Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

BMC Cancer

Research Article

Mayra Furlan-Magaril¹  Edgar González-Buendía¹  Mercedes Dávalos-Salas¹  Félix Recillas-Targa¹  Christian Valdes-Quezada¹  Erandi Ayala-Ortega¹ 

[1] Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, México, D.F., Mexico;

关键词: U87MG Cell;    Stable Cell Line;    Epigenetic Silence;    CTCF Binding;    Repressive Chromatin;   

DOI  :  10.1186/1471-2407-11-232

 received in 2010-10-25, accepted in 2011-06-10,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundLong-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines.MethodsTo assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays.ResultsWe found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation.ConclusionsThis study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.

【 授权许可】

Unknown   
© Dávalos-Salas et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.