Characterization of CTCF protein around the ‐224 A/G single nucleotide polymorphism in the neuropeptide receptor Y2 (NPY2R) gene in predisposition to hypertension (777.4)

Abstract

Hypertension (HTN) is a complex trait resulting from the interactions of multiple genes and environmental factors. Neuropeptide Y receptor 2 (NPY2R) was identified as a candidate gene for HTN in animal models and human populations. Previous work showed that the luciferase activity induced by the NPY2R promoter with the G allele in position ‐224 is reduced by 42% compared to the promoter containing an A in that position. Furthermore, an electrophoretic shift assay (EMSA) showed an allele‐specific binding with the oligonucleotide containing an A nucleotide in ‐224 position. We were also able to detect a super shift using the CTCF antibody. The aim of this study was to characterize the CTCF protein in HEK 293 cells transfected with NPY2R ‐224 A/G variations. Changes in CTCF expression and distribution were analyzed using western blot and immunofluorescence (IF) experiments. Interestingly, the western blot showed higher quantification of CTCF in HEK 293 cells transfected with NPY2R ‐224 A compared to NPY2R ‐224 G (p < 0.001). Analysis of nuclear localization of CTCF by IF revealed that it is highly distributed in HEK 293 cells transfected with NPY2R ‐224 A compare to a lower distribution in NPY2R ‐224 G. CTCF is the only protein identified so far that mediates enhancer‐blocking activity of insulators. These data provides strong evidence of the CTCF role in the transcriptional regulation. Grant Funding Source: Supported by Grant RR003050/MD007579