Binding of Upstream Stimulatory Factor and a Cell-specific Activator to the Calcitonin/Calcitonin Gene-related Peptide Enhancer

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 (K D 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.

Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons

“…The ability of either USF1 or USF2 to activate the promoter and the reduction of activity by siRNAs against either USF1 or USF2 suggest that the CGRP promoter is controlled by a heterodimer of USF1 and USF2. A similar conclusion was reached using in vitro DNA binding data (11). This finding is in agreement with other systems, where USF acts predominantly as a heterodimer (19,38,39).…”

“…2A), whereas TK promoter activity was not reduced. As expected, the activity of the 18-bp-TK promoter was much higher than the TK promoter, consistent with previous reports (11,12). The TK promoter activity was sufficiently high (usually about 5000 light units above background) to have been able to detect a decrease in activity.…”

“…The contribution of USF proteins was suggested by previous in vitro DNA binding studies (11). In this report we have used siRNA treatments of a neuronal-like cell line and rat trigeminal ganglia cultures to demonstrate that USF1 and USF2 enhance expression of the endogenous CGRP gene and CGRP promoter activity.…”

“…We have previously reported that a heterodimer of the transcription factor USF1 and USF2 binds to the 18-bp enhancer of the CGRP gene in vitro (11). In addition to the binding data, USF overexpression increased CGRP promoter activity in a lung carcinoma cell line (12).…”

“…In contrast, in another non-neuronal cell line (COS7) and in the neuronal-like CA77 thyroid C cell line, USF overexpression failed to stimulate promoter activity. 3 Furthermore, the 18-bp enhancer is active only in neuroendocrine thyroid C cell lines (11), yet USF is ubiquitous (13). These discrepancies raised the need to demonstrate whether USF is indeed a regulator of the CGRP 18-bp enhancer in neurons.…”