Methyl-CpG Binding Protein MBD1 Research Articles

PML-RARα/RXR Alters the Epigenetic Landscape in Acute Promyelocytic Leukemia

Many different molecular mechanisms have been associated with PML-RARalpha-dependent transformation of hematopoietic progenitors. Here, we identified high confidence PML-RARalpha binding sites in an acute promyelocytic leukemia (APL) cell line and in two APL primary blasts. We found colocalization of PML-RARalpha with RXR to the vast majority of these binding regions. Genome-wide epigenetic studies revealed that treatment with pharmacological doses of all-trans retinoic acid induces changes in H3 acetylation, but not H3K27me3, H3K9me3, or DNA methylation at the PML-RARalpha/RXR binding sites or at nearby target genes. Our results suggest that PML-RARalpha/RXR functions as a local chromatin modulator and that specific recruitment of histone deacetylase activities to genes important for hematopoietic differentiation, RAR signaling, and epigenetic control is crucial to its transforming potential.

The HP1α–CAF1–SetDB1‐containing complex provides H3K9me1 for Suv39‐mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1alpha (HP1alpha)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1alpha-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1alpha with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

The methyl-CpG binding protein MBD1 is required for PML-RARα function

PML-RARalpha induces a block of hematopoietic differentiation and acute promyelocytic leukemia. This block is based on its capacity to inactivate target genes by recruiting histone deacetylase (HDAC) and DNA methyltransferase activities. Here we report that MBD1, a member of a conserved family of proteins able to bind methylated DNA, cooperates with PML-RARalpha in transcriptional repression and cellular transformation. PML-RARalpha recruits MBD1 to its target promoter through an HDAC3-mediated mechanism. Binding of HDAC3 and MBD1 is not confined to the promoter region but instead is spread over the locus. Knock-down of HDAC3 expression by RNA interference in acute promyelocytic leukemia cells alleviates PML-RAR-induced promoter silencing. We further demonstrate that retroviral expression of dominant-negative mutants of MBD1 in hematopoietic precursors compromises the ability of PML-RARalpha to block their differentiation and thus restored cell differentiation. Our results demonstrate that PML-RARalpha functions by recruiting an HDAC3-MBD1 complex that contributes to the establishment and maintenance of the silenced chromatin state.

DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG

DNA methylation is interpreted by a family of methyl-CpG binding domain (MBD) proteins that repress transcription through recruitment of corepressors that modify chromatin. To compare in vivo binding of MeCP2 and MBD2, we analyzed immunoprecipitated chromatin from primary human cells. Genomic sites occupied by the two MBD proteins were mutually exclusive. As MeCP2 was unable to colonize sites vacated by depletion of MBD2, we tested the hypothesis that methyl-CpG alone is insufficient to direct MeCP2 binding. In vitro selection for MeCP2 bound DNA-enriched fragments containing A/T bases ([A/T] > or = 4) adjacent to methyl-CpG. [A/T] > or = 4 was found to be essential for high-affinity binding at selected sites and at known MeCP2 target regions in the Bdnf and Dlx6 genes. MBD2 binding, however, did not require an A/T run. The unexpected restriction of MeCP2 to a defined subset of methyl-CpG sites will facilitate identification of genomic targets that are relevant to Rett Syndrome.

Transcriptional Repression and Heterochromatin Formation by MBD1 and MCAF/AM Family Proteins

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.

The intracellular domain of teneurin-1 interacts with MBD1 and CAP/ponsin resulting in subcellular codistribution and translocation to the nuclear matrix

Teneurin-1 is a type II transmembrane protein expressed in neurons of the developing and adult central nervous system. To investigate the intracellular signaling of teneurin-1, we searched for proteins interacting with its intracellular domain. One of the proteins identified is the c-Cbl-associated protein CAP/ponsin, an adaptor protein containing SH3 domains. This interaction results on one hand in the recruitment of the soluble intracellular domain of teneurin-1 to the cell membrane enriched in CAP/ponsin. On the other hand, it leads to the translocation of CAP/ponsin to the nucleus, the major site of accumulation of the intracellular domain of teneurin-1. The second interacting protein identified is the methyl-CpG binding protein MBD1. In the nucleus, the intracellular domain of teneurin-1 colocalizes with this transcriptional repressor in foci associated with the nuclear matrix. We propose that these interactions are part of a specific signaling pathway. Evidence for cleavage and nuclear translocation of the intracellular domain has been obtained by the detection of endogenous teneurin-1 immunoreactivity in nuclear speckles in chick embryo fibroblasts. Furthermore, in the nuclear matrix fraction of these cells as well as in cells expressing a hormone-inducible full-length teneurin-1 protein, a teneurin-1 fragment of identical size could be detected as in cells transfected with the intracellular domain alone.

RETRACTED: Methyl-CpG Binding Protein MBD1 Couples Histone H3 Methylation at Lysine 9 by SETDB1 to DNA Replication and Chromatin Assembly

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.

The methyl-CpG binding protein MBD1 interacts with the p150 subunit of chromatin assembly factor 1.

DNA promoter hypermethylation has been shown to be a functional mechanism of transcriptional repression. This epigenetic gene silencing is thought to involve the recruitment of chromatin-remodeling factors, such as histone deacetylases, to methylated DNA via a family of proteins called methyl-CpG binding proteins (MBD1 to -4). MBD1, a member of this family, exhibits transcription-repressive activity, but to this point no interacting protein partners have been identified. In this study, we demonstrate that MBD1 partners with the p150 subunit of chromatin assembly factor 1 (CAF-1), forming a multiprotein complex that also contains HP1alpha. The MBD1-CAF-1 p150 interaction requires the methyl-CpG binding domain of MBD1, and the association occurs in the C terminus of CAF-1 p150. The two proteins colocalize to regions of dense heterochromatin in mouse cells, and overexpression of the C terminus of CAF-1 p150 prevents the targeting of MBD1 in these cells without disrupting global heterochromatin structure. This interaction suggests a role for MBD1 and CAF-1 p150 in methylation-mediated transcriptional repression and the inheritance of epigenetically determined chromatin states.

Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1.

MBD1 is a mammalian protein that binds symmetrically methylated CpG sequences and regulates gene expression in association with DNA methylation. This protein possesses a conserved sequence, named methyl-CpG binding domain (MBD), among a family of methyl-CpG binding proteins that mediate the biological consequences of the methylation. In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the presence of CXXC1, CXXC2, and CXXC3 in MBD1 isoforms v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and -v4. In the present study, we have investigated the significance of MBD, CXXC, and the C-terminal transcriptional repression domain (TRD) in MBD1. A bacterially expressed MBD binds efficiently to densely methylated rather than to sparsely methylated DNAs. In both methylation-deficient Drosophila melanogaster SL2 cells and mammalian CHO-K1 cells, MBD1v1 represses transcription preferentially from both unmethylated and sparsely methylated promoters, while MBD1v3 inhibits densely methylated but not unmethylated promoter activities. The CXXC3 sequence in MBD1v1 is responsible for the ability to bind unmethylated promoter. Furthermore, we have constructed mutant-type MBD1s in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45, and Tyr52 are changed to alanine to investigate the correlation between the structure and function of the MBD in MBD1. Excepting those for Ser45 and Tyr52, none of the recombinant MBD mutants bound to the densely methylated or unmethylated DNAs, and green fluorescent protein-fused MBD1 mutants did not localize properly in the nucleus. All the MBD1v1 and -v3 mutants lost the activity of methylation-dependent gene repression. Based on these findings we have concluded that MBD1 acts as a transcriptional regulator depending on the density of methyl-CpG pairs through the cooperation of MBD, CXXC, and TRD sequences.

Methylation-mediated transcriptional silencing in euchromatin by methyl-CpG binding protein MBD1 isoforms.

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.