Tissue-specific activity of the proximal human calcitonin receptor promoter is mediated by Sp1 and an epigenetic phenomenon.
To identify cis-acting sequences transcriptionally regulating the human calcitonin receptor (hCTR) gene, hCTR promoter/luciferase gene constructs were transiently or stably transfected into hCTR-positive and -negative cell lines. Luciferase assays demonstrated that the proximal hCTR promoter (hCTRP1) was transcriptionally active in all cell lines tested. High-level hCTRP1 activity depended on an 11 bp Sp1/Sp3 binding site. Electrophoretic mobility shift assay showed that this region bound the transcription factors Sp1 and Sp3. We further showed that hCTRP1 was strongly activated by the 11 bp Sp1/Sp3 binding site in hCTRP1/luciferase-, Sp1-transfected Drosophila S2 cells. Bisulphite-mediated sequencing of genomic DNA from hCTR-expressing and -non-expressing cell lines demonstrated that the endogenous hCTRP1 was hypomethylated in all cell lines tested. These results suggest that the hCTRP1 is activated by the tissue-ubiquitous transcription factor Sp1 and that an epigenetic process unrelated to CpG methylation represses its activity in hCTR-negative tissues.
Pondel
M D
MD
Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. mpondel@sghms.ac.uk
Partington
G A
GA
Mould
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004330:Drosophila; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics; D018003:Receptors, Calcitonin / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D018003:Receptors, Calcitonin; D016329:Sp1 Transcription Factor; D008156:Luciferases