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Alfonso Bellacosa - One of the best experts on this subject based on the ideXlab platform.
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biphasic kinetics of the human dna repair protein MED1 mbd4 a mismatch specific dna n glycosylase
Journal of Biological Chemistry, 2000Co-Authors: Fiorella Petronzelli, Antonio Riccio, Steven H Seeholzer, Jay Stoerker, George D Markham, Maurizio Genuardi, Anthony T. Yeung, Yoshihiro Matsumoto, Alfonso BellacosaAbstract:Abstract The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.
Xiaoting Zhang - One of the best experts on this subject based on the ideXlab platform.
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cross talk between her2 and MED1 regulates tamoxifen resistance of human breast cancer cells
Cancer Research, 2012Co-Authors: Jiajun Cui, Katherine Germer, Jiang Wang, Jia Luo, Shao Chun Wang, Qianben Wang, Xiaoting ZhangAbstract:Despite the fact that most breast cancer patients have estrogen receptor (ER) α-positive tumors, up to 50% of the patients are or soon develop resistance to endocrine therapy. It is recognized that HER2 activation is one of the major mechanisms contributing to endocrine resistance. In this study, we report that the ER coactivator MED1 is a novel cross-talk point for the HER2 and ERα pathways. Tissue microarray analysis of human breast cancers revealed that MED1 expression positively correlates most strongly with HER2 status of the tumors. MED1 was highly phosphorylated, in a HER2-dependent manner, at the site known to be critical for its activation. Importantly, RNAi-mediated attenuation of MED1 sensitized HER2-overexpressing cells to tamoxifen treatment. MED1 and its phosphorylated form, but not the corepressors N-CoR and SMRT, were recruited to the ERα target gene promoter by tamoxifen in HER2-overexpressing cells. Significantly, MED1 attenuation or mutation of MED1 phosphorylation sites was sufficient to restore the promoter recruitment of N-CoR and SMRT. Notably, we found that MED1 is required for the expression of not only traditional E2-ERα target genes but also the newly described EGF-ERα target genes. Our results additionally indicated that MED1 is recruited to the HER2 gene and required for its expression. Taken together, these findings support a key role for MED1 in HER2-mediated tamoxifen resistance and suggest its potential usage as a therapeutic target to simultaneously block both ERα and HER2 pathways for the treatment of this type of endocrine resistant breast cancer.
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arginine and glutamate rich 1 arglu1 interacts with mediator subunit 1 MED1 and is required for estrogen receptor mediated gene transcription and breast cancer cell growth
Journal of Biological Chemistry, 2011Co-Authors: Dingxiao Zhang, Pingping Jiang, Qinqin Xu, Xiaoting ZhangAbstract:Estrogen receptor is a nuclear receptor superfamily member of transcriptional activators that regulate gene expression by recruiting diverese transcriptional coregulators. The Mediator complex is a central transcriptional coactivator complex that acts as a bridge between transcriptional activators and RNA polymerase II. MED1 (Mediator subunit 1) is the key Mediator subunit that directly interacts with estrogen receptor to mediate its functions both in vitro and in vivo. Interestingly, our previous biochemical analyses indicated that MED1 exists only in a subpopulation of the Mediator complex that is enriched with a number of distinct Mediator subunits and RNA polymerase II. Here, we report ARGLU1 as a MED1/Mediator-associated protein. We found that ARGLU1 (arginine and glutamate rich 1) not only colocalizes with MED1 in the nucleus, but also directly interacts with a far C-terminal region of MED1. Reporter assays indicate that ARGLU1 is able to cooperate with MED1 to regulate estrogen receptor-mediated gene transcription. Importantly, ARGLU1 is recruited, in a ligand-dependent manner, to endogenous estrogen receptor target gene promoters and is required for their expression. Furthermore, by ChIP-reChIP assay, we confirm that ARGLU1 and MED1 colocalize on the same estrogen receptor target gene promoter upon estrogen induction. Moreover, we found that depletion of ARGLU1 significantly impairs the growth, as well as anchorage-dependent and -independent colony formation of breast cancer cells. Taken together, these results establish ARGLU1 as a new MED1-interacting protein required for estrogen-dependent gene transcription and breast cancer cell growth.
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key roles for MED1 lxxll motifs in pubertal mammary gland development and luminal cell differentiation
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Pingping Jiang, Mitsuhiro Ito, Sara E Meyer, Susan E Waltz, Sohaib A Khan, Robert G Roeder, Xiaoting ZhangAbstract:Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand-dependent interactions of the MED1 subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is little information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Toward this end, we have generated MED1 LxxLL motif-mutant knockin mice. Interestingly, these mice are both viable and fertile and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. Consistent with this phenotype, as well as loss of the strong ligand-dependent estrogen receptor (ER)α-Mediator interaction, expression of a number of known ERα-regulated genes was down-regulated in MED1-mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen-stimulated mammary duct growth in MED1-mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor- and cell-specific in vivo role for MED1 LxxLL motifs, through Mediator-ERα interactions, in mammary gland development.
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the mediator complex functions as a coactivator for gata 1 in erythropoiesis via subunit MED1 trap220
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Melanie Stumpf, Robert G Roeder, Xiaoting Zhang, Claudia Waskow, Marit Krotschel, Dominic Van Essen, Patrick Rodriguez, Boris Guyot, Tilman BorggrefeAbstract:The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit MED1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. MED1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that MED1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. MED1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that MED1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, MED1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit MED1 acts as a pivotal coactivator for GATA-1 in erythroid development.
Giovanni Neri - One of the best experts on this subject based on the ideXlab platform.
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.
Antonio Riccio - One of the best experts on this subject based on the ideXlab platform.
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biphasic kinetics of the human dna repair protein MED1 mbd4 a mismatch specific dna n glycosylase
Journal of Biological Chemistry, 2000Co-Authors: Fiorella Petronzelli, Antonio Riccio, Steven H Seeholzer, Jay Stoerker, George D Markham, Maurizio Genuardi, Anthony T. Yeung, Yoshihiro Matsumoto, Alfonso BellacosaAbstract:Abstract The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.
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the dna repair gene mbd4 MED1 is mutated in human carcinomas with microsatellite instability
Nature Genetics, 1999Co-Authors: Antonio Riccio, Lauri A. Aaltonen, Maurizio Genuardi, Valeria Masciullo, Andrew K Godwin, Anu Loukola, Antonio Percesepe, Reijo Salovaara, Maria Paravatoupetsotas, Daniel E BassiAbstract:The DNA repair gene MBD4 ( MED1 ) is mutated in human carcinomas with microsatellite instability
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.
Maurizio Genuardi - One of the best experts on this subject based on the ideXlab platform.
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biphasic kinetics of the human dna repair protein MED1 mbd4 a mismatch specific dna n glycosylase
Journal of Biological Chemistry, 2000Co-Authors: Fiorella Petronzelli, Antonio Riccio, Steven H Seeholzer, Jay Stoerker, George D Markham, Maurizio Genuardi, Anthony T. Yeung, Yoshihiro Matsumoto, Alfonso BellacosaAbstract:Abstract The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.
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the dna repair gene mbd4 MED1 is mutated in human carcinomas with microsatellite instability
Nature Genetics, 1999Co-Authors: Antonio Riccio, Lauri A. Aaltonen, Maurizio Genuardi, Valeria Masciullo, Andrew K Godwin, Anu Loukola, Antonio Percesepe, Reijo Salovaara, Maria Paravatoupetsotas, Daniel E BassiAbstract:The DNA repair gene MBD4 ( MED1 ) is mutated in human carcinomas with microsatellite instability
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.
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MED1 a novel human methyl cpg binding endonuclease interacts with dna mismatch repair protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.