Abstract
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.
Highlights
The calcitonin/calcitonin gene-related peptide (CT/CGRP)1 gene encodes the hormone CT and the neuropeptide CGRP [1]
These results demonstrate that the CT/CGRP HLH-OB2 enhancer, a key regulatory element of the CT/CGRP gene, is bound by the ubiquitously expressed upstream stimulatory factor (USF) HLH proteins and a cell-specific protein
The mutation was tested in the context of two different 59-flanking fragments of the CT/CGRP gene, a 1250-bp fragment that is sufficient to direct expression to thyroid C cells in transgenic mice [11] and a 205-bp fragment that contains other HLH sites implicated in control of the human CT/CGRP gene [13, 14], as well as consensus sites for SP1 and AP-2 factors
Summary
Cell Culture—The CA77 thyroid C cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (low glucose)/Ham’s F12 (1:1), 10% fetal bovine serum (FBS) (Hyclone). The binding reactions contained 0.02 pmol of labeled probe (50,000 cpm), 3 mg, unless otherwise noted, of nuclear extract prepared by a modified Dignam protocol [16], binding buffer (10 mM Tris, pH 7.5, 5% glycerol, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol), poly(dI-dC) (0.1 mg) (Boehringer Mannheim), and 0.1 pmol of an unrelated double-stranded oligonucleotide (59-GATCCACTATGTCTAGAG-39). UV Cross-linking—The UV cross-linking experiments using modified CT/CGRP HLH-OB2 enhancer element as a probe were performed as described for the electrophoretic mobility shift assays with the exception that 9 mg of nuclear extract was used per reaction. The reactions were resolved on a 10% SDS-polyacrylamide gel
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