Differential recruitment of methyl CpG-binding domain factors and DNA methyltransferases by the orphan receptor germ cell nuclear factor initiates the repression and silencing of Oct4

Abstract

The pluripotency gene Oct4 encodes a key transcription factor that maintains self-renewal of embryonic stem cell (ESC) and is downregulated upon differentiation of ESCs and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, mediates Oct4 gene expression. Here, we show that the orphan nuclear receptor germ cell nuclear factor (GCNF) initiates Oct4 repression and DNA methylation by the differential recruitment of methyl-CpG binding domain (MBD) and DNA methyltransferases (Dnmts) to the Oct4 promoter. When compared with wild-type ESCs and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in retinoic acid (RA)-differentiated GCNF(-/-) ESCs and GCNF(-/-) embryos. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified MBD3, MBD2, and de novo Dnmts as GCNF interacting factors. Upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2 as well as Dnmt3A. In differentiated GCNF(-/-) ESCs, recruitment of MBD3 and MBD2 as well as Dnmt3A to Oct4 promoter is lost and subsequently Oct4 repression and DNA methylation failed to occur. Hypomethylation of the Oct4 promoter is also observed in RA-differentiated MBD3(-/-) and Dnmt3A(-/-) ESCs, but not in MBD2(-/-) and Dnmt3B(-/-) ESCs. Thus, recruitment of MBD3, MBD2, and Dnmt3A 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 ESC differentiation.

Figure 1

Hypomethylation of the Oct4 promoter in differentiated germ cell nuclear factor^−/− (GCNF^−/−) embryonic stem cells (ESCs) and GCNF^−/− embryos at E8.5–E9.5. (A): Expression pattern of Oct4 and GCNF in wild-type (wt) and GCNF^−/− ESCs was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). (B): DNA methylation profile of 16 CpG sites located in the Oct4 proximal promoter from −562 to ATG start code in wt and GCNF^−/− ESCs. The open circles represent unmethylated CpG and the black closed circles represent methylated CpG sites. The red closed square represents GCNF binding site DR0. (C): Comparison of percentage of methylated CpG sites in the Oct4 proximal promoter. Student's t test was used for the statistical analyses. **, p < .01; ***, p < .0001. (D): DNA methylation profile of 16 CpG sites in wt and GCNF^−/− embryos at 8.5 and 9.5 dpc. (E): Comparison of percentage of methylated CpG sites in the Oct4 proximal promoter by dividing the Oct4 promoter into two distal and proximal parts. (F): Reactivation of Oct4 gene expression in GCNF^−/− embryos detected by whole-mount in situ hybridizations. Embryos in panels a, b, and e are wt and in panels c, d, and f are GCNF^−/−. Oct4 cRNA probe was used in panel a, b, c, and d; LRH-1 cRNA probe was used in panel e and f. The number of somites for each embryo is indicated in each panel. Abbreviations: DR0, direct repeat with zero base pair spacing; GCNF, germ cell nuclear factor; LRH-1, liver receptor homolog-1; mu, mutant; RA, retinoic acid; RT-PCR, reverse transcriptase-polymerase chain reaction. so, somites; wt, wild-type.

Figure 2

Interaction of germ cell nuclear factor (GCNF) with MBD3, MBD2, DNA methyltransferases 3A (Dnmt3A), and Dnmt3B in vitro. (A): Putative GCNF interaction partners identified in a Yeast two-hybrid screen. Gal4-nuclear receptor corepressor (NCoR) was used as a positive control and Gal4 DBD and Gal4 AD empty vectors were used as negative controls. (B): Interaction of GCNF and other DNA methylation machinery components was measured by liquid β-gal assay in yeast two-hybrid system. (C): Interaction of GCNF with MBDs and Dnmts was detected by GST-pulldown assay. In vitro translated NCoR was used as a positive control and RXR as a negative control. (D): Detection of interaction of coexpressed recombinant GCNF and MBD3 in COS-1 cells using coimmunoprecipitation (Co-IP) assays. Myc-GCNF signal is indicated with an arrow and the IgG heavy-chain is indicated with a star. (E): Fractions of P19 nuclear extract separated by FPLC were analyzed by gel mobility shift assays (Gelshift) and western blots with anti-GCNF and -MBD3 antibodies. (F): Detection of the interaction of endogenous GCNF and MBD3 in differentiated P19 cell nuclear extracts. (G): Interaction of GCNF with MBD2 in cotransfected COS-1 cell lysates was detected by Co-IP. (H): Illustration of the MBD3 deletions generated and analyzed. The number of amino acids was labeled. The black box denotes the MBD and the open box denotes for the polyglutamic acid motif (poly E). (I): Analysis of interaction of GCNF and different MBD3 deletions by GST-pulldown assay. (J): Comparison of amino acid sequence of MBDs. Abbreviations: Co-IP, coimmunoprecipitation; COS-1, CV-1 (simian) in Origin, and carrying the SV40 genetic material; DBD DNA binding domain; Dnmt, DNA methyltransferases; FPLC fast protein liquid chromatography;GCNF, Germ cell nuclear factor; GST, glutathione S transferase; IP, immunoprepitation; IR, interaction; LBD, ligand binding domain; MBD, methyl CpG binding domain; MD, MegaDaltons; NCoR, nuclear receptor corepressors; RXR, retinoid X receptor; SMRT, silencing mediator of retinioc acid and thyroid hormone receptors; TRIF, transiently retinoid induced factor.

Figure 3

Germ cell nuclear factor (GCNF) recruits MBD2, MBD3, and DNA methyltransferases 3A (Dnmt3A) to the Oct4 promoter in embryonic stem cells (ESCs). (A): Expression of GCNF, Oct4, MBD2, and MBD3 was detected in differentiated wild-type (wt) and GCNF^−/− ESCs by RT-PCR and Western blot. (B): Binding of GCNF, MBD2, and MBD3 to Oct4 promoter in wt and GCNF^−/− ESCs was detected by chromatin immunoprecipitation (ChIP) assay. (C): Quantitation of PCR signals in (B). The strength of GCNF, MBD2, and MBD3 bound signals at the undifferentiated time point was set as one. The bound LRH-1 signal at 72-hour retinoic acid-differentiation was set as 1. (D): Expression of Oct4, Dnmt3A, and Dnmt3B in wt ESC and GCNF^−/− ESCs was detected by Western blot. (E): Binding of Dnmt3A to Oct4 promoter in wt ESC was detected by ChIP assay. (F): Binding of Dnmt3B to major satellite repeat sequence in wt and GCNF^−/− ESCs was detected by ChIP assay. Abbreviations: ChIP, chromatin immunoprecipitation; Dnmt, DNA methyltransferase; GCNF, Germ cell nuclear factor; LRH-1, liver receptor homolog-1; MBD, methyl CpG binding domain; RA, retinoic acid; RT-PCR, reverse transcriptase-polymerase chain reaction.

Figure 4

Comparison of Oct4 expression and Oct4 promoter methylation status in MBD3^−/− and MBD2^−/− embryonic stem cells (ESCs) with wild-type (wt) ESCs and germ cell nuclear factor^−/− (GCNF^−/−) ESCs. Oct4 and GCNF mRNA level (A) and protein level (B) were detected during retinoic acid (RA)-induced differentiated wt and MBD3^−/− ESCs by RT-PCR (A) and Western blot (B). Oct4 and GCNF mRNA (C) and protein expression (D) were detected during RA-induced differentiated wt and MBD2^−/− ESCs by RT-PCR (C) and Western blot (D). (E): Comparison of re-expression of Oct4 in RA-treated ESCs with LIF reactivation in wt ESCs, GCNF^−/−, MBD3^−/−, and MBD2^−/− ESCs. (F): Methylation status of Oct4 promoter in different time point RA treatment and LIF reactivated wt ESCs, MBD3^−/−, and MBD2^−/− ESCs. (G): Comparison of percentage of methylated CpG sites of 16 CpGs in the Oct4 proximal promoter. Student's t test was used for the statistic analyses. *, p < .05; **, p < .01; ***, p < .0001. Abbreviations: ESC, embryonic stem cell; GCNF, germ cell nuclear factor; LIF, leukemia inhibitory factor; MBD, methyl CpG binding domain; RA, retinoic acid; wt, wild-type; RT-PCR, reverse transcriptase-polymerase chain reaction.

Figure 5

Oct4 expression and Oct4 promoter methylation status in different DNA methyltransferases (Dnmt) mutant embryonic stem cells (ESCs). (A): Oct4 and germ cell nuclear factor (GCNF) mRNA level were detected during retinoic acid (RA)-induced differentiated wild-type (wt) ESCs, Dnmt1^−/−, Dnmt3A^−/−, Dnmt3B^−/−, and Dnmt3A^−/− and Dnmt3B^−/− double mutant ESCs by RT-PCR. (B): Methylation status of Oct4 promoter in wt ESC and Dnmt mutant ESCs treated with RA for 3 and 6 days. (C): Quantitation of methylated CpG sites of 16 CpGs in the Oct4 proximal promoter. Student's t test was used for the statistic analyses. **, p < .01; ***, p < .0001. (D): Expression of Oct4, GCNF, MBD2, and MBD3 in wt ESC and Dnmt3A^−/− and Dnmt3B^−/− double mutant ESCs. (E): Recruitment of MBD2 and MBD3 by GCNF in Dnmt3A^−/− and Dnmt3B^−/− double mutant ESCs was analyzed by chromatin immunoprecipitation assay. (F): Quantitation of binding of GCNF, MBD2, and MBD3 to Oct4 promoter in Dnmt3A^−/− and Dnmt3B^−/− ESCs. The strength of GCNF, MBD2, and MBD3 bound signals at the undifferentiated time point was set as 1. Abbreviations: Dnmt, DNA methyltransferase; ESC, embryonic stem cell; GCNF, germ cell nuclear factor; MBD, methyl CpG binding domain; RA, retinoic acid; wt, wild-type; RT-PCR, reverse transcriptase-polymerase chain reaction.

Figure 6

Model of Oct4 gene repression and silencing initiated by germ cell nuclear factor (GCNF)-dependent recruitment of MBD2, MBD3, and DNA methyltransferases 3A (Dnmt3A). The Oct4 promoter with a DR0 and unmethylated CpG sites is activated by LRH-1 under the control of LIF. At the beginning of retinoic acid (RA) induction (1.5 days), induced expression of GCNF hexamer replaces LRH-1 binding at the DR0 site. GCNF recruits MBD3 complex to unmethylated CpG sites and Oct4 repression is initiated. Once de novo DNA methylation is triggered through direct recruitment of Dnmt3A to the Oct4 promoter by GCNF-MBD3 or MBD2/3 complexes recruited to CpG sites and silencing of Oct4 gene occurs (days 1.5–3.0). At late stages of RA-induced differentiation (days 3–6), the expression of GCNF is downregulated and the MBD2 and MBD3 complexes are no longer bound to the Oct4 promoter but DNA methylation is maintained and Oct4 gene is completely silenced (day 6). HP1 complex maybe replaced by GCNF/DNA methylation complex to bind to methylated Oct4 promoter region. Abbreviations: Dnmt, DNA methyltransferase; DR0, direct repeat with zero base pair spacing; GCNF, germ cell nuclear factor; HP1, heterchromatin protein 1; LIF, leukemia inhibitory factor; LRH-1, liver receptor homolog-1; MBD3, methyl CpG binding domain 3; RA, retinoic acid.