (9). A-chain null mice exhibit abnormalities in early embryonic development and formation of lung alveolar myofibroblasts (10, 11), whereas expression of the A-chain and the PDGF ␣-receptor also appear to critical for development of the cardiovascular and nervous systems in mice (12)(13)(14). PDGF also appears to be critical for placental development, with high expression of A-chain observed in multiple cell types of the placenta including trophoblasts (2, 15), as well as extraembryonic membranes and uterine smooth muscle cells during pregnancy (15, 16). Inappropriate expression of the PDGF genes has also been implicated in many disease states involving abnormal cell proliferation, including atherosclerosis, pulmon...

A 5′-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling

“…The limits of the VDR/RXRa-induced footprints were in close agreement with those obtained by DNase I footprinting (Figure 3a). Moreover, JEG-3 nuclear extract elicited strong footprinting patterns over guanine (compare lanes 5 and 6, lanes 13 and 14) and phosphate (compare lanes 7 and 8, lanes 15 and 16) residues in the dr-A and dr-B motifs, consistent with previous results (Maul et al, 1998). Footprinting was observed over essentially all phosphate residues of the DR3 site on the coding strand, including both the half-sites and the 3 bp spacer sequence, while interference on the noncoding strand was seen at the dr-A and dr-B motifs but not within the spacer nucleotides.…”

“…The dominant-negative c-FOS protein, A-FOS, contains a substitution of the basic DNAbinding domain with an acidic amphipathic peptide (Olive et al, 1997), thereby interfering with normal binding of AP1 complexes to DNA. The three tandem copies of the ACE66 element resulted in a greater than 200-fold enhancement (Figure 2b), a transcriptional synergism described previously (Maul et al, 1998). As expected, DMEKK1 and c-JUN superinduced the 3X ACE construct by approximately twofold.…”

“…The ACE66 variants were cloned upstream of a minimal PDGF-A promoter fragment (À261 to þ 8) and their enhancer activities analysed by transient transfection in JEG-3 choriocarcinoma cells, a cell line in which the ACE66 element is especially active (B10-fold enhancement; Figure 1a). While the ACE66 element exhibits basal and vitamin D-inducible enhancer activities from its far upstream location (Maul et al, 1998;Pedigo et al, 2003), relocation of the element to a position much closer to the promoter amplifies that activity. This enables the detection of subtle mutational effects difficult to observe in the context of the fulllength promoter (À7400 to þ 8), which exhibits lower enhancer activity (threefold) and is a poor transfection substrate due to its substantial length.…”

“…On the noncoding strand, proteinnucleotide contacts were apparent not only over the DR3 motif but also extended to the AP1 motif and beyond (lanes 17-20). DNase I footprinting was also conducted with nuclear extract from Saos-2 cells, which exhibit no PDGF-A gene transcription nor ACE66 activity (Maul et al, 1998), but do express significant quantities of VDR (Mulkins et al, 1983). Interestingly, the dimensions and intensity of footprints obtained with the Saos-2 nuclear extract were similar on both strands of the ACE66 element to those obtained with JEG-3 extract (Figure 3a and b, lanes 9-12 and 21-24).…”

“…DNA elements in the far upstream region of the PDGF-A gene also contribute to transcriptional control (Maul et al, 1998). Of particular interest is a 66 bp enhancer element (ACE66) found approximately 7 kb upstream of the transcription start site (À6852 to À6787) that has been shown to be highly activated in many transformed cell lines, including those of choriocarcinoma, hepatoma, osteosarcoma and lung carcinoma origins.…”