Specific association between the methyl-CpG-binding domain protein 2 and the hypermethylated region of the human telomerase reverse transcriptase promoter in cancer cells

Human telomerase reverse transcriptase (hTERT) is expressed in most cancer cells. Paradoxically, its promoter is embedded in a hypermethylated CpG island. A short region escapes to this alteration, allowing a basal level of transcription. However, the methylation of adjacent regions may play a role in the maintenance of low hTERT expression. It is now well established that methyl-CpG binding domain proteins mediate the transcriptional silencing of hypermethylated genes. The potential involvement of these proteins in the control of hTERT expression was firstly investigated in HeLa cells. Chromatin immunoprecipitation assays showed that only methyl-CpG-binding domain protein 2 (MBD2) associated the hypermethylated hTERT promoter. In MBD2 knockdown HeLa cells, constitutively depleted in MBD2, neither methyl CpG binding protein 2 (MeCP2) nor MBD1 acted as substitutes for MBD2. MBD2 depletion by transient or constitutive RNA interference led to an upregulation of hTERT transcription that can be downregulated by expressing mouse Mbd2 protein. Our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT in HeLa cells. This specific transcriptional repression was also observed in breast, liver and neuroblastoma cancer cell lines. Thus, MBD2 seems to be a general repressor of hTERT in hTERT-methylated telomerase-positive cell

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

DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells

The Methyl-CpG Binding Domain protein 2 (MBD2), a DNA methylation-dependent transcriptional repressor : identification and caracterization of MBD2 targets by genome-wide approach

Les protéines à « Methyl-CpG-binding domain » (MBD) jouent un rôle important dans l’interprétationde la méthylation de l’ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted’expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l’échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L’impact sur l’expression génique de l’inhibition de MBD2 par interférence àl’ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n’induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l’ADN, de liaisons de MBD2 et de l’ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l’analyse de l’acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l’ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l’ADN méthylé.The Methyl-CpG-Binding Domain (MBD) proteins represent key molecules in the interpretation ofDNA methylation signals leading to gene silencing through recruitment of chromatin remodelingcomplexes. In cancer, a member of this protein family, MBD2, seems to play an important role in theloss of expression of aberrantly methylated genes. Thus, MBD2 may be a potential target toreestablish their expression. Mapping of MBD2 binding sites and the relationship between MBD2binding and transcriptional activity was, therefore, a crucial step. (1) We investigated the impact ofMBD2 inhibition by RNA interference on gene expression, using microarray analysis, in a normalhuman fibroblastic cell line, MRC-5. MBD2 depletion did not induce global gene overexpression andCpG density of the methylated promoters seems to be an important parameter in the strength of thetranscriptional repression mediated by MBD2. (2) Global profiling for different layers of epigeneticmodifications (DNA methylation, MBD2 association) and RNA polymerase II binding sites in HeLacells was analyzed by a ChIP-chip method using human promoter arrays. This approach, combinedwith an analysis of H3 histone acetylation patterns, was performed in a syngenic model of breastcancer progression. In the models analyzed MBD2 appeared to be a true methylation-dependenttranscriptional repressor. Furthermore, MBD2 binds to a high proportion of methylated silent genes.Comparisons between immortalized and transformed cells did not indicate major changes of DNAmethylation or gene silencing, while a redistribution of MBD2 among these sites was observed,suggesting a redundancy between methylated binding proteins

Étude systémique des cibles génomiques de la methyl-CpG binding domain protein 2 (MBD2), un répresseur transcriptionnel dépendant de la méthylation de l'ADN : évolution de la distribution de MBD2 dans un modèle syngénique de progression tumorale mammaire

The Methyl-CpG-Binding Domain (MBD) proteins represent key molecules in the interpretation ofDNA methylation signals leading to gene silencing through recruitment of chromatin remodelingcomplexes. In cancer, a member of this protein family, MBD2, seems to play an important role in theloss of expression of aberrantly methylated genes. Thus, MBD2 may be a potential target toreestablish their expression. Mapping of MBD2 binding sites and the relationship between MBD2binding and transcriptional activity was, therefore, a crucial step. (1) We investigated the impact ofMBD2 inhibition by RNA interference on gene expression, using microarray analysis, in a normalhuman fibroblastic cell line, MRC-5. MBD2 depletion did not induce global gene overexpression andCpG density of the methylated promoters seems to be an important parameter in the strength of thetranscriptional repression mediated by MBD2. (2) Global profiling for different layers of epigeneticmodifications (DNA methylation, MBD2 association) and RNA polymerase II binding sites in HeLacells was analyzed by a ChIP-chip method using human promoter arrays. This approach, combinedwith an analysis of H3 histone acetylation patterns, was performed in a syngenic model of breastcancer progression. In the models analyzed MBD2 appeared to be a true methylation-dependenttranscriptional repressor. Furthermore, MBD2 binds to a high proportion of methylated silent genes.Comparisons between immortalized and transformed cells did not indicate major changes of DNAmethylation or gene silencing, while a redistribution of MBD2 among these sites was observed,suggesting a redundancy between methylated binding proteins.Les protéines à « Methyl-CpG-binding domain » (MBD) jouent un rôle important dans l’interprétationde la méthylation de l’ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted’expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l’échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L’impact sur l’expression génique de l’inhibition de MBD2 par interférence àl’ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n’induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l’ADN, de liaisons de MBD2 et de l’ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l’analyse de l’acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l’ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l’ADN méthylé