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Robert Dante - One of the best experts on this subject based on the ideXlab platform.
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The "Methyl-CpG Binding Domain protein 2" plays a repressive role in relation to the promoter CpG content in the normal human cell line MRC5.
Current pharmaceutical design, 2014Co-Authors: Laury Perriaud, Joël Lachuer, Robert DanteAbstract:In cancer cells, methylation-dependent gene silencing is at least partly mediated by the “Methyl-CpG-Binding Domain protein 2” (MBD2 protein), via the recruitment of chromatin remodeling complexes. However this repressive role was poorly investigated in normal cells. To identify the genes repressed by MBD2 in these cells, we have determined the impact of MBD2 depletion on gene expression in human embryonic MRC5 fibroblasts, using RNA inference combined with microarray analysis. The up-regulation of some randomly selected genes was confirmed and a direct association between gene repression and MBD2 binding on methylated promoters associated to these genes was subsequently established. This control of gene expression appears to depend on the CpG content of promoters as MBD2 depletion was not sufficient to induce the expression of silent genes associated with High-CpG promoters, but it was required to achieve the methyl-dependent transcriptional locking of the genes associated with promoters exhibiting intermediate CpG content. Therefore, MBD2 seems to play a selective role in gene repression depending on the CpG content of the promoter regions.
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Preferential binding of the methyl-CpG binding Domain protein 2 at methylated transcriptional start site regions.
Epigenetics, 2011Co-Authors: Amandine Chatagnon, Laury Perriaud, Joël Lachuer, Jean Benhattar, Nicolas Nazaret, Séverine Croze, Robert DanteAbstract:Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal sp...
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A role for methyl-CpG binding Domain protein 2 in the modulation of the estrogen response of pS2/TFF1 gene.
PloS one, 2010Co-Authors: Amandine Chatagnon, Esteban Ballestar, Manel Esteller, Robert DanteAbstract:BACKGROUND In human Estrogen Receptor alpha (ERalpha)-positive breast cancers, 5' end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ERalpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation. METHODOLOGY/PRINCIPAL FINDINGS We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ERalpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 5' end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ERalpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ERalpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ERalpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated. CONCLUSIONS/SIGNIFICANCE MBD2 and ERalpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ERalpha and MBD2.
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Specific association between the Methyl-CpG-Binding Domain protein 2 and the hypermethylated region of the human telomerase reverse transcriptase promoter in cancer cells
Carcinogenesis, 2008Co-Authors: Amandine Chatagnon, Stéphanie Bougel, Laury Perriaud, Joël Lachuer, Jean Benhattar, Robert DanteAbstract: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 cells.
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Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of Methyl-CpG-Binding Domain proteins
Oncogene, 2008Co-Authors: Lidia Lopez-serra, Esteban Ballestar, Santiago Ropero, Fernando Setien, Mario F. Fraga, Pilar López-nieva, Miguel Alaminos, D. Guerrero, Robert DanteAbstract:Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of Methyl-CpG-Binding Domain proteins
Esteban Ballestar - One of the best experts on this subject based on the ideXlab platform.
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A role for methyl-CpG binding Domain protein 2 in the modulation of the estrogen response of pS2/TFF1 gene.
PloS one, 2010Co-Authors: Amandine Chatagnon, Esteban Ballestar, Manel Esteller, Robert DanteAbstract:BACKGROUND In human Estrogen Receptor alpha (ERalpha)-positive breast cancers, 5' end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ERalpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation. METHODOLOGY/PRINCIPAL FINDINGS We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ERalpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 5' end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ERalpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ERalpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ERalpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated. CONCLUSIONS/SIGNIFICANCE MBD2 and ERalpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ERalpha and MBD2.
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Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of Methyl-CpG-Binding Domain proteins
Oncogene, 2008Co-Authors: Lidia Lopez-serra, Esteban Ballestar, Santiago Ropero, Fernando Setien, Mario F. Fraga, Pilar López-nieva, Miguel Alaminos, D. Guerrero, Robert DanteAbstract:Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of Methyl-CpG-Binding Domain proteins
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A profile of methyl-CpG binding Domain protein occupancy of hypermethylated promoter CpG islands of tumor suppressor genes in human cancer.
Cancer research, 2006Co-Authors: Lidia Lopez-serra, Esteban Ballestar, Fernando Setien, Mario F. Fraga, Miguel Alaminos, Manel EstellerAbstract:Methyl-CpG binding Domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.
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effects of rett syndrome mutations of the methyl cpg binding Domain of the transcriptional repressor mecp2 on selectivity for association with methylated dna
Biochemistry, 2000Co-Authors: Esteban Ballestar, Timur M Yusufzai, Alan P WolffeAbstract:We have investigated the properties of mutant forms of the methyl-CpG binding transcriptional repressor MeCP2 associated with Rett syndrome, a childhood neurodevelopmental disorder. We find that four Rett syndrome mutations at known sites within the methyl-CpG binding Domain (MBD) impair binding to methylated DNA, but have little effect on nonspecific interactions with unmethylated DNA. Three of these mutations (R106W, R133C, and F155S) have their binding affinities for methylated DNA reduced more than 100-fold; this is consistent with the hypothesis that impaired selectivity for methylated DNA of mutant MeCP2 contributes to Rett syndrome. However, a fourth mutant, T158M, has its binding affinity for methylated DNA reduced only 2-fold, indicative either of additional distinct regulatory functions associated with the MBD or of an exquisite sensitivity of developing neurons to the selective association of MeCP2 with methylated DNA.
Amandine Chatagnon - One of the best experts on this subject based on the ideXlab platform.
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Preferential binding of the methyl-CpG binding Domain protein 2 at methylated transcriptional start site regions.
Epigenetics, 2011Co-Authors: Amandine Chatagnon, Laury Perriaud, Joël Lachuer, Jean Benhattar, Nicolas Nazaret, Séverine Croze, Robert DanteAbstract:Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal sp...
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A role for methyl-CpG binding Domain protein 2 in the modulation of the estrogen response of pS2/TFF1 gene.
PloS one, 2010Co-Authors: Amandine Chatagnon, Esteban Ballestar, Manel Esteller, Robert DanteAbstract:BACKGROUND In human Estrogen Receptor alpha (ERalpha)-positive breast cancers, 5' end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ERalpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation. METHODOLOGY/PRINCIPAL FINDINGS We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ERalpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 5' end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ERalpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ERalpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ERalpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated. CONCLUSIONS/SIGNIFICANCE MBD2 and ERalpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ERalpha and MBD2.
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Spécificité de liaison et de répression de la « Methyl-CpG-Binding Domain protein 2 » (MBD2) : identification de gènes cibles impliqués dans les cancers
2009Co-Authors: Amandine ChatagnonAbstract:De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l’expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l’expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l’analyse globale des profils de distribution de MBD2, de la méthylation de l’ADN, ainsi que de l’ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d’un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle.
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Specific association between the Methyl-CpG-Binding Domain protein 2 and the hypermethylated region of the human telomerase reverse transcriptase promoter in cancer cells
Carcinogenesis, 2008Co-Authors: Amandine Chatagnon, Stéphanie Bougel, Laury Perriaud, Joël Lachuer, Jean Benhattar, Robert DanteAbstract: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 cells.
Manel Esteller - One of the best experts on this subject based on the ideXlab platform.
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A role for methyl-CpG binding Domain protein 2 in the modulation of the estrogen response of pS2/TFF1 gene.
PloS one, 2010Co-Authors: Amandine Chatagnon, Esteban Ballestar, Manel Esteller, Robert DanteAbstract:BACKGROUND In human Estrogen Receptor alpha (ERalpha)-positive breast cancers, 5' end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ERalpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation. METHODOLOGY/PRINCIPAL FINDINGS We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ERalpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 5' end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ERalpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ERalpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ERalpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated. CONCLUSIONS/SIGNIFICANCE MBD2 and ERalpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ERalpha and MBD2.
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A profile of methyl-CpG binding Domain protein occupancy of hypermethylated promoter CpG islands of tumor suppressor genes in human cancer.
Cancer research, 2006Co-Authors: Lidia Lopez-serra, Esteban Ballestar, Fernando Setien, Mario F. Fraga, Miguel Alaminos, Manel EstellerAbstract:Methyl-CpG binding Domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.
Adrian Bird - One of the best experts on this subject based on the ideXlab platform.
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oxidative damage to methyl cpg sequences inhibits the binding of the methyl cpg binding Domain mbd of methyl cpg binding protein 2 mecp2
Nucleic Acids Research, 2004Co-Authors: Victoria Valinluck, Adrian Bird, Hsinhao Tsai, Daniel K Rogstad, Artur Burdzy, Lawrence C SowersAbstract:: Cytosine methylation in CpG dinucleotides is believed to be important in gene regulation, and is generally associated with reduced levels of transcription. Methylation-mediated gene silencing involves a series of DNA-protein and protein-protein interactions that begins with the binding of methyl-CpG binding proteins (MBPs) followed by the recruitment of histone-modifying enzymes that together promote chromatin condensation and inactivation. It is widely known that alterations in methylation patterns, and associated gene activities, are often found in human tumors. However, the mechanisms by which methylation patterns are altered are not currently understood. In this paper, we investigate the impact of oxidative damage to a methyl-CpG site on MBP binding by the selective placement of 8-oxoguanine (8-oxoG) and 5-hydroxymethylcytosine (HmC) in a MBP recognition sequence. Duplexes containing these specific modifications were assayed for binding to the methyl-CpG binding Domain (MBD) of one member of the MBP family, methyl-CpG binding protein 2 (MeCP2). Our results reveal that oxidation of either a single guanine to 8-oxoG or of a single 5mC to HmC, significantly inhibits binding of the MBD to the oligonucleotide duplex, reducing the binding affinity by at least an order of magnitude. Oxidative damage to DNA could therefore result in heritable, epigenetic changes in chromatin organization.
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fas associated death Domain protein interacts with methyl cpg binding Domain protein 4 a potential link between genome surveillance and apoptosis
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Robert A. Screaton, Adrian Bird, Stephan Kiessling, Owen J. Sansom, Catherine B. Millar, Kathryn Maddison, Alan Richard Clarke, Steven M. FrischAbstract:Fas-associated death Domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding Domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD–MBD4 interaction involved the death effector Domain of FADD and a region of MBD4 adjacent to the glycosylase Domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.
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Fas-associated death Domain protein interacts with methyl-CpG binding Domain protein 4
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Robert A. Screaton, Adrian Bird, Stephan Kiessling, Owen J. Sansom, Catherine B. Millar, Kathryn Maddison, Alan Richard Clarke, Steven M. FrischAbstract:Fas-associated death Domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding Domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD–MBD4 interaction involved the death effector Domain of FADD and a region of MBD4 adjacent to the glycosylase Domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.
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DNA Recognition by the Methyl-CpG Binding Domain of MeCP2
The Journal of biological chemistry, 2000Co-Authors: Andrew Free, Robert I.d Wakefield, Brian O. Smith, David T. F. Dryden, Paul N. Barlow, Adrian BirdAbstract:Abstract The methyl-CpG binding Domain (MBD) of the transcriptional repressor MeCP2 has been proposed to recognize a single symmetrically methylated CpG base pair via hydrophobic patches on an otherwise positively charged DNA binding surface. We have tested this binding model by analysis of mutant derivatives of the MeCP2 MBD in electrophoretic mobility shift assays complemented by NMR structural analysis. Exposed arginine side chains on the binding face, in particular Arg-111, were found to be critical for binding. Arg-111 was found to interact with the conserved aspartate side chain Asp-121, which is proposed to orientate the arginine side chain to allow specific contacts with the DNA. The conformational flexibility of the disordered B-C loop region, which forms part of the binding face, was also shown to be important. In contrast, mutation of the exposed hydrophobic side chains had a less severe effect on DNA binding. This suggests that the Arg-111 side chain may contribute to sequence-specific recognition of the CpG site rather than simply making nonspecific contacts with the phosphate backbone. The majority of missense mutations within the MBD found in the human genetic disorder Rett syndrome were shown or predicted to affect folding of the Domain rather than the DNA recognition event directly.
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Dissection of the methyl-CpG binding Domain from the chromosomal protein MeCP2
Nucleic acids research, 1993Co-Authors: Xinsheng Nan, Richard R. Meehan, Adrian BirdAbstract:MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding Domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding Domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M.