title: Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis.
abstract: OBJECTIVE
  The promoter of human interleukin-10 (IL10), a cytokine crucial for suppressing inflammation and regulating immune responses, contains an interspecies-conserved sequence with CpG motifs. The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA).
  
  
  METHODS
  Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human, macaque and mouse IL10 genes. Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected. The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 μmol/L). The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA, respectively. The methylation of CpGs in the IL10 promoter was determined by pyrosequencing. Chromatin immunoprecipitation (ChIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions.
  
  
  RESULTS
  One interspecies-conserved sequence was found within the IL10 promoter. The upstream CpGs at -408, -387, -385, and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls. In contrast, the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016), which was correlated with higher IL10 mRNA and serum levels. In the 5-azacytidine-treated PBMCs, the CpG motifs were demethylated, and the expression levels of IL10 mRNA and protein was significantly increased. CHIP assays revealed increased phospho-CREB binding to the IL10 promoter.
  
  
  CONCLUSIONS
  The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.
authors: Department of Forensic Medicine, Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, Shijiazhuang, China.
authors: Li-hong
authors: LH
authors: Fu
authors: Chun-ling
authors: CL
authors: Ma
authors: Bin
authors: B
authors: Cong
authors: Shu-jin
authors: SJ
authors: Li
authors: Hai-ying
authors: HY
authors: Chen
authors: Jing-ge
authors: JG
authors: Zhang
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000235:genetics*; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics*; D008297:Male; D051379:Mice; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D055815:Young Adult
chemical_list: D016753:Interleukin-10