Binding of upstream stimulatory factor and a cell-specific activator to the calcitonin/calcitonin gene-related peptide enhancer.
The calcitonin/calcitonin gene-related peptide (CT/CGRP) gene is selectively transcribed in thyroid C cells and neurons. We have previously shown that the rat CT/CGRP cell-specific enhancer is synergistically regulated by a helix-loop-helix (HLH) protein and the OB2 octamer-binding protein. In this report, we show that the HLH-OB2 enhancer is required for full promoter activity, even in the context of other HLH elements. Since this enhancer appears to be a major controlling element, we have characterized the HLH and OB2 DNA binding proteins. We have identified the major HLH complex as a heterodimer of the ubiquitous upstream stimulatory factor (USF)-1 and USF-2 proteins. USF bound the enhancer with a reasonably high affinity (KD 1.6 nM), comparable to other genes. Characterization of a series of mutations revealed that a portion of the HLH motif is also recognized by OB2 and confirmed that HLH activity requires OB2. We have shown that OB2 is a single DNA binding protein based on UV cross-linking studies. The 68-kDa protein-DNA complex was detected only in C cell lines, including a human C cell line that has robust HLH-OB2 enhancer activity. These results suggest that the calcitonin/CGRP gene is controlled by the combinatorial activity of a ubiquitous USF HLH heterodimer and an associated cell-specific activator.
Lanigan
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Molecular Biology Program, University of Iowa, Iowa City, Iowa 52242, USA.
Russo
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AF
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002116:Calcitonin / Q000096:biosynthesis* / Q000235:genetics*; D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D016254:Mutagenesis, Insertional; D009474:Neurons / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D013961:Thyroid Gland / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D002116:Calcitonin; D004247:DNA; D008156:Luciferases; D015740:Calcitonin Gene-Related Peptide