Identification of a cis-acting regulatory element conferring inducibility of the atrial natriuretic factor gene in acute pressure overload.

To identify the cis-acting regulatory element(s) which control the induction of the atrial natriuretic factor (ANF) gene in acute pressure overload, DNA constructs consisting of promoter elements linked to a reporter gene were injected into the myocardium of dogs, which underwent aortic banding or were sham-operated. Expression of a reporter gene construct harboring the ANF promoter (-3400ANF) was induced 6-12-fold after 7 d of pressure overload. An internal deletion of 556 bp (nucleotide sequence -693 to -137) completely abrogated the inducibility of the ANF reporter gene construct. An activator protein-1 (AP1)-like site (-496 to -489) and a cAMP regulatory element (CRE) (-602 to -596) are located within the deleted sequence. Site-directed mutagenesis of the AP1-like site but not the CRE completely prevented the induction of this construct to acute pressure overload. Further, the AP1-like site was able to confer inducibility of a heterologous promoter (beta-myosin heavy chain) to higher values than controls. Gel mobility shift assay (GMSA) supershift analysis was performed using a radiolabeled probe of the ANF promoter (-506/-483) that included the AP1-like site (ATGAATCA) sequence, as well as a probe converted to contain an AP1 consensus sequence (ATGACTCA). GMSA analysis demonstrated that the ANF AP1-like element could bind both a constitutively expressed factor and the AP1 proteins, and conversion to a true AP1 site increased its affinity for AP1. However, 7 d after the onset of pressure overload, the AP1 proteins were present only at low levels, and the major complex formed by the ANF AP1-like probe was not supershifted by a jun antibody. Using a large animal model of pressure overload, we have demonstrated that a unique cis-acting element was primarily responsible for the overload induction of the ANF gene.

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