title: Adjacent sequence controls the response polarity of nitric oxide-sensitive Sp factor binding sites.
abstract: Nitric oxide (NO*) and cAMP-dependent protein kinase (PKA) inhibitors up-regulate tumor necrosis factor alpha (TNFalpha) 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 TNFalpha. Like TNFalpha, 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 TNFalpha 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 (TNFalpha-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 TNFalpha 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.
authors.affiliation: Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.forename: Shuibang
authors.initials: S
authors.lastname: Wang
authors.forename: Robert A
authors.initials: RA
authors.lastname: Wesley
authors.forename: Robert L
authors.initials: RL
authors.lastname: Danner
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D013449:Sulfonamides; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007546:Isoquinolines; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D013449:Sulfonamides; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051705:Sp3 Transcription Factor; D009569:Nitric Oxide; D003994:Bucladesine; D004247:DNA; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide