Takaya Ichimura

Methyl-CpG Binding Domain 1 (MBD1) Interacts with the Suv39h1-HP1 Heterochromatic Complex for DNA Methylation-based Transcriptional Repression

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.

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.

Involvement of SUMO Modification in MBD1- and MCAF1-mediated Heterochromatin Formation

Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1β and HP1γ. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.

MCAF mediates MBD1-dependent transcriptional repression

ABSTRACT DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

Rta is a transcription factor encoded by BRLF1 of the Epstein–Barr virus (EBV). This factor is expressed during the immediate-early stage of the lytic cycle to activate the genes required for EBV lytic development. Although transcription activation by Rta is frequently associated with the binding of Rta to the Rta-response element (RRE) in promoters, Rta sometimes activates promoters without an RRE. Here we show that Rta interacts with an Sp1-interacting protein, MBD1-containing chromatin-associated factor 1 (MCAF1). This interaction is critical to the formation of an Sp1–MCAF1–Rta complex at Sp1 sites. Therefore, following lytic induction and the expression of Rta, Rta increases Sp1-mediated transcription. The genes that are thus activated include p16, p21, SNRPN and BRLF1. However, the binding of Rta to RRE prevents the interaction between Rta and MCAF1; therefore, transcription activation by RRE depends only on Rta, and not on MCAF1 or Sp1. Furthermore, this study finds that MCAF1 promotes the expression of Rta and Zta from EBV, indicating that MCAF1 participates EBV lytic activation. Our study documents the critical role of Rta in regulating the transcription of the genes that are mediated by Sp1.

Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

The methyl–CpG dinucleotide containing a symmetrical 5-methylcytosine (mC) is involved in gene regulation and genome stability. We report here that methylation-mediated transcriptional repressor methylated DNA-binding domain 1 (MBD1) interacts with methylpurine–DNA glycosylase (MPG), which excises damaged bases from substrate DNA. MPG itself actively represses transcription and has a synergistic effect on gene silencing together with MBD1. Chromatin immunoprecipitation analysis reveals the molecular movement of MBD1 and MPG in vivo: (i) The MBD1–MPG complex normally exists on the methylated gene promoter; (ii) treatment of cells with alkylating agent methylmethanesulfonate (MMS) induces the dissociation of MBD1 from the methylated promoter, and MPG is located on both methylated and unmethylated promoters; and (iii) after completion of the repair, the MBD1–MPG complex is restored on the methylated promoter. Mobility-shift and structural analyses show that the MBD of MBD1 binds a methyl–CpG pair (mCpG × mCpG) but not the methyl–CpG pair containing a single 7-methylguanine (N) (mCpG × mCpN) that is known as one of the major lesions caused by MMS. We further demonstrate that knockdown of MBD1 by specific small interfering RNAs significantly increases cell sensitivity to MMS. These data suggest that MBD1 cooperates with MPG for transcriptional repression and DNA repair. We hypothesize that MBD1 functions as a reservoir for MPG and senses the base damage in chromatin.

MCAF1/AM is involved in Sp1-mediated maintenance of cancer-associated telomerase activity.

Telomerase maintains telomere length and is implicated in senescence and immortalization of mammalian cells. Two essential components for this enzyme are telomerase reverse transcriptase (TERT) and the telomerase RNA component (encoded by the TERC gene). These telomerase subunit genes are known to be mainly expressed by specificity protein 1 (Sp1). MBD1-containing chromatin-associated factor 1 (MCAF1), also known as ATFa-associated modulator (AM) and activating transcription factor 7-interacting protein (ATF7IP), mediates gene regulation, although the precise function of MCAF1 remains to be elucidated. Here, we report that MCAF1 is involved in Sp1-dependent maintenance of telomerase activity in cancer cells. Two evolutionarily conserved domains of MCAF1 directly interact with Sp1 and the general transcriptional apparatus. Selective depletion of MCAF1 or Sp1 down-regulates TERT and TERC genes in cultured cells, which results in decreased telomerase activity. The transcriptionally active form of RNA polymerase II and the general transcription factor ERCC3 decreased in the TERT promoter under the loss of MCAF1 or Sp1. Consistently, MCAF1 is found to be frequently overexpressed in naturally occurring cancers that originate in different tissues. Our data suggest that transcriptional function of MCAF1 facilitates telomerase expression by Sp1, which may be a common mechanism in proliferative cancer cells.

Overlapping Roles of the Methylated DNA-binding Protein MBD1 and Polycomb Group Proteins in Transcriptional Repression of HOXA Genes and Heterochromatin Foci Formation

Methylated DNA binding domain (MBD) proteins and Polycomb group (PcG) proteins maintain epigenetic silencing of transcriptional activity. We report that the DNA methylation-mediated repressor MBD1 interacts with Ring1b and hPc2, the major components of Polycomb repressive complex 1. The cysteine-rich CXXC domains of MBD1 bound to Ring1b and the chromodomain of hPc2. Chromatin immunoprecipitation analysis revealed that MBD1 and hPc2 were present in silenced Homeobox A (HOXA) genes which could be reactivated by knockdown of either MBD1 or hPc2, suggesting that MBD1 and hPc2 cooperate for transcriptional repression of HOXA genes. In the nuclei of HeLa cells, MBD1 existed in close association with these PcG proteins in some heterochromatin foci, whereas an MBD1 mutant lacking the CXXC domains or an hPc2 mutant lacking the chromodomain lost this colocalization in foci. Use of the DNA demethylating agent 5-azadeoxycytidine abolished the formation of MBD1 foci but not PcG foci. Knockdown of MBD1 by small interfering RNAs did not affect the foci containing hPc2 and Ring1b, whereas the MBD1 foci were not influenced by knockdown of hPc2. These indicate that the heterochromatin foci showing MBD1 and hPc2 colocalization arise through the interaction of MBD1 and hPc2 and that the foci of MBD1 are separable from those of the PcG proteins per se. Our present findings suggest that MBD1 and PcG proteins have overlapping roles in epigenetic gene silencing and heterochromatin foci formation through their interactions.

Epigenetic System: A Pathway to Malignancies and a Therapeutic Target

Cancer cells possess both genetic and epigenetic alterations that dysregulate essential cellular processes, leading to disordered cell proliferation and differentiation. Oncogenes and tumor suppressor genes have been found to be activated and inactivated, respectively, in malignant cells. Epigenetic regulation of the genome is mediated by interactions between DNA methylation, chromatin, and modifications of histones and various transcriptional regulators. Recent studies have shown that some components of the epigenetic system as well as epigenetically mutated genes are diagnostic and therapeutic targets in cancer. We discuss the molecular basis of the epigenetic mechanism in association with the development of cancer.