title: Recruitment of specificity protein 1 by CpG hypomethylation upregulates Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.
abstract: OBJECTIVE
  Promoter hypomethylation leads to upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) in the spontaneously hypertensive rat (SHR). We hypothesized that recruitment of Specificity Protein 1 (Sp1) by CpG hypomethylation would result in upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.
  
  
  METHODS
  Sham-operated Wistar-Kyoto (WKY) rats (sham) and angiotensin II (Ang II)-infused WKY rats, as well as SHRs, were used in this study. We performed real-time PCR and western blot for determination of the expression levels of Nkcc1 mRNA and protein, respectively, and bisulphite sequencing for determination of the methylation status of the proximal promoter; an assay kit was used for assessment of the activity of DNA methyltransferase (DNMT), and the electrophoretic mobility shift assay (EMSA) was used for assessment of binding of Sp1 to cis-element, and promoter function was assessed using the luciferase assay.
  
  
  RESULTS
  Both Ang II-infused WKY rats and SHRs showed higher expression of Nkcc1 mRNA and protein and less DNA methylation, compared with sham. CpG methylation at Sp1 response elements interfered with binding of Sp1, resulting in disabled promoter activity. Both types of hypertensive rats showed hypomethylation of CpG dinucleotides in Sp1 response elements in accordance with the decrease of DNMT activity. DNMT3b and MeCP2 were highly recruited to the more methylated promoter of normotensive rats, whereas the CXXC finger protein 1 (Cfp1), Sp1 and RNA polymerase II were highly recruited to the less methylated promoter of hypertensive rats.
  
  
  CONCLUSIONS
  Our results indicate that recruitment of Sp1 by CpG hypomethylation leads to upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.
authors.affiliation: Department of Pharmacology, Kyungpook National University School of Medicine, Daegu, Republic of Korea.
authors.forename: Hyun-Min
authors.initials: HM
authors.lastname: Cho
authors.forename: Hae-Ahm
authors.initials: HA
authors.lastname: Lee
authors.forename: Hye Young
authors.initials: HY
authors.lastname: Kim
authors.forename: Dong-Youb
authors.initials: DY
authors.lastname: Lee
authors.forename: In Kyeom
authors.initials: IK
authors.lastname: Kim
mesh_terms: D000818:Animals; D001794:Blood Pressure; D018899:CpG Islands; D019175:DNA Methylation; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D060888:Real-Time Polymerase Chain Reaction; D064506:Solute Carrier Family 12, Member 2 / Q000235:genetics / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C578879:Slc12a2 protein, rat; D064506:Solute Carrier Family 12, Member 2; D016329:Sp1 Transcription Factor; D014157:Transcription Factors