INVOLVEMENT OF MULTIPLE CIS-ELEMENTS IN BASAL-INDUCIBLE AND ALPHA-ADRENERGIC AGONIST-INDUCIBLE ATRIAL-NATRIURETIC-FACTOR TRANSCRIPTION - ROLES FOR SERUM RESPONSE ELEMENTS AND AN SP-1-LIKE ELEMENT

In the present study, cis elements in the 5'-flanking sequence (FS) of the rat atrial natriuretic factor (ANF) gene involved in regulating b asal and alpha(1)-adrenergic-inducible transcription were investigated . Truncation analyses using ANF-luciferase reporter constructs transfe cted into primary neonatal rat cardiac myocytes showed that an A/T-ric h serum response element (SRE) at -114 bp of the ANF 5'-FS, which boun d serum response factor (SRF), was required for basal and inducible tr anscription. In constructs composed of 134 bp of rat ANF 5'-FS driving luciferase (ANF-134Luc), mutations in the SRE at -114 bp disrupted SR F binding and ANF promoter activity. However, the same mutations in AN F-638Luc had little effect, suggesting a collaborating role for more d istal sequences, such as the other SRE in ANF-638 at -406 bp. In ANF-6 38Luc, mutations in the SRE at -406 bp that disrupted SRF binding to t hat site decreased ANF reporter activity by only 25%: however, mutatin g both of the SREs completely blocked alpha(1)-adrenergic-inducible ac tivity. Mutation analyses showed that an ...(SP-1)-like site at -69 bp , shown previously to confer inducibility in reporters with 134 bp of ANF 5'-FS, was not required in ANF-638Luc. However, double mutants in the SP-l-like region and either SRE completely blocked alpha(1)-adrene rgic-inducible ANF promoter activity. These findings emphasize that no single element is responsible for alpha(1)-adrenergic agonist-regulat ed ANF transcription but that the SREs at -114 and -406 bp and the SP- 1-like sequence at -69 bp mediate the effect in collaboration.