Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements.
To investigate mechanisms of human angiotensin II type 2 receptor (hAT2) gene regulation we functionally characterized the promoter and downstream regions of the gene. 5'-Terminal deletion mutants from -1417/+100 to -46/+100 elicited significant but low functional activity in luciferase reporter gene assays with PC12W cells. Inclusion into the promoter constructs of intron 1 and the transcribed region of the hAT2 gene up to the translation start enhanced luciferase activity 6.7+/-1.6-fold and 11.6+/-1.7-fold (means+/-S.E.M.) respectively, whereas fusion of the promoter to the spliced 5' untranslated region of hAT2 cDNA did not, which indicated an enhancement caused by intronic sequence elements. Reverse transcriptase-mediated PCR confirmed that the chimaeric hAT2-luciferase mRNA was regularly spliced in PC12W cells. A Northern blot analysis of transfected cells showed levels of luciferase mRNA expression consistent with the respective enzyme activities. Mapping of intron 1 revealed that a 12 bp sequence in the centre of the intron was required for the increase in promoter activity, whereas the 5' adjacent intronic region mediated a decrease in luciferase activity. Mutation of the 12 bp region led to altered protein binding and markedly decreased luciferase activity. Cloned into a promoterless luciferase vector, a 123 bp intron 1 fragment was able to direct reporter gene expression to the same activity as occurred in conjunction with the 5' flanking region. These results indicate that sequence elements in intron 1 are necessary for efficient transcription of hAT2. In reporter gene assays, intron 1 might by itself function as a promoter and initiate transcription from an alternative start point.
Warnecke
C
C
Department of Internal Medicine/Cardiology, Charité, Campus Virchow Clinic, Humboldt University Berlin and Deutsches Herzzentrum Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. warnecke@charite.de
Willich
T
T
Holzmeister
J
J
Bottari
S P
SP
Fleck
E
E
Regitz-Zagrosek
V
V
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics*; D016385:TATA Box / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
D020121:5' Untranslated Regions; D018387:Codon, Initiator; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin