title: CpG site methylation in CRYAA promoter affect transcription factor Sp1 binding in human lens epithelial cells.
abstract: BACKGROUND
  Age-related cataract (ARC) is the leading cause of visual impairment worldwide, and α-crystallin (CRYAA) is the predominant structural protein involved in the maintenance of lens clarity and refractive properties. We previously demonstrated that CRYAA genes undergo epigenetic repression in the lens epithelia in ARC. We further analyze the underlying mechanism in the current study.
  
  
  METHODS
  The transcription factor binding sites of the CpG island of CRYAA promoter were predicted by TESS website. An electrophoretic mobility shift assay (EMSA) was used to analyze the impact of the methylation of CpG sites on transcription factors. Human lens epithelial B-3 (HLE B-3) Cells were treated with demethylation agent zebularine in the concentrations of 0 (PBS as control), 10 μM, 20 μM, 50 μM, 100 μM and 200 μM, respectively. After treatment in the above concentrations for 24 h, 48 h and 72 h, respectively, CRYAA mRNA expression levels were detected by Quantitative Real-Time RT-PCR.
  
  
  RESULTS
  The methylation of the CpG site of the CRYAA promoter decreased the DNA-binding capacity of transcription factor Sp1. Zebularine increased CRYAA expression in HLE B-3 Cells in a dose- dependent and time- dependent pattern.
  
  
  CONCLUSIONS
  The evidence presented suggests that the methylation of the CpG sites of the CRYAA promotor directly affect Sp1 binding, leading to down expression of CRYAA in human lens epithelial cells. Zebularine treatment could restore CRYAA expression in a dose- dependent and time- dependent pattern.
authors: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.|Key Laboratory of Myopia, Ministry of Health, 83 FenYang Road, Shanghai, 200031, People's Republic of China.|Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.
authors: Xin
authors: X
authors: Liu
authors: Department of Ophthalmology, Parkway Health, Specialty and Inpatient Center (Luwan), 170 DanShui Road, Floor 3, Shanghai, 200020, People's Republic of China.|Hong Qiao Medical Center, 2258 HongQiao Road, Shanghai, 200033, People's Republic of China.
authors: Peng
authors: P
authors: Zhou
authors: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.|Key Laboratory of Myopia, Ministry of Health, 83 FenYang Road, Shanghai, 200031, People's Republic of China.|Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.
authors: Fan
authors: F
authors: Fan
authors: Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.
authors: Dan
authors: D
authors: Li
authors: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.|Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.
authors: Jihong
authors: J
authors: Wu
authors: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.|Key Laboratory of Myopia, Ministry of Health, 83 FenYang Road, Shanghai, 200031, People's Republic of China.|Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China.
authors: Yi
authors: Y
authors: Lu
authors: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China. yeeluo1106@163.com.|Key Laboratory of Myopia, Ministry of Health, 83 FenYang Road, Shanghai, 200031, People's Republic of China. yeeluo1106@163.com.|Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, 83 FenYang Road, Shanghai, 200031, People's Republic of China. yeeluo1106@163.com.
authors: Yi
authors: Y
authors: Luo
mesh_terms: D002478:Cells, Cultured; D018899:CpG Islands / Q000235:genetics*; D003459:Crystallins / Q000235:genetics* / Q000378:metabolism; D003562:Cytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D019175:DNA Methylation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*
chemical_list: C511246:CRYAA protein, human; D003459:Crystallins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside
keywords: CRYAA; DNA methylation; Human lens epithelial cells; Transcription factor Sp1; Zebularine