The 6-kilobase c-erbB2 promoter contains positive and negative regulatory elements functional in human mammary cell lines.
A 6-kilobase fragment extending at the 5'-end of the c-erbB2 protooncogene was isolated from a normal human lymphocyte genomic DNA library. The full-length fragment and five subfragments with identical 3'-ends were obtained by progressive unidirectional deletion from the 5'-end and were cloned in front of the luciferase reporter gene. The hybrid genes were analyzed for transcriptional activity in human mammary cell lines synthesizing low (HBL-100 and T-47D), moderate (MDA-MB-453), or high (BT-474) amounts of the c-erbB2 mRNA and were also analyzed in HeLa cells. Gene-specific expression was observed, indicating the presence of multiple cis-acting sequences in the c-erbB2 promoter. A major negative element is located in the -2- to -4-kilobase region. It is flanked on both sides by positive elements that display enhanced transcriptional activity in the BT-474 tumor cells only. While predominant in the low-expressing cells, the effect of the repressor appears to be overcome by the distal transactivator in the high-expressing BT-474 cells, resulting in a 15 to 50 times increase in luciferase activity relative to the HBL-100 and T-47D cells, respectively. Cell-specific expression relies on the trans-acting factors present in the different cell lines. The formation of cell-specific protein-DNA complexes was demonstrated by gel retardation assay.
Grooteclaes
M
M
Département de Radioimmunologie, Université de Liège, Sart Tilman, Belgium.
Pasleau
F
F
Dijkmans
H
H
Berzi
P
P
Albert
A
A
Winkler-Gol
R
R
D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics* / Q000502:physiology; D005809:Genes, Regulator / Q000235:genetics* / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D011519:Proto-Oncogenes / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D066246:ErbB Receptors; D008156:Luciferases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases