The Experts below are selected from a list of 13416 Experts worldwide ranked by ideXlab platform
Moshe Szyf - One of the best experts on this subject based on the ideXlab platform.
-
transcription onset of genes critical in liver carcinogenesis is epigenetically regulated by methylated dna binding protein mbd2
Carcinogenesis, 2013Co-Authors: Barbara Stefanska, Matthew Suderman, Ziv Machnes, Bishnu Bhattacharyya, Michael Hallett, Moshe SzyfAbstract:: We previously delineated genes whose promoters are hypomethylated and induced in hepatocellular carcinoma (HCC) patients. The purpose of this study was to establish the players that regulate these genes in liver cancer cells. We performed chromatin immunoprecipitation with methyl-CpG-binding domain protein 2 (MBD2), RNA polymerase II (RNA pol II), CCAAT/enhancer-binding protein alpha (CEBPA) antibodies and methylated DNA immunoprecipitation in HepG2 liver cancer cells treated with scrambled small interfering RNA (siRNA) and siRNA to MBD2 or CEBPA. We then hybridized DNA to microarrays spanning the entire coding sequences, introns and regulatory regions of several hundred HCC-hypomethylated genes. These analyses reveal that MBD2 binds a significant fraction of the hypomethylated genes, determines RNA pol II binding and DNA methylation state. MBD2 binding can result in promoter activation and hypomethylation or in repression. In activated target genes, MBD2 colocalizes with the transcription factor CEBPA, and MBD2 binding at these positions is reduced upon CEBPA depletion. Significant fraction of MBD2 effects on DNA methylation and transcription appears to be indirect since changes occur upon MBD2 depletion in genes where no MBD2 binding was detected. Our study delineates the rules governing the interaction of MBD2 with its targets and the consequences to RNA pol II binding and DNA methylation states. This has important implications for understanding the role of DNA methylation in cancer and targeting DNA methylation proteins in cancer therapy.
-
methylated dna binding protein 2 antisense inhibitors suppress tumourigenesis of human cancer cell lines in vitro and in vivo
Carcinogenesis, 2003Co-Authors: Paul M Campbell, Veronica Bovenzi, Moshe SzyfAbstract:Methylated DNA-binding protein 2 (MBD2) has been proposed to function both as a silencer of methylated genes and as a DNA demethylase. Our previous data indicated that knockdown of MBD2 inhibited tumourigenesis of human cancer lines and MBD2-deficient mice were recently shown to be resistant to intestinal tumourigenesis. MBD2 is an attractive anticancer target since MBD2-deficient mice were previously shown to be viable and fertile and knockdown of MBD2 was reported to have no effect on cellular growth parameters of non-transformed cells. In this paper we test the hypothesis that pharmacological inhibition of MBD2 inhibits cancer growth in vivo using human tumour lines implanted in mice as a model. We develop sequence-specific antisense inhibitors of MBD2 and we show that these agents inhibit anchorage-independent growth of human lung (A549) and colorectal (HCT116) cancer cell lines in vitro and tumourigenic growth of human cancer cell xenografts in vivo. MBD2 antisense oligonucleotide does not inhibit the growth of normal and transformed cell lines and does not alter cell cycle parameters in vitro and does not exhibit overt toxicity in vivo in comparison with a scrambled control oligonucleotide, as determined by measuring body mass, blood cell parameters and liver and kidney enzymes. Our data provide a proof of principle that MBD2 is a new anticancer target and that pharmacological inhibition of MBD2 by agents such as the antisense inhibitors described in this paper is a potential new anticancer therapy, which in contrast to the vast majority of current approaches does not target normal progression of the cell cycle.
-
antisense mbd2 gene therapy inhibits tumorigenesis
Journal of Gene Medicine, 2002Co-Authors: Andrew Slack, Veronica Bovenzi, Pascal Bigey, M A Ivanov, Shyam Ramchandani, Sanjoy K Bhattacharya, Benjamin R Tenoever, B Lamrihi, Daniel Scherman, Moshe SzyfAbstract:Background Aberration in the pattern of DNA methylation is one of the hallmarks of cancer. We present data suggesting that dysregulation of MBD2, a recently characterized member of a novel family of methylated DNA binding proteins, is involved in tumorigenesis. Two functions were ascribed to MBD2, DNA demethylase activity and repression of methylated genes. Methods Multiple antisense expression and delivery systems, transfection, electrotransfer and adenoviral were employed to demonstrate that MBD2 is essential in tumorigenesis, both ex vivo and in vivo. Results Inhibition of MBD2 by antisense expression resulted in inhibition of anchorage-independent growth of antisense transfected cancer cells or cells infected with an adenoviral vector expressing MBD2 antisense. Xenograft tumors treated with an adenoviral vector expressing MBD2 antisense or xenografts treated with electrotransferred plasmids expressing MBD2 antisense showed reduced growth. Conclusions These results support the hypothesis that one or both of the functions described for MBD2 are critical in tumorigenesis and that MBD2 is a potential anticancer target. Copyright © 2002 John Wiley & Sons, Ltd.
Adrian Bird - One of the best experts on this subject based on the ideXlab platform.
-
mbd2 contributes to dna methylation directed repression of the xist gene
Molecular and Cellular Biology, 2007Co-Authors: Helen Barr, Brian Hendrich, Andrea Hermann, Jennifer Berger, Hsinhao Tsai, Karen Adie, Anna Prokhortchouk, Adrian BirdAbstract:Transcription of the Xist gene triggers X chromosome inactivation in cis and is therefore silenced on the X chromosome that remains active. DNA methylation contributes to this silencing, but the mechanism is unknown. As methylated DNA binding proteins (MBPs) are potential mediators of gene silencing by DNA methylation, we asked whether MBP-deficient cell lines could maintain Xist repression. The absence of Mbd2 caused significant low-level reactivation of Xist, but silencing was restored by exogenous Mbd2. In contrast, deficiencies of MBD1, MeCP2, and Kaiso had no detectable effect, indicating that MBPs are not functionally redundant at this locus. Xist repression in Mbd2-null cells was hypersensitive to the histone deacetylase inhibitor trichostatin A and to depletion of the DNA methyltransferase Dnmt1. These synergies implicate Mbd2 as a mediator of the DNA methylation signal at this locus. The presence of redundant mechanisms to enforce repression at Xist and other loci is compatible with the hypothesis that “stacking” of imperfect repressive tendencies may be an evolutionary strategy to ensure leakproof gene silencing.
-
mbd2 is a transcriptional repressor belonging to the mecp1 histone deacetylase complex
Nature Genetics, 1999Co-Authors: Huckhui Ng, Brian Hendrich, Colin A Johnson, Bryan M Turner, Hediye Erdjumentbromage, Paul Tempst, Yi Zhang, Adrian BirdAbstract:Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins1,2,3,4. The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases5,6,7. Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro 4 and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex1,9. An affinity-purified HDAC1 corepressor complex10,11 also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylation-dependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes.
-
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.
-
identification and characterization of a family of mammalian methyl cpg binding proteins
Molecular and Cellular Biology, 1998Co-Authors: Brian Hendrich, Adrian BirdAbstract:Methylation at the DNA sequence 5*-CpG is required for mouse development. MeCP2 and MBD1 (formerly PCM1) are two known proteins that bind specifically to methylated DNA via a related amino acid motif and that can repress transcription. We describe here three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain. MBD2 and MBD4 bind specifically to methylated DNA in vitro. Expression of MBD2 and MBD4 tagged with green fluorescent protein in mouse cells shows that both proteins colocalize with foci of heavily methylated satellite DNA. Localization is disrupted in cells that have greatly reduced levels of CpG methylation. MBD3 does not bind methylated DNA in vivo or in vitro. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem cells which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo and are therefore likely to be mediators of the biological consequences of the methylation signal. DNA methylation is the major modification of eukaryote genomes. In vertebrates, this occurs predominantly at position 5 of cytosines when followed by guanosine (CpG). DNA methylation can repress transcription and for this reason has been implicated in stable alterations of gene expression in development (3). Whereas the genomes of certain invertebrates appear to contain “compartments” of either mostly methylated or mostly unmethylated DNA (43), the somatic genomes of vertebrates are globally methylated, with the exception of socalled CpG islands (6). CpG islands are GC-rich regions of DNA, stretching for an average of about 1 kb, which are coincident with the promoters of approximately 60% of human RNA polymerase II-transcribed genes (1). Methylation of CpG islands and subsequent silencing of associated transcription units have been found to occur in genes located on the inactive X chromosome (39), genes silenced by genomic imprinting (36, 38), and genes silenced in transformed cell lines and tumors (2, 8, 16, 18, 40). DNA methylation is known to play an essential role in mammalian development because mice lacking a functional gene encoding the maintenance DNA methyltransferase (DNMT) are developmentally retarded and die at midgestation (29). In contrast to the situation in somatic cells, undifferentiated embryonic stem (ES) cells lacking a functional DNMT gene apparently grow normally despite containing approximately 5% of the wild-type DNA
Mitsuyoshi Nakao - One of the best experts on this subject based on the ideXlab platform.
-
transcriptional repression and heterochromatin formation by MBD1 and mcaf am family proteins
Journal of Biological Chemistry, 2005Co-Authors: Takaya Ichimura, Naoyuki Fujita, Sugiko Watanabe, Yasuo Sakamoto, Takahiro Aoto, Mitsuyoshi NakaoAbstract:Abstract DNA methylation cooperates with methylation at lysine 9 of histone H3 (H3-K9), a modified histone molecule that is targeted by heterochromatin protein 1, to form a transcriptionally silent chromatin. Methyl CpG-binding protein MBD1 recognizes methylated CpG dinucleotide and recruits H3-K9 methyltransferases such as SETDB1 to genomic regions. Here we show that MBD1-containing chromatin-associated factor (MCAF) 1, also known as the human homologue of murine ATFa-associated modulator (AM), is required for transcriptional repression and heterochromatin formation by MBD1, together with the involvement of SETDB1. Moreover, the amino acid sequence of MCAF1 shows similarity to a number of sequences of the MCAF/AM-related proteins, resulting in the identification of a new member of the protein family, termed MCAF2. Immunoprecipitation and in vitro binding analyses reveal that both MCAF proteins interact with MBD1, SETDB1, and Sp1 via two evolutionarily conserved distinct domains. Furthermore, MCAF1 enhances transcriptional repression by MBD1 together with SETDB1, and exogenous expression of MCAF2 partly compensates for the repressive activity in MCAF1 knockdown HeLa cells. The expression of MBD1 mutant, which lacks interaction with MCAF proteins, perturbs heterochromatin protein 1-enriched heterochromatin formation at the MBD1-containing chromosomal loci. These data suggest that MBD1·MCAF1·SETDB1 complex facilitates the formation of heterochromatic domains, emphasizing the role of MCAF/AM family proteins in epigenetic control.
-
Methyl-CpG binding domain 1 (MBD1) interacts with the Suv39h1-HP1 heterochromatic complex for DNA methylation-based transcriptional repression
The Journal of biological chemistry, 2003Co-Authors: Naoyuki Fujita, Sugiko Watanabe, Takaya Ichimura, Shu Tsuruzoe, Yoichi Shinkai, Makoto Tachibana, Tsutomu Chiba, Mitsuyoshi NakaoAbstract:Cytosine methylation and posttranslational modifications of the amino termini of the core histones in the nucleosome provide epigenetic codes for genome regulation. In the nucleus, not only is the DNA methylated, but the methylated DNA is also interpreted by methyl-CpG binding domain (MBD) proteins. MBD1 possesses an MBD involved in mediating DNA methylation-dependent transcriptional repression. The MBD of MBD1 binds a symmetrically methylated CpG sequence, but the precise roles of this domain have not been investigated. In addition, little is understood about the state of histone modifications within MBD1-containing heterochromatin on methylated gene promoters. Here we show that histone H3 methylase Suv39h1 and the methyl lysine-binding protein HP1 directly interact with MBD of MBD1 in vitro and in cells. Suv39h1 was found to enhance MBD1-mediated transcriptional repression via MBD but not via the C-terminal transcriptional repression domain of MBD1. Furthermore, MBD1 links to histone deacetylases through Suv39h1, resulting in methylation and deacetylation of histones for gene inactivation. These data indicate that MBD1 may tether the Suv39h1-HP1 complex to methylated DNA regions, suggesting the presence of a pathway from DNA methylation to the modifications of histones for epigenetic gene regulation.
-
Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.
Cell, 2001Co-Authors: Izuru Ohki, Naoyuki Fujita, Mitsuyoshi Nakao, Nobuya Shimotake, Jun-goo Jee, Takahisa Ikegami, Masahiro ShirakawaAbstract:In vertebrates, the biological consequences of DNA methylation are often mediated by protein factors containing conserved methyl-CpG binding domains (MBDs). Mutations in the MBD protein MeCP2 cause the neurodevelopmental disease Rett syndrome. We report here the solution structure of the MBD of the human methylation-dependent transcriptional regulator MBD1 bound to methylated DNA. DNA binding causes a loop in MBD1 to fold into a major and novel DNA binding interface. Recognition of the methyl groups and CG sequence at the methylation site is due to five highly conserved residues that form a hydrophobic patch. The structure indicates how MBD may access nucleosomal DNA without encountering steric interference from core histones, and provides a basis to interpret mutations linked to Rett syndrome in MeCP2.
-
METHYLATION-MEDIATED TRANSCRIPTIONAL SILENCING IN EUCHROMATIN BY METHYL-CPG BINDING PROTEIN MBD1 ISOFORMS
Molecular and cellular biology, 1999Co-Authors: Naoyuki Fujita, Tsutomu Chiba, Shin-ichiro Takebayashi, Katsuzumi Okumura, Shinichi Kudo, Hideyuki Saya, Mitsuyoshi NakaoAbstract:DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.
Beth A. Weaver - One of the best experts on this subject based on the ideXlab platform.
-
A Golgi-Localized Pool of the Mitotic Checkpoint Component Mad1 Controls Integrin Secretion and Cell Migration
Current biology : CB, 2014Co-Authors: Jun Wan, Fen Zhu, Lauren M. Zasadil, Lei Wang, Adam Johnson, Erwin Berthier, David J. Beebe, Anjon Audhya, Beth A. WeaverAbstract:Summary Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the major cell-cycle checkpoint that regulates chromosome segregation during mitosis, the mitotic checkpoint (also known as the spindle assembly checkpoint). During mitosis, Mad1 recruits Mad2 to unattached kinetochores [1, 2], where Mad2 is converted into an inhibitor of the anaphase-promoting complex/cyclosome bound to its specificity factor, Cdc20 [1, 3–6]. During interphase, Mad1 remains tightly bound to Mad2 [2, 3, 7, 8], and both proteins localize to the nucleus and nuclear pores [9, 10], where they interact with Tpr (translocated promoter region). Recently, it has been shown that interaction with Tpr stabilizes both proteins [11] and that Mad1 binding to Tpr permits Mad2 to associate with Cdc20 [12]. However, interphase functions of Mad1 that do not directly affect the mitotic checkpoint have remained largely undefined. Here we identify a previously unrecognized interphase distribution of Mad1 at the Golgi apparatus. Mad1 colocalizes with multiple Golgi markers and cosediments with Golgi membranes. Although Mad1 has previously been thought to constitutively bind Mad2, Golgi-associated Mad1 is Mad2 independent. Depletion of Mad1 impairs secretion of α5 integrin and results in defects in cellular attachment, adhesion, and FAK activation. Additionally, reduction of Mad1 impedes cell motility, while its overexpression accelerates directed cell migration. These results reveal an unexpected role for a mitotic checkpoint protein in secretion, adhesion, and motility. More generally, they demonstrate that, in addition to generating aneuploidy, manipulation of mitotic checkpoint genes can have unexpected interphase effects that influence tumor phenotypes.
-
Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Sean D. Ryan, Lauren M. Zasadil, Anjon Audhya, Eric M. C. Britigan, Kristen Witte, Avtar Roopra, Beth A. WeaverAbstract:The mitotic checkpoint is the major cell cycle checkpoint acting during mitosis to prevent aneuploidy and chromosomal instability, which are hallmarks of tumor cells. Reduced expression of the mitotic checkpoint component Mad1 causes aneuploidy and promotes tumors in mice [Iwanaga Y, et al. (2007) Cancer Res 67:160–166]. However, the prevalence and consequences of Mad1 overexpression are currently unclear. Here we show that Mad1 is frequently overexpressed in human cancers and that Mad1 up-regulation is a marker of poor prognosis. Overexpression of Mad1 causes aneuploidy and chromosomal instability through weakening mitotic checkpoint signaling caused by mislocalization of the Mad1 binding partner Mad2. Cells overexpressing Mad1 are resistant to microtubule poisons, including currently used chemotherapeutic agents. These results suggest that levels of Mad1 must be tightly regulated to prevent aneuploidy and transformation and that Mad1 up-regulation may promote tumors and cause resistance to current therapies.
Irina Stancheva - One of the best experts on this subject based on the ideXlab platform.
-
h19 lncrna controls gene expression of the imprinted gene network by recruiting MBD1
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Paul Monnier, Clemence Martinet, Julien Pontis, Slimane Aitsiali, Irina Stancheva, Luisa DandoloAbstract:The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG–binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA–MBD1 complex is required for the control of five genes of the IGN. For three of these genes—Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)—both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA–MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.
-
recruitment of MBD1 to target genes requires sequence specific interaction of the mbd domain with methylated dna
Nucleic Acids Research, 2010Co-Authors: Thomas Clouaire, Jose I De Las Heras, Cara Merusi, Irina StanchevaAbstract:MBD1, a member of the methyl-CpG-binding domain family of proteins, has been reported to repress transcription of methylated and unmethylated promoters. As some MBD1 isoforms contain two DNA-binding domains—an MBD, which recognizes methylated DNA; and a CXXC3 zinc finger, which binds unmethylated CpG—it is unclear whether these two domains function independently of each other or if they cooperate in facilitating recruitment of MBD1 to particular genomic loci. In this report we investigate DNA-binding specificity of MBD and CXXC3 domains in vitro and in vivo. We find that the methyl-CpG-binding domain of MBD1 binds more efficiently to methylated DNA within a specific sequence context. We identify genes that are targeted by MBD1 in human cells and demonstrate that a functional MBD domain is necessary and sufficient for recruitment of MBD1 to specific sites at these loci, while DNA binding by the CXXC3 motif is largely dispensable. In summary, the binding preferences of MBD1, although dependent upon the presence of methylated DNA, are clearly distinct from those of other methyl-CpG-binding proteins, MBD2 and MeCP2.
-
methyl cpg binding protein MBD1 couples histone h3 methylation at lysine 9 by setdb1 to dna replication and chromatin assembly
Molecular Cell, 2004Co-Authors: Shireen A Sarraf, Irina StanchevaAbstract:In mammals, heterochromatin is characterized by DNA methylation at CpG dinucleotides and methylation at lysine 9 of histone H3. It is currently unclear whether there is a coordinated transmission of these two epigenetic modifications through DNA replication. Here we show that the methyl-CpG binding protein MBD1 forms a stable complex with histone H3-K9 methylase SETDB1. Moreover, during DNA replication, MBD1 recruits SETDB1 to the large subunit of chromatin assembly factor CAF-1 to form an S phase-specific CAF-1/MBD1/SETDB1 complex that facilitates methylation of H3-K9 during replication-coupled chromatin assembly. In the absence of MBD1, H3-K9 methylation is lost at multiple genomic loci and results in activation of p53BP2 gene, normally repressed by MBD1 in HeLa cells. Our data suggest a model in which H3-K9 methylation by SETDB1 is dependent on MBD1 and is heritably maintained through DNA replication to support the formation of stable heterochromatin at methylated DNA.