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CpG site methylation in CRYAA promoter affect transcription factor Sp1 binding in human lens epithelial cells.

ABSTRACT: Age-related cataract (ARC) is the leading cause of visual impairment worldwide, and ?-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.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 ?M, 20 ?M, 50 ?M, 100 ?M and 200 ?M, 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.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.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.

SUBMITTER: Liu X

PROVIDER: S-EPMC4979130 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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CpG site methylation in CRYAA promoter affect transcription factor Sp1 binding in human lens epithelial cells.

Liu Xin X Zhou Peng P Fan Fan F Li Dan D Wu Jihong J Lu Yi Y Luo Yi Y

BMC ophthalmology 20160809

<h4>Background</h4>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.<h4>Methods</h4>The transcription factor binding sites of the CpG island of CRYAA promoter were pr ...[more]

PMID: 27507241

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Project description:BackgroundPodocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene.ResultsThe promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines.ConclusionOur results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.

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CpG site methylation in CRYAA promoter affect transcription factor Sp1 binding in human lens epithelial cells.

Publications

CpG site methylation in CRYAA promoter affect transcription factor Sp1 binding in human lens epithelial cells.

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