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J. Biol. Chem., Vol. 278, Issue 31, 29192-29200, August 1, 2003

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Adjacent Sequence Controls the Response Polarity of Nitric Oxide-sensitive Sp Factor Binding Sites^*

Jianhua Zhang, Shuibang Wang, Robert A. Wesley and Robert L. Danner 

From the Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892

Nitric oxide (NO^•) and cAMP-dependent protein kinase (PKA) inhibitors up-regulate tumor necrosis factor (TNF) by decreasing Sp1 binding to a proximal GC box element. Here, elements flanking GC boxes were tested for their role in determining whether Sp sites act as activators or repressors. Promoter studies in receptive human cell lines demonstrated that NO^• down-regulated endothelial NO^• synthase (eNOS) but up-regulated TNF. Like TNF, Sp1 binding to the eNOS promoter was decreased by NO^• and a PKA inhibitor, H89, and increased by a PKA activator, dibutyryl cAMP (Bt₂cAMP). For either promoter, mutation of Sp sites abolished NO^• responses. In contrast, mutation of an upstream AP1 site in the TNF promoter (not present in eNOS) maintained NO^• responsiveness, but reversed the direction of NO^• and cAMP effects. Using artificial constructs, NO^• increased transcription when Sp and AP1 sites were both present (TNF-like response), but decreased it when the adjacent AP1 site was disrupted (eNOS-like response). NO^•, H89, and Bt₂cAMP were found to produce reciprocal protein binding changes at contiguous AP1 and Sp sites (p < 0.0001 for an interaction). Chromatin immunoprecipitation assays demonstrated that Sp1 and to a lesser extent Sp3 bound to the GC box regions of eNOS and TNF in intact cells. Thus, this NO^•- and cAMP-responsive regulatory module has a Sp site sensor variably coupled to an adjacent element that determines response polarity. These results define a composite element that can utilize secondary inputs to convert off signals to on, thereby conferring complex functionalities to the same DNA binding motif.

Received for publication, December 20, 2002 , and in revised form, May 6, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Critical Care Medicine Dept., Bldg. 10, Rm. 7D43, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, Tel.: 301-496-9320; Fax: 301-402-1213; E-mail: rdanner{at}nih.gov.

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