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
Figure 3
DNA methylation silences hsa-mir-496 and ectopic MBD2 induces methylated hsa-mir-496 by transient transfection luciferase assay.
(A) Physical map of the hsa-mir-496- pCpGl Luciferase reporter. The position of CG dinucleotide sequences are indicated as balloons which are all located in the hsa-mir-496 5′ region. Arrows indicate position of primers used for Q-Chip. The position of primer used for pyrosequencing is indicated by a horizontal arrows under the scheme. (B) Relative luciferase activity in HEK 293 cells transiently transfected with hsa-mir-496 promoter cloned into pCpGl in Sense [S], Antisense [AS] and in vitro methylated sense hsa-mir-496- pCpGl [mS]. (C) Relative luciferase activity in HEK293 cells co-transfected with methylated hsa-mir-496- pCpGl and empty pEF6 vector [Control], MBD2 expression vector [MBD2] or MBD2 mutant without the MBD domain [mtMBD2]. (D) Ectopic MBD2 binding to methylated hsa-mir-496 region in the hsa-mir-496- pCpGl plasmid in transiently transfected HEK 293 cells as determined by QPCR of a ChIP assay with antiMBD2 antibody. The position of primers used for amplification is indicated in (A). (E) Bisulfite pyrosequencing of methylated hsa-mir-496-pCpGl (grey), MBD2 immunoprecipitation and bisulfite sequencing of hsa-mir-496 -pCpGl following transient-co-transfection experiment of methylated hsa-mir-496 with either pEF6 plasmids (empty) or pEF-MBD2 expression vector (black) in HEK-293 cells.