Functional relevance of promoter CpG island of human Angiotensin II type 1 receptor (AT1R) gene

Abstract

Angiotensin II type 1 receptor can activate number of signalling pathways upon stimulation and consequently its involvement in cancer progression have also been revealed. But which epigenetic mechanisms are involved in its regulation, need to be further explored. In-silico analysis revealed a promoter CpG island (CGI) which was cloned and assayed for functional activity using reporter gene system. The effect of methylation on this CGI was analysed through varying degree of methyltransferase treatment of cloned fragment. Results thus obtained were validated by direct sequencing. To further establish the status of this effect, in-vivo analysis was done through screening of methylation pattern in the targeted region among hypertensives (HTN) and normotensives\xa0(NTN) using PCR-Bisulphite sequencing. Additionally, clinical details of all participants, biochemical parameters and lifestyle related information was also collected and statistically evaluated. Reporter gene assay assigned functional activity to the cloned promoter CpG island. Increased dose and durations of methyltransferase treatment decreased the expression of reporter gene indicating the role of promoter DNA methylation. Among all the human samples screened, only one of the HTN individual was found to have single hemi-methylated CpG site at a position which happens to be a part of Sp1 transcription factor binding site. To conclude, CpG island in the promoter region of AT1R (CpG.P.AT1R) gets regulated through epigenetic mechanism of DNA methylation.