Involvement of multiple cis elements in basal- 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 basal and alpha 1-adrenergic-inducible transcription were investigated. Truncation analyses using ANF-luciferase reporter constructs transfected into primary neonatal rat cardiac myocytes showed that an A/T-rich serum response element (SRE) at -114 bp of the ANF 5'-FS, which bound serum response factor (SRF), was required for basal and inducible transcription. In constructs composed of 134 bp of rat ANF 5'-FS driving luciferase (ANF-134Luc), mutations in the SRE at -114 bp disrupted SRF binding and ANF promoter activity. However, the same mutations in ANF-638Luc had little effect, suggesting a collaborating role for more distal sequences, such as the other SRE in ANF-638 at -406 bp. In ANF-638Luc, mutations in the SRE at -406 bp that disrupted SRF binding to that site decreased ANF reporter activity by only 25%; however, mutating both of the SREs completely blocked alpha 1-adrenergic-inducible activity. 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-1-like region and either SRE completely blocked alpha 1-adrenergic-inducible ANF promoter activity. These findings emphasize that no single element is responsible for alpha 1-adrenergic agonist-regulated 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.
Sprenkle
A B
AB
Department of Biology, San Diego State University, CA 92182, USA.
Murray
S F
SF
Glembotski
C C
CC
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000032:analysis; D004586:Electrophoresis; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D015345:Oligonucleotide Probes; D011485:Protein Binding; D051381:Rats; D026362:Serum Response Factor; D014157:Transcription Factors / Q000032:analysis; D014158:Transcription, Genetic
D000316:Adrenergic alpha-Agonists; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D026362:Serum Response Factor; D014157:Transcription Factors; D009320:Atrial Natriuretic Factor