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Mol Cell Biol. Dec 2006; 26(24): 9471–9483.
Published online Oct 9, 2006. doi:  10.1128/MCB.00898-06
PMCID: PMC1698546

This article has been retractedRetraction in: Mol Cell Biol. 2009 April; 29(7): 1987    See also: PMC Retraction Policy

Differential Recruitment of Methylated CpG Binding Domains by the Orphan Receptor GCNF Initiates the Repression and Silencing of Oct4 Expression[down-pointing small open triangle]

Abstract

The pluripotent factor Oct4 is a key transcription factor that maintains embryonic stem (ES) cell self-renewal and is down-regulated upon the differentiation of ES cells and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, are involved in the regulation of Oct4 gene expression. Here we show that the orphan nuclear receptor GCNF initiates Oct4 repression and DNA methylation by the differential recruitment of MBD (methylated CpG binding domain) factors to the promoter. Compared with wild-type ES cells and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in RA-differentiated GCNF−/− ES cells. The Oct4 gene is reexpressed in some somatic cells of GCNF−/− embryos, showing that it has not been properly silenced coincident with reduced DNA methylation of its promoter. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified methyl-DNA binding proteins, MBD3 and MBD2, as GCNF-interacting factors. In P19 and ES cells, upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2. In differentiated GCNF−/− ES cells, recruitment of MBD3 and MBD2 to the Oct4 promoter is lost, and repression of Oct4 expression and DNA methylation fails to occur. RNA interference-mediated knockdown of MBD3 and/or MBD2 expression results in reduced Oct4 repression in differentiated P19 and ES cells. Repression of Oct4 expression and recruitment of MBD3 are maintained in de novo DNA methylation-deficient ES cells (Dnmt3A/3B-null cells), while MBD2 recruitment is lost. Thus, recruitment of MBD3 and MBD2 by GCNF links two events, gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ES cell differentiation.

Embryonic stem (ES) cell self-renewal is maintained by a core of key transcription factors, Oct4, Sox2, and Nanog, which through feed-forward and feedback transcriptional mechanisms maintain pluripotent gene expression (5, 6). However, specific mechanisms are required to disrupt this novel pattern of gene expression upon ES cell differentiation to inhibit the pluripotent phenotype and allow the acquisition of a differentiated cell fate. One of the key questions in the regulation of mammalian transcription is that of how silencing of pluripotency genes is regulated. The Oct4 gene is an excellent transcriptional model for understanding the regulation of pluripotency gene expression because its expression and cis regulation both in the mouse and in ES cells have been well defined (5, 7, 32). Oct4 is a member of the POU family of transcription factors and is one of the most important transcription factors that regulate a pluripotent pattern of gene expression during early embryonic development and ES cell renewal (31-33, 35). Expression of the Oct4 gene is maintained in the blastocyst and epiblast and then is restricted to primordial germ cells in mouse embryos and silenced in all somatic cell lineages (14, 25, 29). Oct4 is also expressed in ES cells and embryonic carcinoma (EC) cells, such as P19 cells (30, 32), and its expression is rapidly down-regulated during differentiation of these cells (7, 14, 36).

The orphan nuclear receptor GCNF has been shown to play a central role in the repression of the Oct4 gene upon differentiation of ES cells through binding to the DR0 element located in the Oct4 proximal promoter (9, 14, 17, 45). GCNF is highly expressed during the gastrula and neurula stages of mouse embryonic development, corresponding to the time when the Oct4 gene is repressed in vivo (8). Inactivation of the GCNF gene in mice by gene targeting results in embryonic lethality due, in part, to the loss of the restricted expression of Oct4 (8, 14, 26). In ES and EC cells, GCNF is transiently induced during early stages of retinoic acid (RA) differentiation, and its expression is subsequently rapidly down-regulated (14, 17, 28). GCNF−/− ES cells fail to turn off Oct4 expression upon differentiation and maintain pluripotent gene expression during RA treatment (17).

Methylation of the DNA in Oct4 gene regulatory regions and histone modifications have been reported to contribute to the silencing of the Oct4 gene during mouse and human ES and EC cell differentiation and embryo development (2, 10, 13, 15, 39, 41). DNA methylation occurs after repression of the Oct4 gene, and loss of DNA methylation and chromatin remodeling have no effects on the repression of the Oct4 gene (13). The regulation of Oct4 DNA methylation is currently not understood. The DNA methylation machinery consists of a family of DNA methyltransferases and a family of methyl-DNA binding domain proteins (MBDs) (3, 19, 20). Two such proteins, MBD2 and MBD3, are closely related to each other in their primary structure and belong to the MeCP1 and NuRD/Mi-2 transcriptional repression complexes, respectively (12, 38, 43, 46). MBD2 binds CpG dinucleotides in a methylation-dependent manner, and MBD2 knockout mice display abnormal maternal methylation patterns (21). In contrast, mammalian MBD3 can bind to unmethylated CpG dinucleotides (23, 34). Inactivation of the MBD3 gene leads to embryonic lethality before gastrulation, and MBD3−/− ES cells maintain pluripotent gene expression in the absence of leukemia inhibitory factor (LIF) (21, 24).

The important question remains, what links the sequence-specific repression initiated by binding of the nuclear receptor GCNF to the Oct4 proximal promoter and epigenetic covalent modifications that lead to gene silencing? To address this question, we investigated the molecular mechanism of Oct4 silencing by GCNF to identify the mediators of its repression activity. Our results demonstrate that the interaction of GCNF with MBD2 and MBD3 during differentiation initiates the repression of the Oct4 gene and DNA methylation by means of sequential recruitment of these novel nuclear receptor corepressors.

MATERIALS AND METHODS

Cell culture and antibodies.

P19 cells were maintained as monolayers in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 100 units of penicillin and streptomycin. Wild-type and GCNF−/− ES cells were cultured on gelatinized tissue culture dishes and maintained in an undifferentiated state with 1,000 units/ml of LIF (Chemicon, Temecula, PA) in the ES cell medium (DMEM supplemented with 15% FCS tested for ES cell culture, 100 mM nonessential amino acids, 2 mM glutamine, 100 U of penicillin-streptomycin/ml [Invitrogen], and 0.55 μM β-mercaptoethanol [Sigma]). For differentiation of ES cells, monolayer-cultured ES cells at low density were treated with 1 μM all-trans-retinoic acid (RA) (Sigma) daily in LIF without ES medium and harvested at different time points. Anti-GCNF antibody was produced by our laboratory. Anti-Myc, antihemagglutinin (anti-HA), anti-Oct4, and anti-MBD3 antibodies were purchased from Santa Cruz (Santa Cruz, CA). Anti-Flag and anti-β-actin antibodies were purchased from Sigma. Anti-MBD2 antibody was purchased from Upstate Biotechnologies.

Bisulfite genomic sequencing.

The genomic DNA from ES cells was extracted with a QIAGEN DNeasy kit (Valencia, CA). The genomic DNA from embryos was extracted by boiling embryos in 20 mM NaOH and then neutralizing with 100 mM Tris-Cl, pH 7.5. Embryos were genotyped as previously reported (8). Purified genomic DNA (500 ng) was denatured and converted with an EZ DNA methylation kit (Zymo Research, Orange, CA). The bisulfite-modified DNA was purified and used as a template for nested PCR. The second-round PCR products were subcloned into the TOPO cloning vector, and individual clones were randomly selected for DNA sequencing with SP6 or T7 primers. The primer sequences are listed in Table S1 in the supplemental material at http://www.nursa.org/retrieveFile.cfm?type=datasets&fileLoc=02006&file=Gu%20et%20al%20Supplementary%20data.pdf.

Yeast two-hybrid screen and assays.

DNA extracted from an amplified 7-day mouse embryo cDNA library in the yeast vector pACT2 (Clontech; catalog no. 638844) was cotransfected with GCNF bait plasmid pGBKT7-GCNF (ligand binding domain [LBD]) into yeast AH109 cells according to the manufacturer's protocols. First-round selection was performed with 7.5 mM 3-amino-1,2,4-triazole (3-AT), and second-round selection was performed with 25 mM 3-AT. Plasmids extracted from putative positive yeast colonies were cotransfected back into yeast cells with the bait plasmid. The interaction was confirmed by colony lift and liquid β-galactosidase (β-Gal) assays according to Clontech's protocol.

GST pull-down and coimmunoprecipitation assays (Co-IP).

Glutathione S-transferase (GST), GST-GCNF, and GST-MBD3b proteins were expressed in Escherichia coli BL21(DE3) and purified with glutathione-agarose beads (Amersham Bioscience). In vitro-translated proteins were labeled with [35S]methionine (ICN Pharmaceuticals) using a TNT T7 in vitro translation kit (Promega) and incubated with equivalent amounts of purified GST- or GST fusion protein-coated glutathione agarose beads in TBST buffer (20 mM Tris-Cl [pH 8.0], 136 mM NaCl, 0.1% Tween-20) and washed with TBST. Transfected or untransfected COS-1 cell total proteins and RA-differentiated P19 cell nuclear proteins were extracted in buffer D (25 mM HEPES [pH 7.9], 150 mM KCl, 0.4 mM EDTA, 2 mM dithiothreitol [DTT], 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1× proteinase inhibitor cocktail [Roche]). Protein A/G-agarose bead slurry (20 μl; Santa Cruz) was incubated with 1 μg of normal immunoglobulin G (IgG) or specific antibodies and 50 μl of cell lysate overnight and washed with TBST buffer. Bound proteins were eluted by boiling in sodium dodecyl sulfate (SDS) loading buffer and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Western blots were used to detect proteins with specific antibodies and visualized using the ECL system (Amersham Bioscience).

ChIP and Western blots.

Undifferentiated or RA-differentiated P19 or ES cells were cross-linked with 1% formaldehyde (Sigma) and soluble chromatin was extracted and sonicated according to the protocol provided by Upstate Biotechnology (Lake Placid, NY). Protein expression prior to chromatin immunoprecipitation (ChIP) assays was detected by Western blot analysis according to the protocol provided by Amersham Pharmacia (RPN2209). Sonicated chromatin proteins (250 μg) were incubated with 0.5 to 2 μg of various antibodies or normal IgGs and immunoprecipitated with protein A/G agarose beads (Santa Cruz). The bound DNA was eluted by incubation with SDS-proteinase K solution overnight at 65°C and extracted with phenol-CHCl3. PCR was performed as described previously (17).

siRNA treatment.

P19 cells were seeded (1 × 105) in six-well plates 1 day before transfection. Each small interfering RNA (siRNA) duplex (20 nM) was transfected with 5 μl of Lipofectamine 2000 (Invitrogen) in 2 ml of medium per well. RA was added to a final concentration of 1 μM, and plates were incubated for the indicated time points. siRNA (80 nM) was transfected with 10 μl of Lipofectamine 2000 into 2 ml of a 5 × 105 ES cell suspension in six-well plates, and plates were incubated for 24 or 40 h in ES cell medium supplemented with 1 × 104 units of LIF or 1 μM RA. Triplicate samples were combined. Total protein was extracted with 100 μl of 1× passive lysis buffer (Promega), and the amount of protein was determined using a Bio-Rad protein assay kit. Total RNA was extracted with Trizol reagent (Invitrogen) and in vitro reverse transcribed into cDNA (Clontech Advantage RT kit). The efficiency of knockdown and expression of GCNF and Oct4 was determined by using PCR and Western blots. Sequences of siRNA were designed according to the asymmetry rule (37) and synthesized by Dharmacon (Lafayette, CO). Sequences of siRNAs and PCR primers are described in Table S1 in the supplemental material (see above).

Whole-mount in situ hybridizations, RT-PCR, and 32P-labeled PCR.

Embryos from matings between GCNF heterozygous mice were harvested between embryonic day 8.5 (E8.5) and E8.75 and fixed in 4% paraformaldehyde. Whole-mount in situ hybridizations were carried out as described previously (16, 17). Total RNA was extracted from ES and P19 cells with the Trizol reagent (Invitrogen) and reverse transcribed into cDNA with an Advantage RT PCR kit (Clontech, Mountain View, CA). Semiquantitative PCR was performed with Taq DNA polymerase (see Fig. S1 in the supplemental material [http://www.nursa.org/retrieveFile.cfm?type=datasets&fileLoc=02006&file=Gu%20et%20al%20Supplementary%20data.pdf]). 32P-labeling PCR was performed under similar PCR conditions but at reduced cycle numbers. dATP, dTTP, dGTP, and [α-32P]dCTP were used as the four deoxynucleoside triphosphate substrates in 32P-labeled PCR. The 32P-labeled PCR products were resolved in 4 to 5% polyacrylamide gels in 0.5× Tris-borate-EDTA buffer. The radioactive signals were captured by direct exposure of dried gels to phosphorimage systems and quantitated with the ImageQuant 5.2 program (Molecular Dynamics).

RESULTS

Hypomethylation of the Oct4 promoter in differentiated GCNF−/− ES cells.

Repression of the Oct4 gene is lost in GCNF−/− ES cells induced to differentiate with RA (Fig. (Fig.1B)1B) (17). DNA methylation is involved in the process of Oct4 gene silencing in RA-induced EC and ES cell differentiation and in somatic cells (2, 15). Thus, we analyzed the DNA methylation profile of the Oct4 gene during RA-induced differentiation in GCNF−/− ES cells compared to wild-type (WT) cells. The methylation status of 16 CpG sites in the Oct4 gene between −562 and the translation start site was scanned at different time points of RA treatment by bisulfate treatment, PCR, and DNA sequencing (Fig. (Fig.1A).1A). In undifferentiated WT ES cells, the CpG sites maintained an unmethylated status. The onset of DNA methylation could be clearly detected when the WT ES cells were treated with RA for 3 days. Upon treatment for 4 days, more than 50% of the CpG dinucleotides were methylated, and the methylation status was maintained between days 4 and 6. In the GCNF−/− ES cells, the CpG sites were also unmethylated in the undifferentiated state. Interestingly, on day 3 of RA treatment, CpG methylation was not initiated in the GCNF−/− ES cells, and even at day 6, less than 10% of CpG dinucleotides were methylated. The DNA methylation analysis clearly showed that the proximal promoter of the Oct4 gene was hypomethylated in GCNF−/− ES cells. When we compared the GCNF expression profile (Fig. (Fig.1B)1B) and previous DNA binding studies (17) with the quantitated CpG methylation profile (Fig. (Fig.1C),1C), it was clear that methylation of the Oct4 promoter occurred concomitantly with the period of elevated GCNF expression and DNA binding activity, between days 1 and 3 of RA treatment in WT ES cells. Although Oct4 repression was detectable as early as day 2 of RA treatment (Fig. (Fig.1B;1B; see also Fig. S2 in the supplemental material [http://www.nursa.org/retrieveFile.cfm?type=datasets&fileLoc=02006&file=Gu%20et%20al%20Supplementary%20data.pdf]), there was no significant (P < 0.1) difference in percent methylated CpG dinucleotides between the WT and GCNF−/− ES cells. After 3 days of differentiation, the percent methylation in GCNF−/− ES cells was significantly lower (P < 0.01) than that in WT ES cells. At later time points of differentiation, greater significant differences were maintained (P < 0.0001) between WT and GCNF−/− ES cells.

FIG. 1.
Hypomethylation of the Oct4 promoter in differentiated GCNF−/− ES cells. (A) DNA methylation profile of 16 CpG sites located in the Oct4 proximal promoter from −562 to the ATG start codon during differentiation induced by RA for ...

Hypomethylation of the Oct4 promoter in GCNF−/− embryos.

Previous studies demonstrated that DNA methylation of the Oct4 gene took place by 6.5 days postcoitus (dpc) in normal mouse embryos (15, 44). There was loss of repression of the Oct4 gene in many of the somatic cells in 8.5- to 8.75-dpc GCNF−/− embryos (14). Thus, the DNA methylation status of the Oct4 promoter was analyzed in GCNF−/− embryos at 8.5 and 9.5 dpc. As expected, the Oct4 promoter was heavily methylated (40% overall) in WT embryos, and DNA methylation was also observed in the GCNF−/− embryos (overall, 20% at 8.5 dpc and 30% at 9.5 dpc) (Fig. 2A and B). However, when the Oct4 promoter was divided into two parts—the region proximal to the GCNF DR0 element (from the first CpG to the seventh CpG) and the region distal to the DR0 (from the eighth to the sixteenth CpG)—significant differences in DNA methylation levels in the proximal region close to DR0 between WT and GCNF−/− embryos at both 8.5 dpc and 9.5 dpc were observed (Fig. (Fig.2C).2C). In contrast, there was no difference in the region distal to DR0 at 9.5 dpc between WT and GCNF−/− embryos. These results demonstrated that hypomethylation of the Oct4 promoter also occurred in GCNF−/− embryos in a region spanning the transcriptional start site, close to the GCNF binding site. The hypomethylation of the Oct4 gene suggested that it was not properly silenced in the GCNF−/− embryos, which was observed. Whole-mount in situ analysis showed that the Oct4 gene is silenced in somatic cells of WT embryos (Fig. (Fig.2C,2C, panels a and b) and there is widespread loss of repression in somatic cells of the GCNF−/− embryos. Careful analysis of the expression of Oct4 in GCNF−/− embryos at multiple time points indeed showed that some of the Oct4 expression observed at the five-somite stage, compared to the two-somite stage, was due to reexpression of Oct4 in the neuroepithelium (Fig. (Fig.2C,2C, panels c and d) as opposed to loss of repression, which is observed in other regions of the embryo. Reexpression of the Oct4 gene in the neuroepithelium spatially matched the expression pattern of LRH-1, which was previously shown to regulate Oct4 expression (Fig. (Fig.2C,2C, panels e and f) (16). These results supported the contention that loss of GCNF binding and DNA methylation leads not only to loss of repression of Oct4 but also to loss of silencing, making it possible for a transcriptional activator, like LRH-1, to reverse Oct4 gene repression and reactivate the gene.

FIG. 2.
Hypomethylation of the Oct4 promoter in GCNF−/− embryos. (A) DNA methylation profile of 16 CpG sites located in the Oct4 proximal promoter from −521 to the ATG start codon in WT and GCNF−/− (Mu) embryos at 8.5 dpc ...

Identification of GCNF-interacting factors that mediate Oct4 repression.

The hypomethylation of the Oct4 promoter in GCNF−/− ES cells and embryos suggested that GCNF-mediated repression was probably directly or indirectly linked to DNA methylation of the promoter. To address this hypothesis, we set out to identify mediators of GCNF repression of target gene expression. We carried out a yeast two-hybrid screen to identify GCNF-interacting partners. More than 3 × 106 independent mouse E7.0 embryo cDNA clones were screened with a Gal4 activation domain (Gal4-AD)-GCNF-LBD fusion protein as bait. The screen identified several groups of positive colonies after two rounds of selection. One group of cDNAs that produced positive colonies shared identical sequences and encoded the short form of mouse MBD3b. Another group of cDNAs encoded partial sequences for the corepressor NCoR, which was expected based on previous reports of interaction between these two factors (14). To confirm the interaction, a Gal4-DBD-MBD3b prey plasmid was cotransfected with a Gal-AD-GCNF bait plasmid and analyzed using colony-lift and β-Gal liquid assays. The results showed that yeast colonies cotransfected with GCNF and MBD3b vectors turned blue on synthetic dropout selection plates (data not shown). In the liquid assays, Gal4-GCNF-dependent activation of β-Gal reporter activity was augmented by MBD3b cotransfection, to levels comparable to that observed with NCoR cotransfection (Fig. (Fig.3A),3A), which confirmed the interaction between GCNF and MBD3b. GST pull-down assays further confirmed a specific interaction between MBD3b and GCNF in vitro (Fig. (Fig.3B).3B). E. coli-expressed GST-GCNF pulled down in vitro-translated and radiolabeled MBD3b, while GST alone did not pull down either MBD3b or GCNF. As a positive control, in vitro-translated NCoR was also pulled down by GST-GCNF. As expected, in vitro-translated RXR did not show any interaction with GST-GCNF, establishing a negative control.

FIG. 3.
Interaction of GCNF with MBD3 in vitro. (A) Interaction of GCNF and MBD3 was detected by liquid β-Gal assay in a yeast two-hybrid system. Gal4-NCoR was used as a positive control, and Gal4 DBD and Gal4 AD empty vectors were used as negative controls. ...

The association of MBD3b with GCNF was further corroborated in a mammalian system using Co-IP assays (Fig. (Fig.3C).3C). When Myc-tagged GCNF and Flag-tagged MBD3a (full-length MBD3) or MBD3b were coexpressed in COS-1 cells by transient transfection, anti-Myc antibody could coimmunoprecipitate the Flag-MBD3a or Flag-MBD3b (Fig. (Fig.3C,3C, lanes 5 and 6, Co-IP-GCNF). Similarly, anti-Flag antibody could coimmunoprecipitate Myc-GCNF (Fig. (Fig.3C,3C, lanes 5 and 6, Co-IP-MBD3). In the untransfected COS-1 cells (Fig. (Fig.3C,3C, lane 1) and singly transfected COS-1 cells (Fig. (Fig.3C,3C, lanes 2 to 4), no Co-IP signal was observed even though expression of the transfected protein was detected in the input and single immunoprecipitates. These results established that in mammalian cells, GCNF could associate with MBD3.

GCNF, however, is not expressed in COS1 cells; rather, it is transiently expressed in differentiating P19 and ES cells (14, 17). GCNF is induced to maximal levels after 36 h of RA treatment in P19 cells (14). Therefore, the association of endogenous GCNF and MBD3 was assayed in differentiating P19 cell nuclear extracts (Fig. (Fig.3D).3D). We found that the two forms of MBD3 (MBD3a and MBD3b) are expressed in P19 cells (Fig. (Fig.3D,3D, lanes 1 and 2 and lanes 5 and 6), a fraction of which is associated with GCNF and could be coimmunoprecipitated with anti-GCNF antibody (lanes 7 and 8). Under the same Co-IP conditions, no signal was detected when regular IgGs were used in the place of the anti-GCNF antibody. The Co-IP results established that endogenous GCNF and MBD3 interact in differentiating P19 cells.

GCNF interacts with a subset of the MBD family via the MBD domain.

The in vitro and in vivo experiments to this point confirmed a protein-protein interaction between GCNF and MBD3. As expected, the yeast two-hybrid assays defined the GCNF LBD as the MBD3-interacting domain. To define the interaction domain in MBD3, several N- and C-terminal deletions were generated (Fig. (Fig.4A),4A), and their interaction with GCNF was analyzed by GST pull-down assays (Fig. (Fig.4B).4B). Deletion of the extreme C terminus of MBD3, including the poly-glutamic acid domain and the coiled-coil motif (deletions 3 and 4), did not affect the interaction of MBD3 with GCNF (Fig. (Fig.4B,4B, lanes 5 and 6). However, when the MBD domain was completely deleted (deletion 1) or partially deleted (deletion 2), the interaction between GCNF and MBD3 was either lost (Fig. (Fig.4B,4B, lane 3) or considerably weakened (lane 4). The deletion experiments showed that the GCNF interaction domain in MBD3 overlapped with the methyl-DNA binding domain (amino acids 38 to 91).

FIG. 4.
GCNF-LBD interacts with methyl-DNA binding domains of MBD proteins. (A) Illustration of the MBD3 deletions generated and analyzed. The amino acid numbers are shown. The black box denotes the methyl-DNA binding domain (MBD), and the open box denotes the ...

The MBD3 gene is part of a discrete family of genes which includes genes for MeCP2 and MBD1, -2, and -4 (19). Alignment of the MBD domains of MBD1, -2, -3, and -4 and MeCP2 revealed that MBD3 shared the highest amino acid sequence homology with MBD2 in the GCNF interaction region (especially between amino acids 38 and 70) and relatively low homology with MBD1, MBD4, and MeCP2 in the same region (19) (Fig. (Fig.4C).4C). The interaction of GCNF with MBD2 was analyzed by yeast two-hybrid and GST pull-down assays. We found that GCNF also interacted with in vitro-translated MBD2 but not MBD1 (Fig. (Fig.4D),4D), and the interaction between GCNF and MBD2 was recapitulated in yeast as well (Fig. (Fig.4E).4E). The interaction of MBD2 with GCNF was further verified by Co-IP in mammalian cells (Fig. (Fig.4F).4F). In cotransfected COS-1 cells, anti-HA antibody (specific for HA-GCNF) coimmunoprecipitated Myc-MBD2, and anti-Myc antibody (specific for Myc-MBD2) also coimmunoprecipitated HA-GCNF. Thus, GCNF can interact with a subset of MBD family members, specifically, MBD3 and MBD2, via the MBD domain.

GCNF-dependent recruitment of MBD2 and MBD3 to the Oct4 promoter during P19 cell differentiation.

To explore the physiological significance of the interactions between GCNF and MBD2 and MBD3, repression of Oct4 was studied in P19 cells. The expression of GCNF, Oct4, MBD2, and MBD3 was detected during RA treatment of P19 cells by RT-PCR and Western blot analysis. As shown in Fig. Fig.5A5A (see also Fig. S3 and Fig. S4 [http://www.nursa.org/retrieveFile.cfm?type=datasets&fileLoc=02006&file=Gu%20et%20al%20Supplementary%20data.pdf]), GCNF protein was maximally expressed after 36 h of RA treatment (36-fold induction), coinciding with the dramatic repression of Oct4, which was determined to be an 80% decrease at that time point. Both RNA and protein levels for MBD3 were maintained at constant levels during RA-induced differentiation, while the RNA level of MBD2 slightly increased with RA treatment (Fig. (Fig.5A;5A; also Fig. S4). The direct binding of MBD2 and MBD3 to the Oct4 promoter in P19 cells was detected using ChIP assays (Fig. 5B and C). The analyzed region spanned the GCNF binding site DR0 from the first to seventh CpG sites (proximal to the DR0 region). Binding of MBD3 to the Oct4 promoter in vivo occurred with the same RA-induced temporal pattern as that observed with GCNF. Both factors showed stronger binding after 36 h of RA treatment and decreased binding after 72 h of treatment. When 32P-labeling PCR results were quantitated (Fig. (Fig.5C),5C), they clearly showed that binding of GCNF and MBD3 to the Oct4 proximal promoter occurred at 36 h of RA treatment and were enriched fivefold and threefold, respectively, over background levels observed at time zero. Similar to MBD3, the binding of MBD2 to the Oct4 promoter was not detected in undifferentiated P19 cells. In contrast to MBD3, MBD2 binding to the Oct4 promoter was minimal at 36 h of RA treatment. Interestingly, recruitment of MBD2 to the Oct4 promoter was delayed to 72 h of RA treatment, with approximately a fivefold increase in binding over background. At this time point, the binding of GCNF had decreased somewhat. These results demonstrated that endogenous GCNF and MBD2 and MBD3 were sequentially recruited to the Oct4 promoter upon differentiation of P19 cells.

FIG. 5.
Recruitment of MBD2 and MBD3 to the Oct4 promoter and decrease of Oct4 repression in differentiated P19 cells treated with MBD2 and MBD3 siRNA. (A) Expression of GCNF, Oct4, MBD2, and MBD3 in differentiated P19 cells was detected by RT-PCR and Western ...

To address the role of MBD2 and MBD3 in Oct4 repression we used siRNA to knock down their expression in P19 cells. Expression of endogenous MBD2 and MBD3a and -3b was significantly reduced after treatment with specific siRNAs for 24 and 36 h in undifferentiated P19 cells P19 cells that had undergone RA-induced differentiation when cells were transfected with MBD2 siRNA (siMBD2) alone (Fig. (Fig.5D,5D, lanes 2, 6, and 10) and siMBD3 alone (lanes 3, 7, and 11) or with siMBD2/siMBD3 cotransfection (lanes 4, 8, and 12). According to quantitative data analysis of the results (Fig. S5 [http://www.nursa.org/retrieveFile.cfm?type=datasets&fileLoc=02006&file=Gu%20et%20al%20Supplementary%20data.pdf]), single siMBD3 treatment resulted in a 70 to 90% loss of MBD3 expression at the RNA and protein levels. The decrease in MBD2 levels was difficult to estimate due to weak signals in the Western blot; however, expression of MBD2 RNA was reduced 70 to 87% after transfection of siMBD2. RT-PCR and Western blots showed that MBD siRNA treatments did not affect the induction of endogenous GCNF or the decrease of endogenous SF-1, which activates Oct4 expression in undifferentiated P19 cells (1, 14). As expected, after 24 h of RA treatment, expression of GCNF was induced, with a concomitant decrease in the expression of SF-1 and Oct4. Neither single nor double MBD siRNA transfection significantly altered the expression of Oct4 in undifferentiated P19 cells, which is not surprising since although both MBD2 and MBD3 are expressed, the results of the ChIP assays showed that they were not recruited to the Oct4 promoter in undifferentiated P19 cells (Fig. (Fig.5D,5D, lanes 1 to 4). Significantly, siMBD3 alone or combined with siMBD2 obviously impaired the repression of Oct4 in differentiated P19 cells at 24 h of RA treatment (Fig. (Fig.5D,5D, lanes 7 and 8). However, siMBD2 alone had no significant effect on the Oct4 expression at this time point (Fig. (Fig.5D,5D, lane 6). After 36 h of RA treatment, knockdown of MBD2 and MBD3 expression by siRNA treatment was still significant, but reduced repression of Oct4 was not as obvious as that at 24 h of treatment, and only simultaneous knockdown of MBD2 and MBD3 caused reduced Oct4 repression (lane 12). Quantitation of the expression data (Fig. (Fig.5E)5E) clearly showed that Oct4 was dramatically reduced with the treatment of RA (90% at 24 h and 96% at 36 h, compared to undifferentiated levels). Treatment with siMBD2 alone did not affect Oct4 levels at 24 h (Fig. (Fig.5E,5E, lanes 5 and 6); meanwhile, it caused a 2.5-fold increase of Oct4 after 36 h of treatment (lanes 9 and 10). Treatment with siMBD3 alone reduced Oct4 repression fivefold at 24 h (Fig. (Fig.5E,5E, lanes 5 and 7); however, this effect was significantly reduced after 36 h of treatment, resulting in threefold-reduced Oct4 repression compared to controls (lanes 9 and 11). The effect of combined siMBD2 and siMBD3 treatment was similar to that of the siMBD3 single treatment at 24 h (fivefold reduction in Oct4 repression; Fig. Fig.5E,5E, lanes 5 and 8), and this treatment was more effective than single treatment at 36 h (fivefold-reduced Oct4 repression; Fig. Fig.5E,5E, lanes 9 and 12). The quantitative data of RT-PCR and Western blotting also showed similar changes in Oct4 RNA and protein levels during siRNA treatment (see Fig. S5 at the URL given above). These observations demonstrated that significant effects of MBD3 siRNA knockdown on Oct4 repression could be detected only between 24 and 36 h, because of technical limitations, such as dilution of the siRNA, while MBD2 knockdown alone did affect Oct4 expression because its recruitment occurs later than MBD3, at a stage when Oct4 repression can no longer be affected, due to the loss of expression of the activator SF-1 during P19 cell differentiation and the ability to maintain Oct4 expression (14).

GCNF-dependent recruitment of MBD2 and MBD3 to the Oct4 promoter during ES cell differentiation.

The results from ChIP assays and siRNA treatments indicated that the effect of MBD2 or MBD3 on Oct4 repression was dependent on GCNF expression and binding to the Oct4 promoter. These observations raised the question of whether GCNF is the critical component in the recruitment of MBD2 and MBD3 to the Oct4 promoter. To address this question, WT and GCNF−/− ES cells were utilized to compare the binding of GCNF, MBD2, and MBD3 to the Oct4 gene in vivo. The expression of MBD2, MBD3a, and MBD3b at the RNA and protein levels was not significantly altered upon RA-induced differentiation in either WT or GCNF−/− ES cells (Fig. (Fig.6A;6A; also Fig. S3 and Fig. S5 [see above]). As expected, GCNF expression was up-regulated (24-fold induction) and Oct4 expression was down-regulated (90% loss of Oct4 protein) by the treatment of ES cells with RA; however, in GCNF−/− ES cells, the repression of Oct4 was lost.

FIG. 6.
GCNF recruits MBD2 and MBD3 to bind to the Oct4 promoter in ES cells. (A) Expression of GCNF, Oct4, MBD2, and MBD3 was detected in differentiated WT and GCNF−/− ES cells by RT-PCR and Western blotting. (B) Binding of GCNF, MBD2, and MBD3 ...

The direct binding of MBD2 and MBD3 to the Oct4 promoter in WT and GCNF−/− ES cells was analyzed by ChIP assays (Fig. (Fig.6B).6B). To quantitatively estimate the binding signals of GCNF, MBD2, and MBD3 in WT and GCNF−/− ES cells, the ChIP samples were analyzed by 32P-labeled PCR (Fig. (Fig.6B)6B) and the results were plotted (Fig. (Fig.6C).6C). To validate the ChIP samples prepared from GCNF−/− ES cells, another nuclear receptor, LRH-1, was used as a control to check its binding to Oct4 promoter (16). Binding of LRH-1 to the Oct4 promoter was clearly detected in GCNF−/−, similar to observations made with WT ES cells under the control of LIF. In undifferentiated ES cells, no GCNF, MBD2, or MBD3 binding was detected, as expected. Upon differentiation, GCNF and MBD3 binding was enriched at the Oct4 promoter after 40 h of RA treatment in WT ES cells (sevenfold and fivefold increases in DNA binding were detected, respectively). In contrast to P19 cells, at the 72-hour RA differentiation time point, GCNF protein was still detected (Fig. (Fig.6A,6A, lanes 3 and 9) and bound to the Oct4 promoter (Fig. (Fig.6B,6B, lanes 3 and 9); meanwhile, MBD2 replaced MBD3 at the Oct4 promoter, displaying a ninefold increase in DNA binding. As expected, binding of GCNF to the Oct4 promoter was lost in the GCNF−/− ES cells, which, significantly, resulted in loss of recruitment of both MBD2 and MBD3 to the Oct4 promoter (Fig. (Fig.6B).6B). Thus, GCNF is essential for the recruitment of MBD2 and MBD3 to the Oct4 promoter.

To investigate the role of the interaction of GCNF with MBD2 and MBD3 in ES cells, undifferentiated or RA-differentiated cells were treated with siRNA specific for MBD2 or MBD3 for 24 and 40 h (Fig. (Fig.6D).6D). The relative expression level of Oct4 was quantitated (Fig. (Fig.6E),6E), and in the WT ES cells, GCNF was induced and Oct4 was repressed by RA treatment (Fig. (Fig.6D,6D, lanes 1, 4, and 7). Oct4 expression dropped to 30% at 24 h and 20% at 40 h of RA induction in the absence of siRNA treatment (Fig. (Fig.6E,6E, lanes 1, 4, and 7). MBD2 and MBD3 were specifically and significantly knocked down by the treatment with siRNA (Fig. (Fig.6D,6D, lanes 2, 5, and 8 and lanes 3, 6, and 9). The efficiency of siRNA knockdown in ES cells was determined to be 75 to 90% for MBD2 or MBD3 RNA upon treatment with siMBD2 or siMBD3 (Fig. S5). As expected, in undifferentiated ES cells, knockdown of MBD2 and/or MBD3 did not affect Oct4 expression (Fig. (Fig.6E,6E, lanes 1, 2, and 3). After 24 h of cotreatment with siMBD2 and RA, the expression of Oct4 was reduced but there was no significant difference between the control (Fig. (Fig.6E,6E, lane 4) and siRNA treatment (Fig. (Fig.6E,6E, lane 5). Interestingly, when the treatment of siMBD2 and RA was extended to 40 h, the repression of Oct4 was significantly impaired and a 2.8-fold decrease in the repression of Oct4 was observed, relative to the control (Fig. (Fig.6E,6E, lanes 7 and 9). The effect of siMBD3 in inhibiting Oct4 repression was much more efficient than siMBD2 treatment at 24 h and 40 h. About a threefold decrease in Oct4 repression was achieved with the treatment of siMBD3 (Fig. (Fig.6E,6E, compare lanes 4 and 6 and lanes 7 and 9). We have attempted to knock down both MBD2 and MBD3 in ES cells, as in P19 cells; however, ES cells are so sensitive to the transfection reagent, Lipofectamine 2000, that they failed to grow normally with the higher concentration of transfection reagent used to match the doubled siRNA levels. Thus, transient decrease of MBD2 and MBD3 expression in differentiating ES cells reduced Oct4 repression. The time point corresponded with the GCNF expression peak in RA-induced ES cells.

Recruitment of MBD3 is independent of de novo DNA methylation.

According to the DNA methylation profile in WT ES cells, de novo Oct4 DNA methylation lagged behind the repression by GCNF (Fig. (Fig.1).1). To address the question of whether the differential recruitment of MBD3 and MBD2 is dependent on DNA methylation, we analyzed the repression and DNA methylation of the Oct4 gene in de novo DNA methylation-deficient ES cells, i.e., Dnmt3A−/− Dnmt3B−/− ES cells. Although DNA methylation is completely lost at day 6 of RA treatment in Dnmt3A−/− Dnmt3B−/− ES cells (Fig. (Fig.7A),7A), repression of the Oct4 gene maintained the temporal pattern seen in WT ES cells, which was mediated by induced expression of GCNF in these cells (Fig. (Fig.7B).7B). Western blot assay further confirmed the maintenance of Oct4 repression and the induction of GCNF and expression of MBD3 and MBD2 in the Dnmt3A−/− Dnmt3B−/− ES cells (Fig. (Fig.7C;7C; quantitated in Fig. S3 [see above]). Binding of GCNF, MBD2, and MBD3 to the Oct4 promoter was confirmed at various time points between 0 and 3 days of RA treatment; interestingly, neither MBD2 nor MBD3 is bound to the Oct4 promoter at day 6 (Fig. 7D and E). GCNF expressed in the Dnmt3A−/− Dnmt3B−/− ES cells bound to the Oct4 promoter in the same pattern as in WT ES cells. Interestingly, recruitment of MBD2 to the Oct4 promoter was lost in the Dnmt3A−/− Dnmt3B−/− ES cells; however, the GCNF-dependent binding of MBD3 was maintained with the same pattern in Dnmt3A−/− Dnmt3B−/− ES cells as in WT ES cells. These results demonstrated that the recruitment of MBD3 was independent of de novo DNA methylation but required GCNF and that such recruitment was necessary for Oct4 repression. In contrast, MBD2 recruitment was dependent on both CpG methylation and GCNF recruitment.

FIG. 7.
Recruitment of MBD3 is independent of Dnmt3A and Dnmt3B in ES cells. (A) Loss of Oct4 DNA methylation in Dnmt3A−/− Dnmt3B−/− ES cells after RA differentiation for 6 days. (B) Repression of Oct4 gene in differentiated ES ...

DISCUSSION

In this paper we demonstrate that repression of the Oct4 gene is mediated by recruitment of the novel nuclear receptor corepressors MBD3 and MBD2 to the Oct4 promoter via direct interactions with the orphan receptor GCNF in mouse ES cells in which differentiation was induced by RA. The recruitment of MBD3 to the Oct4 promoter by GCNF is concomitant with binding of GCNF to the DR0 in the Oct4 promoter (36 to 40 h), and MBD3 is subsequently replaced by MBD2 (72 h). The time course of differential recruitment of MBD2 and MBD3 to the Oct4 promoter correlates with the DNA methylation status of the promoter (Fig. (Fig.11 and and2).2). The temporal recruitment of MBD2 and MBD3 to the Oct4 promoter is also consistent with the differential effects of siMBD2 and siMBD3 on Oct4 repression (Fig. (Fig.55 and and6).6). Although MBD2 and MBD3 are structurally closely related to each other, the knockout mouse models for each gene clearly established that they are not functionally redundant. MBD2 was shown to bind methylated DNA and interact with MBD3 in the NuRD repression complex (40, 46) or with Sin3A in the Sin3A repression complex (4). The MBD3 knockout mouse model and MBD3−/− ES cells demonstrate important roles for the MBD3-NuRD complex during early embryonic development and ES cell differentiation, respectively (21, 24). The dependence of MBD2 and independence of MBD3 recruitment to the Oct4 promoter on DNA methylation underscores the mechanistic differences of these factors and their complexes. GCNF-dependent MBD3 recruitment to the Oct4 promoter is still observed in the Dnmt3A−/− Dnmt3B−/− ES cells, showing that it is independent of DNA methylation. In contrast, recruitment of MBD2 in these cells is lost, and thus, stable recruitment of MBD2 to the Oct4 promoter is dependent on both GCNF and DNA methylation. The differential dependence of MBD3 and MBD2 on DNA methylation may provide a rationale for the significant differences observed in the phenotypes of the two knockout mouse models. One could speculate that because MBD3 is important for the repression of genes such as the Oct4 gene, its inactivation would result in dramatic defects due to loss of repression of target genes. In contrast, inactivation of MBD2 would have less acute effects, because it appears to be involved in the silencing of genes rather than their repression.

Our findings clearly define a role for MBD3 at the molecular level in the repression of Oct4 expression during ES cell differentiation. The delay between Oct4 repression and DNA methylation also indicates that Oct4 repression occurs at the unmethylated stage, in which GCNF binding and interaction with MBD3 take place. The DR0-specific binding of GCNF to its target gene, Oct4, leads to recruitment of MBD3, which has the ability to bind to unmethylated CpG dinucleotides. Oligomerization of GCNF in differentiated P19 and ES cells likely accelerates the recruitment of MBD3 and spreading of the repression complex throughout the Oct4 promoter (18). Such promoter occupation by GCNF and MBD3 may protect the DNA from transactivator binding (e.g., LRH-1) and facilitate subsequent histone deacetylation, DNA methylation and recruitment of MBD2, leading ultimately to gene silencing (13). Impairment of Oct4 repression by siMBD2 occurred only at later stages, when DNA methylation had been initiated (36 to 40 h), which means that binding of MBD2 to the Oct4 promoter is involved not in the initiation of Oct4 repression but rather in downstream events such as Oct4 silencing. Thus, direct recruitment of MBD2 and MBD3 represents a novel repression model for nuclear receptors and may link epigenetic modification to gene-specific repression by nuclear receptors. Our results are also consistent with recent observations showing that MBD2 and MBD3 belong to distinct NuRD repression complexes, which agrees with the distinct knockout phenotypes observed for MBD2 and MBD3 (27). The Oct4 gene represents an excellent model system to understand the differential functions of MBD2 and MBD3 during gene specific repression and GCNF plays a pivotal role in initiating and coordinating the Oct4 gene silencing in RA-induced differentiated ES cells.

The utilization of MBD family members as corepressors of nuclear receptor function in a pathological state has been recently described. Analysis of the oncogenic function of a PML-RAR chimera in leukemogenesis showed that it involved aberrant gene silencing that was mediated, in part, by MBD1. Di Croce's group established a model in which the oncogenic transcription factor PML is fused with the nuclear receptor RAR to form a PML-RAR chimera in the hematopoietic precursor cell U937-PR9 (11, 42). In that model system, induction of the PML-RAR fusion protein directly recruits DNA methyltransferase Dnmt1 and/or Dnmt3A to the RAR target gene, the RARβ2 promoter, and caused its hypermethylation. The RAR moiety of the fusion protein interacts with the C-terminal part of MBD1 to recruit it to methylated CpG sites around the RARE sites in the RARβ2 promoter; however, the interaction is indirect, as it requires HDAC3 as a bridge. Recruitment of Dnmts and association with MBD1 take place simultaneously with repression and DNA methylation of the RARβ2 gene. GCNF-induced Oct4 repression and DNA methylation are mechanistically different from the PML-RAR chimera-dependent repression of RARβ2 via DNA methylation. In our model, which represents a normal physiological function, GCNF directly interacts with MBD3 and MBD2 via the MBD domain and the recruitment of MBD3 and MBD2 to the Oct4 promoter by GCNF occurs progressively. The GCNF-dependent repression and DNA methylation of Oct4 occur normally during ES cell differentiation and embryonic development.

The repression and silencing of a gene constitute a step process. Various epigenetic covalent modifications (DNA and histone methylation) are required to repress and silence the Oct4 gene (10, 15, 22, 39, 41). Each modification requires that specific factors and complexes be brought to the promoter to facilitate efficient repression. Loss of any one of these factors or failure to recruit the repressor complex will likely lead to loss of proper silencing of the gene; however, repression of Oct4 can still be observed (13, 22). Cases in point are Dnmt3A and -3B, which are clearly required for the de novo methylation of the Oct4 gene and its silencing (Fig. (Fig.6)6) (13, 22); however, Oct4 expression is still repressed when the double-knockout ES cells are treated with RA. The same phenomenon occurs in G9α−/− ES cells (13). G9α is a histone methyltransferase that has been implicated in the silencing of the Oct4 gene (14). Repression of Oct4 still occurs in these mutant ES cells, most likely because GCNF is still expressed and is able to recruit other factors to the promoter. Likewise, MBD3−/− ES cells have been isolated, and the repression of the Oct4 gene has been analyzed. In the presence of RA but in the absence of MBD3, the Oct4 gene is still repressed (24), likely because expression of activators, like LRH-1, is down-regulated and GCNF is still induced and binds to the Oct4 DR0 to displace LRH-1 (16). It would be interesting to determine if indeed GCNF is expressed in MBD3-knockout ES cells and whether MBD2 is still recruited to the promoter. Thus, inactivation of individual corepressors that mediate repression and silencing of the Oct4 gene does not lead to loss of repression; only when the initiator and coordinator of Oct4 repression, GCNF, is inactivated is there a complete loss of repression and silencing of the Oct4 gene.

In summary, our results have established GCNF as an important initiator of Oct4 gene repression and silencing, and we propose the following model as the important key steps in Oct4 repression in ES cells (Fig. (Fig.8).8). In undifferentiated and early differentiating ES cells, transactivators such as LRH-1 and its coactivator complex bind to the DR0 site in the promoter and sites in the proximal enhancer to maintain Oct4 gene expression (16). Upon induction with RA, a GCNF hexamer displaces LRH-1 from DR0, causing passive loss of activation. Concomitant with binding to the DR0, GCNF recruits MBD3 and likely the Mi-NuRD complex, which binds to unmethylated CpGs to initiate active repression of Oct4. Subsequently, de novo DNA methylation occurs, and MBD2 is recruited by GCNF to bind to methylated CpG dinucleotides. Corecruitment of MBD3 and MBD2 to the Oct4 promoter can be detected in ES cells between 36 and 72 h. The apparent corecruitment could reflect asynchrony in the ES cell cultures or may be due to direct interactions between MBD2 and MBD3 (46), which cannot be ruled out. Alternatively, simple intermediate complexes containing both MBD2 and MBD3 maybe present simultaneously on Oct4 (Fig. (Fig.8).8). Subsequently, GCNF expression itself is repressed but Oct4 DNA methylation is maintained, reflective of true gene silencing, as MBD2 complexes and/or other repression complexes are docked on the Oct4 promoter. This model is the first to define the initiation steps of Oct4 gene repression and DNA methylation mediated by GCNF and proposes a function for MBD3 prior to de novo DNA methylation and MBD2 after DNA methylation.

FIG. 8.
Model of Oct4 gene repression and silencing initiated by GCNF-dependent recruitment of MBD2 and MBD3. The Oct4 promoter with a DR0 and unmethylated CpG sites is activated by LRH-1 under the control of LIF. At the beginning of RA induction (1.5 days), ...

Acknowledgments

We thank Adrian Bird for kindly providing the MBD1, MBD2, MBD3, and MBD4 expression vectors. We are also grateful to En Li for Dnmt3A−/− Dnmt3B−/− ES cells. We thank Zafar Nawaz for help with the yeast two-hybrid screen.

This research was supported by grant 07352401 from the NIH NIDDK to A.J.C. and NIH training grant HD07165.

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 October 2006.

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