PGC7 suppresses TET3 for protecting DNA methylation

Abstract

Ten-eleven translocation (TET) family enzymes convert 5-methylcytosine to 5-hydroxylmethylcytosine. However, the molecular mechanism that regulates this biological process is not clear. Here, we show the evidence that PGC7 (also known as Dppa3 or Stella) interacts with TET2 and TET3 both in vitro and in vivo to suppress the enzymatic activity of TET2 and TET3. Moreover, lacking PGC7 induces the loss of DNA methylation at imprinting loci. Genome-wide analysis of PGC7 reveals a consensus DNA motif that is recognized by PGC7. The CpG islands surrounding the PGC7-binding motifs are hypermethylated. Taken together, our study demonstrates a molecular mechanism by which PGC7 protects DNA methylation from TET family enzyme-dependent oxidation.

Figure 1.

PGC7 interacts with TET2 and TET3. (A) PGC7 interacts with TET2 and TET3, but not with TET1. Co-IP was performed in 293T cells. PGC7 and TET1-3 were examined with indicated antibodies. The whole cell lysates (WCL) of 293T was used as the input. An irrelevant IgG was used as the IP control. (B) The catalytic DSBH (CD) domain of TET2 interacts with PGC7. Flag-tagged deletion mutants of TET2 were expressed in 293T cells. Co-IP assays were performed with indicated antibodies. Mock transfected cells were used as the control. (C) The CD domain of TET3 also interacts with PGC7. (D) The fragment of PGC7 with a.a. 20–95 interacts with TET3 catalytic domain (TET3CD) and TET2 catalytic domain (TET2CD). Myc-tagged N-terminus or C-terminus deletion mutants of PGC7 were generated and expressed in 293T cells. Co-IP assays were performed with indicated antibodies.

Figure 2.

PGC7 suppresses the enzymatic activity of TET2 or TET3 in vitro. (A) Recombinant PGC7 suppresses TET2 or TET3, but not TET1, for 5mC to 5hmC conversion in vitro. The in vitro 5hmC assays were performed and described in ‘Materials and Methods’ section. (B) The interaction between PGC7 and TET3 is required for the inhibition of TET3 in vitro. Recombinant PGC7 mutants were generated and examined in the in vitro 5hmC assays. (C) Non-specific DNA-binding of PALB2 does not suppress the enzymatic activity of TET3CD. (D), (E) Wild-type PGC7 and D9 mutant, but not the D3 mutant, suppress TET2CD- or TET3CD-induced 5hmC. 5hmC catalyzed by TET2CD or TET3CD was examined by PvuRts1I digestion assays as described in ‘Materials and Methods’ section. DNA samples were analyzed by gel electrophoresis (D) or qPCR (E). Error bars indicate standard deviation (SD) (n = 3).

Figure 3.

PGC7 suppresses the enzymatic activity of TET2CD and TET3CD in vivo. (A) The 5hmC level is examined by dot blotting assays in 293T or 293-PGC7 cells overexpressing TET2CD or TET3CD. (B) Immunofluorescence staining shows that TET2CD- or TET3CD-induced 5hmC is suppressed by wild-type PGC7 or the D9 mutant, but not the D3 or D8 mutant. (C) The ratio of 5hmC positively stained cells in TET2CD or TET3CD positively transfected cells was summarized.

Figure 4.

PGC7 protects CpG methylation at imprinting loci from TET3-induced DNA demethylation. (A) shRNA treatment specifically down-regulates the mRNA level of PGC7 in HBL100 cells. The level of PGC7 mRNA was examined by RT–PCR (left panel) and RT–qPCR (right panel). Error bars indicate SD (n = 3). Scramble shRNA was used as negative control. (B) SBP-TET3 is stably expressed in HBL100 cells. IP and western blotting were examined by indicated antibodies. (C) Bisulfite sequencing shows methylated CpG at Peg1, Peg3, Peg10 and H19 loci in the presence or absence of PGC7 depletion and/or TET3 overexpression. (D) Expression of RNAi-resistant PGC7, but not the D3 mutant, rescues the loss of DNA methylation at Peg1 locus in HBL100 cells with endogenous PGC7 depletion and TET3 overexpression.

Figure 5.

PGC7 co-localizes with TET3 at a set of genes. (A) Venn diagram shows a significant overlap between PGC7 and TET3 target genes. (B) 5mC level in the genes occupied by PGC7 and TET3 (Group I genes) is significantly higher than that in the genes occupied by only TET3 (Group II genes). (C) Mean distribution of 5mC at TSS (± 1 kb) of Group I or Group II genes. (D) PGC7 and TET3 co-occupancy is associated with 5mC at whole-chromosome level. Significant overlap is shown in chromosome 13. (E) ChIP-Seq results show the co-occupancy of PGC7 and TET3 at methylated DNA region of Peg1, Peg3, Peg10 and H19 loci. (F) The consensus DNA-binding motif of PGC7 is analyzed according to ChIP-Seq result. The binding sequences in Peg1, Peg3, Peg10 and H19 loci are shown in red.

Figure 6.

Analysis of non-imprinting loci co-occupied by PGC7 and TET3. (A) ChIP-Seq results show the PGC7 and TET3 co-occupancy at Piwil1, Spaca4, Tssk2, Fyb and Rrh loci, which is associated with high 5mC level. (B) ChIP–qPCR results confirm the localization of PGC7, TET3 and 5mC in these loci. (C) A model shows that PGC7 suppresses TET3-dependent DNA demethylation.