Journal: Nucleic Acids Research

Article Title: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

doi: 10.1093/nar/gkv508

Figure Lengend Snippet: Endogenous MBD2 proteins bind methylated DNA regions. ( A ) Experimental scheme; the transformed mesenchymal HMLER cell line is derived from the immortalized epithelial HMEC-hTERT cells by oncogenic transformation using H-RAS V12 and SV40 T/t oncogenes. Sequencings were performed on a pool of five independent experiments. ( B ) Reads visualization at a MBD2 peak present in each cell line; MBD2 reads in black, input reads in gray, MBD2 peaks in black bars. Reads matching the + and − strands are on top and bottom, respectively. ( C and F ) Read density of MeDPseq, MBD2 ChIPseq and Input at each MBD2 peak. ( D and G ) Read density of MeDPseq, MBD2 ChIPseq and input at each MeDP peak. ( E and H ) Read density of MeDPseq, MBD2 ChIPseq and input at each CpG island (CGI), sorted by their size. (C to E) HMLER cell line. (F to H) HMEC-hTERT cell line.

Article Snippet: ChIP were then performed with a custom-made rabbit polyclonal antiserum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) ( ) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer's instruction.

Techniques: Methylation, Transformation Assay, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

doi: 10.1093/nar/gkv508

Figure Lengend Snippet: Concordance between ChIP-qPCR and ChIP-MBD2 at various genomic locations. ( A ) ChIP-qPCR experiments mapping MBD2 binding sites, in HMEC-hTERT cells (green) and HMLER cells (red). Each experiment was performed in duplicate. ( B – H ) Genome coverage for MeDPseq (black), MBD2 ChIPseq (orange) and Input-seq (gray) in HMEC-hTERT (upper tracks) and HMLER (bottom tracks) at the seven genomic location tested in A. Scales are in Fragments Per Million (FPM); Genes in blue, CpG islands in red; qPCR amplicon localization in green.

Article Snippet: ChIP were then performed with a custom-made rabbit polyclonal antiserum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) ( ) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer's instruction.

Techniques: Binding Assay, Amplification

Journal: Nucleic Acids Research

Article Title: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

doi: 10.1093/nar/gkv508

Figure Lengend Snippet: MBD2 is a methylation-dependent transcriptional repressor in HMLER cells. ( A ) Immunoblot analysis of MBD2 proteins in siRNA treated HMLER cells. ( B ) Box plots depicting the fold changes (FC) of the transcription levels of genes upregulated upon MBD2 depletion (siMBD2) and the FC of the same genes in 5-aza-deoxycytidine treated cells (DAC). The upregulated genes were defined as genes exhibiting a FC ≥ 2 with a P value ≤ 0.01. ( C ) Box plots depicting the FC of the transcription levels of genes upregulated upon DAC treatments and the FC of the same genes in MBD2-depleted HMLER cells. ( D ) Heat maps of genes upregulated or downregulated upon siRNA targeting MBD2. Most genes upregulated by MBD2 depletion were also upregulated by DAC treatments. ( E ) Heat maps of genes upregulated or downregulated upon DAC treatments. About 44% of the genes upregulated upon DAC treatments were also upregulated by MBD2 depletion. ( F to I ) Integrative analysis of ChIPseq and RNAseq. Mean coverages at gene promoters were plotted according to their responses to MBD2 siRNA or DAC treatments. Genes were classified into seven groups according to their responses to either MBD2 depletion or DNA methylation inhibition. N genes: number of genes in each group. For each gene cluster, mean MBD2-read densities and mean MeDP-read densities at the TSS (± 3 kb) were determined. (F) MBD2-read density at TSS regions of genes classified by their response to siMBD2. (G) MeDP-read density at TSS regions of genes classified by their response to siMBD2. (H) MBD2-read density at TSS regions of genes classified by their response to DAC treatments. (I) MeDP-read density at TSS regions of genes classified by their response to DAC treatments.

Article Snippet: ChIP were then performed with a custom-made rabbit polyclonal antiserum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) ( ) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer's instruction.

Techniques: Methylation, Western Blot, DNA Methylation Assay, Inhibition

Journal: Nucleic Acids Research

Article Title: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

doi: 10.1093/nar/gkv508

Figure Lengend Snippet: Dynamics of MBD2 distribution during oncogenic transformation. ( A ) Venn diagram depicting the overlap between MBD2 binding sites of HMEC-HTERT and HMLER cells; in green: regions that significantly lose MBD2 during transformation ( P value ≤ 0.01), in red: regions that significantly gain MBD2 during transformation ( P value ≤ 0.01), in brown: common regions. ( B ) Venn diagram depicting the overlap between methylated DNA regions of HMEC-HTERT and HMLER cells; in green: regions that significantly lose DNA methylation during transformation ( P value ≤ 0.01), in red: regions that significantly gain DNA methylation during transformation ( P value ≤ 0.01), in brown: common methylated DNA regions. ( C ) DNA methylation changes at MBD2 binding sites. Differences in MeDP coverage (FPM, Fragments Per Million) at MBD2 peaks were computed for regions bound by MBD2 in both cell lines, losing MBD2 or gaining MBD2 in HMLER when compared with HMEC-hTERT. ( D and E ) Genes were classified according to their differential expression level (FC) between HMEC-HTERT and HMLER cells. (D) Genes downregulated during oncogenic transformation gain MBD2-reads near their TSS (red curve), in contrast to unaffected (black curve) or downregulated genes (green curve). (E) Genes downregulated during oncogenic transformation exhibited gain of MeDP-reads near their TSS (red curve), in contrast to unaffected (black curve) or downregulated genes (green curve) ( F ) Venn diagram depicting the overlap between genes upregulated upon MBD2 depletion, DAC treatments and genes downregulated in HMLER cells when compared with HMEC-hTERT cells. A large proportion of genes downregulated during oncogenic transformation was also upregulated upon MBD2 depletion and DNA methylation inhibition in the transformed HMLER cell line. ( G ) Transcription level modification between HMEC-hTERT and HMLER, of the 2,164 genes upregulated after a siMBD2 treatment in HMLER. Around 1,168 of these genes are expressed at least two times less in HMLER than in HMEC-hTERT. ( H ) Read densities from MeDPseq, MBD2 ChIPseq and Input-seq in HMEC-hTERT and HMLER, at the closest MBD2 peaks in HMLER cells form the TSS of the 1,168 genes identified in (G). Around half of these regions shown an increase MBD2 ChIPseq read density in HMLER as compared to HMEC-hTERT.

Article Snippet: ChIP were then performed with a custom-made rabbit polyclonal antiserum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) ( ) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer's instruction.

Techniques: Transformation Assay, Binding Assay, Methylation, DNA Methylation Assay, Expressing, Inhibition, Modification

Journal: Nucleic Acids Research

Article Title: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

doi: 10.1093/nar/gkv508

Figure Lengend Snippet: Examples of genes repressed by MBD2 during oncogenic transformation. ( A – D ) MBD2 ChIP-qPCR at four genomic regions corresponding to five gene promoters in the HMEC-hTERT cell line and the transformed HMLER, HME-ZEB1-RAS and HME-shP53-RAS cell lines. These regions exhibited an increase of MBD2 binding during oncogenic transformation. Each experiment was performed two times independently. ( E – I ) Relative expression of five genes in HMEC-hTERT, HMLER treated with a control siRNA (siCtrl), a siRNA targeting MBD2 (siMBD2) or 5-aza-deoxycytidine (DAC), HME-ZEB1-RAS treated with a control siRNA or a siRNA targeting MBD2 and in HME-shP53-RAS treated with a control siRNA or a siRNA targeting MBD2 . Expression levels were estimated from RNA sequencing duplicates.

Article Snippet: ChIP were then performed with a custom-made rabbit polyclonal antiserum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) ( ) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer's instruction.

Techniques: Transformation Assay, Binding Assay, Expressing, RNA Sequencing Assay

Journal: Nucleic Acids Research

Article Title: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

doi: 10.1093/nar/gkv508

Figure Lengend Snippet: MBD2 repressed a common set of genes during oncogenic transformation. ( A ) Experimental scheme of generation of two cell lines from HMEC-hTERT cells by oncogenic transformation. HME-ZEB1-RAS cells were obtained by the expression of the embryonic transcription factor ZEB1 and H-RAS V12 , and HME-shP53-RAS cells were obtained by the expression of a shRNA targeting P53 and H-RAS V12 genes. ( B ) Immunoblot analysis of MBD2 protein level in HME-ZEB1-RAS and HME-shP53-RAS upon siRNA treatment targeting MBD2 . ( C and D ) Heat maps summarizing genes downregulated (green) or upregulated (red). (C) A set of 548 genes was upregulated in MBD2 knockdown cell lines. (D) Most of these genes upregulated by siRNA against MBD2 in transformed cell lines were genes that are downregulated during oncogenic transformation.

Article Snippet: ChIP were then performed with a custom-made rabbit polyclonal antiserum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) ( ) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer's instruction.

Techniques: Transformation Assay, Expressing, shRNA, Western Blot

Journal: Nucleic Acids Research

Article Title: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

doi: 10.1093/nar/gkv508

Figure Lengend Snippet: MBD2 ChIP-qPCR experiments from HMEC-HTERT, HMLER, HME-ZB1-RAS and HME-shP53 cell lines. Error bars represent average deviation from two independent experiments. ( A ) MBD2 binding at several positive and negative regions. ( B ) MBD2 redistribution in the genome during oncogenic transformation was frequently concordant in the different transformed cell lines.

Article Snippet: ChIP were then performed with a custom-made rabbit polyclonal antiserum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) ( ) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer's instruction.

Techniques: Binding Assay, Transformation Assay

Journal:

Article Title: Heterogeneity in the modification and involvement of chromatin components of the CpG island of the silenced human CDH1 gene in cancer cells

doi:

Figure Lengend Snippet: Differential MBP binding to the CpG island of cancer cells. (A) Presence of MBPs in the nuclei of each cells. Nuclear extract of cells (15 µg of protein) was subjected to western blot analysis using antibodies specific to each MBP. (B) Duplex PCR analysis of DNA fragments derived from the CpG island in input dilution (Input) or chromatin immunoprecipitates of anti-MBP antibodies. Panels depicted are representative of four independent trials. Amplified regions are identical to those used for analysis of acetylated histones. The antibodies that were used are 07-013 (MeCP2), S751 (MBD1) and S923 (MBD2).

Article Snippet: Antibodies against MBD1 protein (M254) and MBD2 protein (07-199) were obtained from Santa Cruz and Upstate Biotechnology, respectively.

Techniques: Binding Assay, Western Blot, Derivative Assay, Amplification

Journal:

Article Title: Heterogeneity in the modification and involvement of chromatin components of the CpG island of the silenced human CDH1 gene in cancer cells

doi:

Figure Lengend Snippet: Differential MBP bindings to the CpG island revealed by ChIP analysis using isolated nuclei of cancer cells. (A) PCR analysis of DNA fragments derived from the methylated promoter of CDKN2A gene in chromatin immunoprecipitates of anti-MeCP2 or anti-MBD2 antibodies from cell or nuclei. (B) Duplex PCR analysis of DNA fragments derived from the CpG island in input dilution (Input) or chromatin immunoprecipitates of anti-MBP antibodies from nuclei. Panels depicted are representative of duplicate trials. Amplified regions are identical to those used for analysis of cells. Input dilution was prepared from cross-linked chromatin of nuclei. Antibodies that were used are 07-013 (MeCP2), M254 (MBD1) and 07-199 (MBD2). The antibody S751 that was used in the experiments shown in Figure ​Figure44 was replaced with the antibody M254 in this experiment due to the lack of the material. We confirmed that similar results were obtained as shown in Figure ​Figure44 by using the antibody M254 (data not shown).

Article Snippet: Antibodies against MBD1 protein (M254) and MBD2 protein (07-199) were obtained from Santa Cruz and Upstate Biotechnology, respectively.

Techniques: Isolation, Derivative Assay, Methylation, Amplification