Identification of TET1 Partners That Control Its DNA-Demethylating Function

Abstract

Several recent reports have identified TET1 as the main enzyme modulating DNA methylation and gene transcription via hydroxylation of 5-methylcytosine. However, little is known about the protein network that controls TET1 activity. By using a new proximity ligation in situ assay, we identified MeCP2, HDAC1/6/7, EZH2, mSin3A, PCNA, and LSD1 as TET1-interacting proteins. We also discerned that TET1/PCNA acts as a demethylator of the cyclical methylation/demethylation process, the perturbation of which promotes the aberrant methylation hallmarks frequently observed in cancer cells.

Keywords: DNA methylation; TET1; demethylation; glioma.

Figure 1.

Description of TET1-interacting proteins. (A) Monitoring of TET1-interacting proteins in U251 cells by using P-LISA. The nucleus appears in blue after DAPI coloration, and each red dot represents one interaction between TET1 and the considered protein. For each interaction, we quantified the number of interactions by using the freeware BlobFinder. Each bar on the graph represents the mean ± SD of considered interactions quantified from 100 nuclei in 3 independent experiments. (B) Monitoring of TET1-interacting proteins in U251 cells by using the immunoprecipitation (IP) method. “NE” represents gel loading with 50% of the nuclear extract used to perform the IP of TET1. “–” indicates the IP performed in the absence of the TET1 antibody, and “+” indicates the IP performed in the presence of the TET1 antibody. Gel pictures are representative of 3 independent experiments. (C) Monitoring of TET1-interacting proteins by using the pull-down method. The His-tag TET1 recombinant protein was used as bait, while indicated glutathione S-transferase proteins were used as prey in the pull-down assay. “I” (input) represents the deposit of 30% of the total amount of protein used in the considered assay. “–” indicates the pull-down performed in the absence of the His-tag TET1 recombinant protein, and “+” indicates the pull-down performed in the presence of the His-tag TET1 recombinant protein. Gel pictures are representative of 3 independent experiments.

Figure 2.

Nonredundant functionality of TET1/EZH2- and TET1/Sin3A-including complexes to the NES1 and HOXD12 genes. (A) Impact of TET1, EZH2, or Sin3A down-regulation on the formation of TET1/EZH2 and TET1/Sin3A interactions. P-LISA experiments were performed 48 hours after siRNA treatment. Graphs represent the mean ± SD of considered interactions quantified by using the freeware BlobFinder from 100 nuclei in 3 independent experiments. (B) Impact of TET1, EZH2, or Sin3A down-regulation on TET1/EZH2 and TET1/Sin3A recruitment on the NES1^{−366/−296} and HOXD12^{−507/−389} genes by ChIP and reChIP experiments. Graphs represent the mean ± SD of 3 independent experiments. (C) Impact of TET1, EZH2, or Sin3A down-regulation on methylation of the NES1^{−366/−296} and HOXD12^{−507/−389} genes by using the MeDIP method. Graphs represent the mean ± SD of 3 independent experiments. (D) Impact of TET1, EZH2, or Sin3A down-regulation on methylation of the NES1^{−366/−296} and HOXD12^{∓507/∓389} genes by using the bisulfite sequencing method. A black circle represents a CpG having a methylation percentage >50%, and an open circle represents a CpG having a methylation percentage <50% (according to data illustrated by Suppl. Fig. S3). (E) Impact of TET1, EZH2, or Sin3A down-regulation on methylation of the NES1^{−366/−296} and HOXD12^{−507/−389} genes by using the qPCR method. Graphs represent the mean ± SD of 3 independent experiments.

Figure 3.

Cyclical epigenetic regulation of the MUC2 and RRM1 genes. (A) The kinetics of MeDIP, hMeDIP, and ChIP experiments were analyzed at the indicated time. The numbers of immunoprecipitated genes or methylated genes were quantified by qPCR and normalized to inputs according to the previous description. The number of generations (F0, F1, and F2) was determined by counting the cells after an initial synchronization in the G0/G1 phase (serum deprivation, 72 hours) (Suppl. Fig. S3). S phases (gray areas) were determined by measuring the ³H-thymidine incorporation in the DNA of U251 cells (Suppl. Fig. S3). (B) qPCR monitoring of expression levels of the MUC2 and RRM1 genes at certain times (as explained in A).

Figure 4.

Implication of cyclical methylation/demethylation on the methylation hallmarks of glioma. (A) Identification of genes aberrantly methylated in glioma and a target of TET1/PCNA. (B) Impact on the methylation status of the AHR and SH3BP2 genes of DNMT3B or TET1 down-regulation. CC = cell cycle. (C) Correlation between the number of PCNA/TET1 interactions (according to P-LISA experiments) and the methylation level of FZD9. Each open circle represents 1 cell line or 1 PCTC.