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Molecular and Cellular Biology, June 1999, p. 4191-4199, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Two-Hit Mechanism for Vitamin D3-Mediated Transcriptional Repression of the Granulocyte-Macrophage Colony-Stimulating Factor Gene: Vitamin D Receptor Competes for DNA Binding with NFAT1 and Stabilizes c-Jun

Terri L. Towers,1 Teodora P. Staeva,2 and Leonard P. Freedman1,*

Cell Biology Program1 and Immunology Program,2 Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Cornell University Graduate School of Medical Sciences, New York, New York 10021

Received 29 October 1998/Returned for modification 3 March 1999/Accepted 15 March 1999

We previously described a control element in the granulocyte-macrophage colony-stimulating factor (GM-CSF) enhancer that is necessary and sufficient to mediate both transcriptional activation in response to T-cell stimuli and transcriptional repression by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through the vitamin D3 receptor (VDR). This DNA element is a composite site that is recognized by both Fos-Jun and NFAT1; it is directly bound by VDR in the absence of a retinoid X receptor as an apparent monomer, and it is bound in a unique tertiary conformation. We describe here the mechanism by which VDR elicits its transcriptional inhibitory effect. Firstly, VDR outcompetes NFAT1 for binding to the composite site. Overexpression of NFAT1 in vivo by transient transfection is able to relieve the 1,25(OH)2D3-dependent repression. Secondly, VDR stabilizes the binding of a Jun-Fos heterodimer to the adjacent AP-1 portion of the element. This appears to occur through a direct interaction between VDR and c-Jun, as demonstrated in vitro by direct glutathione S-transferase coprecipitation assays. In vivo, overexpression of c-Jun, but not c-Fos, leads to a rescue of the 1,25(OH)2D3-mediated repression. Transfected FLAG-VDR bound to the NFAT1-AP-1 DNA binding element can be selectively precipitated from nuclear extracts that are made from cells treated with activating agents in the presence of 1,25(OH)2D3. VDR is not detected in the complex in the absence of the ligand. Thus, VDR acts selectively on the two components required for activation of this promoter/enhancer: it competes with NFAT1 for binding to the composite site, positioning itself adjacent to Jun-Fos on the DNA. Co-occupancy apparently leads to an inhibitory effect on c-Jun's transactivation function. These two events mediated by VDR effectively block the NFAT1-AP-1 activation complex, resulting in an attenuation of activated GM-CSF transcription.

* Corresponding author. Mailing address: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Cornell University Graduate School of Medical Sciences, 1275 York Ave., New York, NY 10021. Phone: (212) 639-2976. Fax: (212) 717-3298. E-mail: l-freedman{at}ski.mskcc.org.

Molecular and Cellular Biology, June 1999, p. 4191-4199, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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