Table 1.
Primers used for bisulfite mapping and ChIP of mir-496.
Figure 1.
Depletion of MBD2 in mammary breast cancer cell lines leads to induction of hsa-mir-496 expression.
(A) qPCR of endogenous MBD2 mRNA in MCF-10A, MCF-7 and MDA-231. (B) A track showing the position of demethylated probes (descending grey bars) in the hsa-mir-496 promoter region as determined by MeDIP enrichment for methylated DNA and hybridization to a genome wide promoter array. (C) qPCR of MBD2 mRNA levels in MBD2 transfected (black) MCF-10A and controls (empty), and siRNA-MBD2 treated MCF-7 and MDA-231 cells (empty boxes) and controls (black boxes). Bottom panel is a Western blot analysis with an anti MBD2 antibody (D) qPCR quantification of hsa-mir-496 in MBD2 transfected (black) MCF-10A and controls (empty), and siRNA-MBD2 treated MCF-7 and MDA-231 cells (empty) and controls (black).
Table 2.
microRNAs that become demethylated upon overexpression of MBD2 in MCF-10A as determined by meDIP array.
Figure 2.
MBD2 overexpression in MCF-10A cells induces hsa-mir-496 expression and demethylation through binding to the TSS.
(A) Physical map of the 5′ region of hsa-mir-496. The balloons represent CG dinucleotide sequences. The transcription start site TSS is indicated. The position of primers used to amplify in qChIP analysis the are indicated. (B) Results of bisulfite mapping of the 5′ hsa-mir-496 in MCF-10A (empty), MCF-7 (grey) and MDA-MB-231 cells (dark) (C) qPCR ChIP of MBD2 in MCF-10A, MCF-7 and MDA-MB-231 with primers as outlined in panel A. MCF-10A [C], MCF-10A expressing ectopic MBD2 [+M], MCF-7 Control [C] and MBD2 depleted MCF-7 cells [-M], Control [C] and MBD2 depleted [-M] MDA-MB-231 cells with primers as outlined in panel A (D) Results of bisulfite mapping of the 5′ hsa-mir-496 in MCF-10A (empty) [C] transfected with MBD2 (black). (E) Results of bisulfite mapping of the 5′ hsa-mir-496 in MCF-7 cells (empty) or MBD2 depleted MCF-7 cells (Dark). (F) Results of bisulfite mapping of the 5′ hsa-mir-496 in MDA-MB-231 cells (empty) or MBD2 depleted MDA-MB-231 cells (Dark).
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.
Figure 4.
Repressed targets of MBD2 in MBD2 overexpressing cells are putative targets of hsa-mir-496.
(A) CTSH expression in MBD2 overexpressing MCF-10A [+M] and siMBD2 depleted MCF-7 [−M ] and MDA-231 cell lines and controls [C]. (B) POU2F3 expression in MBD2 overexpressing MCF-10A [+M] and in response to transient depletion of MBD2 [−M] in MCF-7 and MDA-231 and controls [C]. (C) PTGS1 expression in MBD2 overexpressing MCF-10A [+M] and in response to transient depletion of MBD2 [−M] in MCF-7 and MDA-231 and controls [C]. (D) hsa-mir-496 expression as determined by QPCR analysis in LNA treated MCF-10A, MCF-7 and MDA-MB-231 cells. (E) CTSH expression in response to transient knockdown of hsa-mir-496 in MCF-10A overexpressing MBD2 , MCF-7 and MDA-231 and controls . (F) POU2F3 expression in a transient knockdown of hsa-mir-496 in MCF-10A overexpressing MBD2 , MCF-7 and MDA-231 and controls. (G) PTGS1 expression in a transient knockdown of hsa-mir-496 in MCF-10A overexpressing MBD2 , MCF-7 and MDA-231 and controls.
Figure 5.
Ingenuity pathway analysis of putative targets of the MBD2-hsa-mir-496 pathway in MCF-10A cells overexpressing MBD2.
(A) A list of genes repressed by MBD2 overexpression in MCF-10A cells was compared to a computed list of hsa-mir-496 targets (miRANDA) and subjected to Ingenuity pathway analysis. (B) Associated network functions identified a network with a role in cell migration and haptotaxis. Down regulated mRNA and putative hsa-mir-496 targets are highlighted in bold and light blue outline. Data were analyzed through the use of IPA (Ingenuity® Systems, www.ingenuity.com).
Figure 6.
MBD2 mediated repression through the direct activation of microRNA a model.
MBD2 represses methylated genes in cis by recruiting chromatin repressor complexes. It can also induce gene expression through demethylation or recruitment of chromatin activation complexes. A new pathway for long-range repression mediated through activation and demethylation of microRNA is supported by the data presented in this paper.