title: Methylated DNA binding domain protein 2 (MBD2) coordinately silences gene expression through activation of the microRNA hsa-mir-496 promoter in breast cancer cell line.
abstract: Methylated DNA binding protein 2 (MBD2) binds methylated promoters and suppresses transcription in cis through recruitment of a chromatin modification repressor complex. We show here a new mechanism of action for MBD2: suppression of gene expression indirectly through activation of microRNA hsa-mir-496. Overexpression of MBD2 in breast epithelial cell line MCF-10A results in induced expression and demethylation of hsa-mir-496 while depletion of MBD2 in a human breast cancer cell lines MCF-7 and MDA-MB231 results in suppression of hsa-mir-496. Activation of hsa-mir-496 by MBD2 is associated with silencing of several of its target genes while depletion of MBD2 leads to induction of hsa-mir-496 target genes. Depletion of hsa-mir-496 by locked nucleic acid (LNA) antisense oligonucleotide leads to activation of these target genes in MBD2 overexpressing cells supporting that hsa-mir-496 is mediating in part the effects of MBD2 on gene expression. We demonstrate that MBD2 binds the promoter of hsa-mir-496 in MCF-10A, MCF-7 and MDA-MB-231 cells and that it activates an in vitro methylated hsa-mir-496 promoter driving a CG-less luciferase reporter in a transient transfection assay. The activation of hsa-mir-496 is associated with reduced methylation of the promoter. Taken together these results describe a novel cascade for gene regulation by DNA methylation whereby activation of a methylated microRNA by MBD2 that is associated with loss of methylation triggers repression of downstream targets.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Alvarado
authors.forename: Joanne
authors.initials: J
authors.lastname: Wyglinski
authors.forename: Matthew
authors.initials: M
authors.lastname: Suderman
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Andrews
authors.forename: Moshe
authors.initials: M
authors.lastname: Szyf
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; C115368:MBD2 protein; C000590213:MIRN496 microRNA, human; D035683:MicroRNAs