Regulation of the cell-specific calcitonin/calcitonin gene-related peptide enhancer by USF and the Foxa2 forkhead protein.
An 18-bp enhancer controls cell-specific expression of the calcitonin/calcitonin gene-related peptide gene. The enhancer is bound by a heterodimer of the bHLH-Zip protein USF-1 and -2 and a cell-specific factor from thyroid C cell lines. In this report we have identified the cell-specific factor as the forkhead protein Foxa2 (previously HNF-3beta). Binding of Foxa2 to the 18-bp enhancer was demonstrated using electrophoretic mobility shift assays. The cell-specific DNA-protein complex was selectively competed by a series of Foxa2 DNA binding sites, and the addition of Foxa2 antiserum supershifted the complex. Likewise, a complex similar to that seen with extracts from thyroid C cell lines was generated using an extract from heterologous cells expressing recombinant Foxa2. Interestingly, overexpression of Foxa2 activated the 18-bp enhancer in heterologous cells but only in the presence of the adjacent helix-loop-helix motif. Likewise, coexpression of USF proteins with Foxa2 yielded greater activation than by Foxa2 alone. Unexpectedly, Foxa2 overexpression repressed activity in the CA77 thyroid C cell line, suggesting that Foxa2 may interact with additional cofactors. The stimulatory role of Foxa2 at the calcitonin/calcitonin gene-related peptide gene enhancer was confirmed by short interfering RNA-mediated knockdown of Foxa2. As seen with Foxa2 overexpression, the effect of Foxa2 knockdown also required the adjacent helix-loop-helix motif. These results provide the first evidence for combinatorial control of gene expression by bHLH-Zip and forkhead proteins.
Viney
Tim J
TJ
Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA.
Schmidt
Thomas W
TW
Gierasch
William
W
Sattar
A Wahed
AW
Yaggie
Ryan E
RE
Kuburas
Adisa
A
Quinn
John P
JP
Coulson
Judy M
JM
Russo
Andrew F
AF
D000818:Animals; D002116:Calcitonin / Q000235:genetics* / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000502:physiology; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C496504:Foxa2 protein, rat; D009687:Nuclear Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D051542:Hepatocyte Nuclear Factor 3-beta; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide