Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements

Abstract

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.

Poly(ADP-ribose) polymerase-1 (PARP-1) transcriptionally regulates angiotensin AT2 receptor (AT2R) and AT2R binding protein (ATBP) genes

“…Our experiments, consistent with a previous publication [33], also demonstrated the important regulatory function of the exon 1-intron 1 region of the AT2R gene is necessary and sufficient for AT2R promoter activity, whereas upstream promoter elements (> 316 bp relative to exon 2) do not contribute to promoter activity to a major degree (Fig. 2&3).…”

“…In this context, our result of a single transcriptional start site within the TATA box positive AT2R promoter is consistent with the promoter architecture of a tissue-specific gene [36] in contrast to strong housekeeping genes associated with multiple start sites and the absence of a TATA motif [29,35]. It is noteworthy that two transcriptional start sites upstream the TATA box were identified in human uterus RNA by primer extension analysis [33] (Fig. 1A) which might reflect tissue specific differences and could affect mRNA stability.…”

Association of Angiotensin II Type 2 Receptor Gene Variant with Hypertension

“…The AT2-R gene has three exons, with the entire coding region on the third exon (13). Regulatory elements are located in the first intron in addition to the promoter region (14). The complete nucleotide sequence of the human AT2-R gene including the promoter region has been elucidated (13) and is publicly available via the World Wide Web in the NCBI sequence database (http://www.ncbi.nlm.nih.gov/) (12).…”

Homozygous intronic mutation leading to inefficient transcription combined with a novel frameshift mutation in F13A1 gene causes FXIII deficiency

“…[12][13][14][15][16] In this study, reduced levels of FXIIIA antigen and mRNA were observed in the patients (Supplementary Table 3 and Supplementary Figure 5), suggesting that the Int1(+12)C4A mutation affects the expression of the F13A1 gene. To determine the functional effects of the Int1(+12)C4A mutation in the F13A1 gene, luciferase reporters were transiently expressed in U937 cells.…”