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Axel Behrens - One of the best experts on this subject based on the ideXlab platform.
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c jun n terminal phosphorylation antagonises recruitment of the MBD3 nurd repressor complex
Nature, 2011Co-Authors: Cristina Aguilera, Brian Hendrich, Kentaro Nakagawa, Rocío Sancho, Atanu Chakraborty, Axel BehrensAbstract:AP-1 (activator protein 1) activity is strongly induced in response to numerous signals, including growth factors, cytokines and extracellular stresses. The proto-oncoprotein c-Jun belongs to the AP-1 group of transcription factors and it is a crucial regulator of intestinal progenitor proliferation and tumorigenesis. An important mechanism of AP-1 stimulation is phosphorylation of c-Jun by the Jun amino-terminal kinases (JNKs). N-terminal phosphorylation of the c-Jun transactivation domain increases target gene transcription, but a molecular explanation was elusive. Here we show that unphosphorylated, but not N-terminally phosphorylated c-Jun, interacts with MBD3 and thereby recruits the nucleosome remodelling and histone deacetylation (NuRD) repressor complex. MBD3 depletion in colon cancer cells increased histone acetylation at AP-1-dependent promoters, which resulted in increased target gene expression. The intestinal stem cell marker lgr5 was identified as a novel target gene controlled by c-Jun/MBD3. Gut-specific conditional deletion of MBD3 (MBD3(ΔG/ΔG) mice) stimulated c-Jun activity and increased progenitor cell proliferation. In response to inflammation, mdb3 deficiency resulted in colonic hyperproliferation and MBD3(ΔG/ΔG) mice showed markedly increased susceptibility to colitis-induced tumorigenesis. Notably, concomitant inactivation of a single allele of c-jun reverted physiological and pathological hyperproliferation, as well as the increased tumorigenesis in MBD3(ΔG/ΔG) mice. Thus the transactivation domain of c-Jun recruits MBD3/NuRD to AP-1 target genes to mediate gene repression, and this repression is relieved by JNK-mediated c-Jun N-terminal phosphorylation.
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c-Jun N-terminal phosphorylation antagonises recruitment of the MBD3/NuRD repressor complex
Nature, 2011Co-Authors: Cristina Aguilera, Brian Hendrich, Kentaro Nakagawa, Rocío Sancho, Atanu Chakraborty, Axel BehrensAbstract:AP-1 (activator protein 1) activity is strongly induced in response to numerous signals, including growth factors, cytokines and extracellular stresses. The proto-oncoprotein c-Jun belongs to the AP-1 group of transcription factors and it is a crucial regulator of intestinal progenitor proliferation and tumorigenesis. An important mechanism of AP-1 stimulation is phosphorylation of c-Jun by the Jun amino-terminal kinases (JNKs). N-terminal phosphorylation of the c-Jun transactivation domain increases target gene transcription, but a molecular explanation was elusive. Here we show that unphosphorylated, but not N-terminally phosphorylated c-Jun, interacts with MBD3 and thereby recruits the nucleosome remodelling and histone deacetylation (NuRD) repressor complex. MBD3 depletion in colon cancer cells increased histone acetylation at AP-1-dependent promoters, which resulted in increased target gene expression. The intestinal stem cell marker lgr5 was identified as a novel target gene controlled by c-Jun/MBD3. Gut-specific conditional deletion of MBD3 (MBD3(ΔG/ΔG) mice) stimulated c-Jun activity and increased progenitor cell proliferation. In response to inflammation, mdb3 deficiency resulted in colonic hyperproliferation and MBD3(ΔG/ΔG) mice showed markedly increased susceptibility to colitis-induced tumorigenesis. Notably, concomitant inactivation of a single allele of c-jun reverted physiological and pathological hyperproliferation, as well as the increased tumorigenesis in MBD3(ΔG/ΔG) mice. Thus the transactivation domain of c-Jun recruits MBD3/NuRD to AP-1 target genes to mediate gene repression, and this repression is relieved by JNK-mediated c-Jun N-terminal phosphorylation.
Thomas G. Fazzio - One of the best experts on this subject based on the ideXlab platform.
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An Embryonic Stem Cell-Specific NuRD Complex Functions through Interaction with WDR5
Stem cell reports, 2017Co-Authors: Kurtis N. Mccannell, Yang Tang, Nancy Fernandes, W. Rod Hardy, Michael R. Green, Feixia Chu, Thomas G. FazzioAbstract:The Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin regulatory complex that functions as a transcriptional co-repressor in metazoans. The NuRD subunit MBD3 is essential for targeting and assembly of a functional NuRD complex as well as embryonic stem cell (ESC) pluripotency. Three MBD3 isoforms (MBD3A, MBD3B, and MBD3C) are expressed in mouse. Here, we find that the MBD3C isoform contains a unique 50-amino-acid N-terminal region that is necessary for MBD3C to specifically interact with the histone H3 binding protein WDR5. Domain analyses of WDR5 reveal that the H3 binding pocket is required for interaction with MBD3C. We find that while MBD3c knockout ESCs differentiate normally, MBD3C is redundant with the MBD3A and MBD3B isoforms in regulation of gene expression, with the unique MBD3C N terminus required for this redundancy. Together, our data characterize a unique NuRD complex variant that functions specifically in ESCs.
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DNA methylation directs genomic localization of Mbd2 and MBD3 in embryonic stem cells.
eLife, 2016Co-Authors: Sarah J. Hainer, Kurtis N. Mccannell, Lihua Julie Zhu, Oliver J. Rando, Thomas G. FazzioAbstract:Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and MBD3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or MBD3. We show that chromatin localization of Mbd2 and MBD3 is highly overlapping and, unexpectedly, we find Mbd2 and MBD3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and MBD3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers.
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MBD3/NURD Complex Regulates Expression of 5-Hydroxymethylcytosine Marked Genes in Embryonic Stem Cells
Cell, 2011Co-Authors: Ozlem Yildirim, Oliver J. Rando, Jui-hung Hung, Poshen B Chen, Xianjun Dong, Zhiping Weng, Thomas G. FazzioAbstract:Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that MBD3 and Brg1 antagonistically regulate a common set of genes by regulating promoter nucleosome occupancy. Furthermore, both MBD3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): MBD3 colocalizes with Tet1 and 5hmC in vivo, MBD3 knockdown preferentially affects expression of 5hmC-marked genes, MBD3 localization is Tet1-dependent, and MBD3 preferentially binds to 5hmC relative to 5-methylcytosine in vitro. Finally, both MBD3 and Brg1 are themselves required for normal levels of 5hmC in vivo. Together, our results identify an effector for 5hmC, and reveal that control of gene expression by antagonistic chromatin regulators is a surprisingly common regulatory strategy in ES cells.
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MBD3 nurd complex regulates expression of 5 hydroxymethylcytosine marked genes in embryonic stem cells
Cell, 2011Co-Authors: Ozlem Yildirim, Oliver J. Rando, Jui-hung Hung, Poshen B Chen, Xianjun Dong, Zhiping Weng, Thomas G. FazzioAbstract:Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that MBD3 and Brg1 antagonistically regulate a common set of genes by regulating promoter nucleosome occupancy. Furthermore, both MBD3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): MBD3 colocalizes with Tet1 and 5hmC in vivo, MBD3 knockdown preferentially affects expression of 5hmC-marked genes, MBD3 localization is Tet1-dependent, and MBD3 preferentially binds to 5hmC relative to 5-methylcytosine in vitro. Finally, both MBD3 and Brg1 are themselves required for normal levels of 5hmC in vivo. Together, our results identify an effector for 5hmC, and reveal that control of gene expression by antagonistic chromatin regulators is a surprisingly common regulatory strategy in ES cells.
Brian Hendrich - One of the best experts on this subject based on the ideXlab platform.
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c jun n terminal phosphorylation antagonises recruitment of the MBD3 nurd repressor complex
Nature, 2011Co-Authors: Cristina Aguilera, Brian Hendrich, Kentaro Nakagawa, Rocío Sancho, Atanu Chakraborty, Axel BehrensAbstract:AP-1 (activator protein 1) activity is strongly induced in response to numerous signals, including growth factors, cytokines and extracellular stresses. The proto-oncoprotein c-Jun belongs to the AP-1 group of transcription factors and it is a crucial regulator of intestinal progenitor proliferation and tumorigenesis. An important mechanism of AP-1 stimulation is phosphorylation of c-Jun by the Jun amino-terminal kinases (JNKs). N-terminal phosphorylation of the c-Jun transactivation domain increases target gene transcription, but a molecular explanation was elusive. Here we show that unphosphorylated, but not N-terminally phosphorylated c-Jun, interacts with MBD3 and thereby recruits the nucleosome remodelling and histone deacetylation (NuRD) repressor complex. MBD3 depletion in colon cancer cells increased histone acetylation at AP-1-dependent promoters, which resulted in increased target gene expression. The intestinal stem cell marker lgr5 was identified as a novel target gene controlled by c-Jun/MBD3. Gut-specific conditional deletion of MBD3 (MBD3(ΔG/ΔG) mice) stimulated c-Jun activity and increased progenitor cell proliferation. In response to inflammation, mdb3 deficiency resulted in colonic hyperproliferation and MBD3(ΔG/ΔG) mice showed markedly increased susceptibility to colitis-induced tumorigenesis. Notably, concomitant inactivation of a single allele of c-jun reverted physiological and pathological hyperproliferation, as well as the increased tumorigenesis in MBD3(ΔG/ΔG) mice. Thus the transactivation domain of c-Jun recruits MBD3/NuRD to AP-1 target genes to mediate gene repression, and this repression is relieved by JNK-mediated c-Jun N-terminal phosphorylation.
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c-Jun N-terminal phosphorylation antagonises recruitment of the MBD3/NuRD repressor complex
Nature, 2011Co-Authors: Cristina Aguilera, Brian Hendrich, Kentaro Nakagawa, Rocío Sancho, Atanu Chakraborty, Axel BehrensAbstract:AP-1 (activator protein 1) activity is strongly induced in response to numerous signals, including growth factors, cytokines and extracellular stresses. The proto-oncoprotein c-Jun belongs to the AP-1 group of transcription factors and it is a crucial regulator of intestinal progenitor proliferation and tumorigenesis. An important mechanism of AP-1 stimulation is phosphorylation of c-Jun by the Jun amino-terminal kinases (JNKs). N-terminal phosphorylation of the c-Jun transactivation domain increases target gene transcription, but a molecular explanation was elusive. Here we show that unphosphorylated, but not N-terminally phosphorylated c-Jun, interacts with MBD3 and thereby recruits the nucleosome remodelling and histone deacetylation (NuRD) repressor complex. MBD3 depletion in colon cancer cells increased histone acetylation at AP-1-dependent promoters, which resulted in increased target gene expression. The intestinal stem cell marker lgr5 was identified as a novel target gene controlled by c-Jun/MBD3. Gut-specific conditional deletion of MBD3 (MBD3(ΔG/ΔG) mice) stimulated c-Jun activity and increased progenitor cell proliferation. In response to inflammation, mdb3 deficiency resulted in colonic hyperproliferation and MBD3(ΔG/ΔG) mice showed markedly increased susceptibility to colitis-induced tumorigenesis. Notably, concomitant inactivation of a single allele of c-jun reverted physiological and pathological hyperproliferation, as well as the increased tumorigenesis in MBD3(ΔG/ΔG) mice. Thus the transactivation domain of c-Jun recruits MBD3/NuRD to AP-1 target genes to mediate gene repression, and this repression is relieved by JNK-mediated c-Jun N-terminal phosphorylation.
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MBD3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells
Development (Cambridge England), 2007Co-Authors: Keisuke Kaji, Jennifer Nichols, Brian HendrichAbstract:MBD3 is a core component of the NuRD (Nucleosome Remodeling and Histone Deacetylation) co-repressor complex, and NuRD-mediated silencing has been implicated in cell fate decisions in a number of contexts. MBD3-deficient embryonic stem (ES) cells made by gene targeting are viable but fail to form a stable NuRD complex, are severely compromised in the ability to differentiate, and show LIF-independent self-renewal. MBD3 is known to be essential for postimplantation embryogenesis in mice, but the function of MBD3 in vivo has not previously been addressed. Here we show that the inner cell mass (ICM) of MBD3-deficient blastocysts fails to develop into mature epiblast after implantation. Unlike MBD3-null ES cells, MBD3-deficient ICMs grown ex vivo fail to expand their Oct4-positive, pluripotent cell population despite producing robust endoderm outgrowths. Additionally, we identify a set of genes showing stage-specific expression in ICM cells during preimplantation development, and show that MBD3 is required for proper gene expression patterns in pre- and peri-implantation embryos and in ES cells. These results demonstrate the importance of MBD3/NuRD for the development of pluripotent cells in vivo and for their ex vivo progression into embryonic stem cells, and highlight the differences between ES cells and the ICM cells from which they are derived.
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Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development
Genes & development, 2001Co-Authors: Brian Hendrich, Jacqueline Guy, Bernard Ramsahoye, Valerie Wilson, Adrian BirdAbstract:MBD2 and MBD3 are closely related proteins with consensus methyl-CpG binding domains. MBD2 is a transcriptional repressor that specifically binds to methylated DNA and is a component of the MeCP1 protein complex. In contrast, MBD3 fails to bind methylated DNA in murine cells, and is a component of the Mi-2/NuRD corepressor complex. We show by gene targeting that the two proteins are not functionally redundant in mice, as MBD3-/- mice die during early embryogenesis, whereas Mbd2-/- mice are viable and fertile. Maternal behavior of Mbd2-/- mice is however defective and, at the molecular level, Mbd2-/- mice lack a component of MeCP1. Mbd2-mutant cells fail to fully silence transcription from exogenous methylated templates, but inappropriate activation of endogenous imprinted genes or retroviral sequences was not detected. Despite their differences, MBD3 and Mbd2 interact genetically suggesting a functional relationship. Genetic and biochemical data together favor the view that MBD3 is a key component of the Mi-2/NuRD corepressor complex, whereas MBD2 may be one of several factors that can recruit this complex to DNA.
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Genomic structure and chromosomal mapping of the murine and human Mbd1, Mbd2, MBD3, and Mbd4 genes.
Mammalian genome : official journal of the International Mammalian Genome Society, 1999Co-Authors: Brian Hendrich, Catherine M. Abbott, Heather A. Mcqueen, Doreen M. Chambers, Sally H. Cross, Adrian BirdAbstract:DNA methylation is essential for murine development and is implicated in the control of gene expression. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of mammalian, nuclear proteins related by the presence in each of an amino acid motif called the methyl-CpG binding domain (MBD). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA. MeCP2, MBD1 and MBD2 can also repress transcription. We describe the genomic structure and chromosomal localization of the human and murine Mbd1, Mbd2, MBD3, and Mbd4 genes. We find that the highly similar MBD2 and MBD3 proteins are encoded by genes that map to different chromosomes in humans and mice but show a similar genomic structure. The Mbd1 and Mbd2 genes, in contrast, map together to murine and human Chromosomes (Chrs)18. The MBD3 and Mbd4 genes map to murine Chrs 10 and 6, respectively, while the human MBD3 and MBD4 genes map to Chrs 19 and 3, respectively.
Akira Horii - One of the best experts on this subject based on the ideXlab platform.
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ret finger protein enhances mbd2 and mbd4 dependent transcriptional repression
Biochemical and Biophysical Research Communications, 2006Co-Authors: Shinichi Fukushige, Emiko Kondo, Zhaodi Gu, Hideyuki Suzuki, Akira HoriiAbstract:We recently demonstrated that MBD4 possesses the ability to repress transcription through methyl-CpG and is associated with methylated promoters in the CDKN2A and MLH1 genes. In order to further investigate the role of MBD4 in methylation-based transcriptional repression, a yeast two-hybrid screening was performed, and the RET finger protein (RFP) was found to be one of the major proteins that interact with the transcriptional repression domain in MBD4. The effect of the MBD4-mediated transcriptional repression in methylated CDKN2A and MLH1 promoters was extremely enhanced by the overexpression of RFP. Furthermore, RFP forms a protein complex not only with MBD4 but also with MBD2 or MBD3 and was shown to enhance transcriptional repression through MBD2. These results suggest that RFP is a mediator connecting several MBD proteins and allowing the formation of a more potent transcriptional repressor complex. Because RFP has been detected at high levels in a variety of tumor cell lines as well as testis, and embryos, RFP may have an important role in the enhancement of transcriptional repression through MBD proteins in tumorigenesis, spermatogenesis, and embryogenesis.
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ret finger protein mediates mbd2 and mbd4 dependent transcriptional repression
Cancer Research, 2006Co-Authors: Shinichi Fukushige, Emiko Kondo, Zhaodi Gu, Akira HoriiAbstract:21 The methyl-CpG binding domain protein 4, MBD4, has long been thought to be a thymine DNA glycosylase that repairs G-T mismatches originated by deamination of 5mC at CpG sites. However, recently we demonstrated that MBD4 also has the ability to repress transcription through methyl-CpG sequences and is associated with methylated promoters in p16INK4a and hMLH1 genes. In order to further investigate the role of MBD4 in methylation-based transcriptional repression, a yeast two-hybrid screen was performed and Ret finger protein (RFP) was found to interact with the transcriptional repression domain of MBD4. Because it has been reported that RFP was highly expressed in a variety of human and rodent tumor cell lines, and was involved in transcriptional repression through Enhancer of Polycomb 1 (EPC1) and Mi-2β, we assessed the functional implication of the MBD4-RFP association by examining the effect of RFP on repression activity of MBD4. Expression of RFP extremely enhanced the MBD4-mediated transcriptional repression in both methylated p16INK4a and hMLH1 promoters. Furthermore, RFP forms a protein complex with MBD2 and MBD3 through Mi-2β, and was also shown to be involved in transcriptional repression through MBD2. Therefore, these results suggest that RFP is one of essential components involved in transcriptional repression through MBD2, MBD3, and MBD4.
Fuyuki Ishikawa - One of the best experts on this subject based on the ideXlab platform.
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MBD3 and HDAC1, two components of the NuRD complex, are localized at Aurora-A-positive centrosomes in M phase.
The Journal of biological chemistry, 2002Co-Authors: Hirotaka Sakai, Motoki Saito, Takeshi Urano, Kayoko Ookata, Mi-hyun Kim, Yugo Hirai, Yoshihisa Nojima, Fuyuki IshikawaAbstract:Abstract MBD3, a component of the histone deacetylase NuRD complex, contains the methyl-CpG-binding domain (MBD), yet does not possess appreciable mCpG-specific binding activity. The functional significance of MBD3 in the NuRD complex remains enigmatic, partly because of the limited availability of biochemical approaches, such as immunoprecipitation, to analyze MBD3. In this study, we stably expressed the FLAG-tagged version of MBD3 in HeLa cells. We found that MBD3-FLAG was incorporated into the NuRD complex, and the MBD3-FLAG-containing NuRD complex was efficiently immunoprecipitated by anti-FLAG antibodies. By exploiting this system, we found that MBD3 is phosphorylated in vivo in the late G2and early M phases. Moreover, we found that Aurora-A, a serine/threonine kinase active specifically in the late G2and early M phases, phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase. Interestingly, HDAC1 is distributed at the centrosomes in a manner similar to MBD3. These results suggest the highly dynamic nature of the temporal and spatial distributions, as well as the biochemical modification, of the NuRD complex in M phase, probably through an interaction with kinases, including Aurora-A. These observations will contribute significantly to the elucidation of the yet-uncharacterized cell cycle-controlled functions of the NuRD complex.
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the mcpg binding domain of human MBD3 does not bind to mcpg but interacts with nurd mi2 components hdac1 and mta2
Journal of Biological Chemistry, 2002Co-Authors: Motoki Saito, Fuyuki IshikawaAbstract:Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.
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The mCpG-binding Domain of Human MBD3 Does Not Bind to mCpG but Interacts with NuRD/Mi2 Components HDAC1 and MTA2
The Journal of biological chemistry, 2002Co-Authors: Motoki Saito, Fuyuki IshikawaAbstract:Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.
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mbd2 MBD3 complex binds to hemi methylated dna and forms a complex containing dnmt1 at the replication foci in late s phase
Genes to Cells, 2000Co-Authors: Ken‐ichiro Tatematsu, Tetsu Yamazaki, Fuyuki IshikawaAbstract:Background In vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently, a family of methyl-CpG-binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive. Results It was shown that MBD2 and MBD3 form homo- and hetero-dimers (or multimers) in vitro and in vivo. Significantly, the MBD2-MBD3 complex showed an affinity to hemi-methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co-localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a co-immunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle. Conclusions The results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2-MBD3 complex recognizes hemi-methylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.
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MBD2‐MBD3 complex binds to hemi‐methylated DNA and forms a complex containing DNMT1 at the replication foci in late S phase
Genes to cells : devoted to molecular & cellular mechanisms, 2000Co-Authors: Ken‐ichiro Tatematsu, Tetsu Yamazaki, Fuyuki IshikawaAbstract:Background In vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently, a family of methyl-CpG-binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive. Results It was shown that MBD2 and MBD3 form homo- and hetero-dimers (or multimers) in vitro and in vivo. Significantly, the MBD2-MBD3 complex showed an affinity to hemi-methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co-localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a co-immunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle. Conclusions The results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2-MBD3 complex recognizes hemi-methylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.