Fig 2: TET2 Is Recruited by SNIP1 to Regulate c-MYC Target Gene Expression(A) RNA sequencing analysis reveals that SNIP1 knockdown causes significant alterations in mRNA expression of genes that are c-MYC targets or involved in cell apoptosis. More detailed information is provided in STAR Methods.(B) Real-time qPCR confirms that c-MYC target genes are commonly downregulated by SNIP1 knockdown or TET2 knockout in U2OS cells. Data were normalized by mRNA expression in control cells. A gray-to-blue color scale indicates values from no change to downregulation, and row scaling is used to facilitate visualization.(C) SNIP1 knockdown and TET2 knockout do not have an additive effect on downregulating c-MYC target genes. In SNIP1-knockdown and/or TET2-knockout U2OS cells, mRNA expression of the indicated c-MYC direct target genes was determined using real-time qRT-PCR, as described in STAR Methods.(D) TET2 association is indispensable for SNIP1 to activate c-MYC target genes. Wild-type or TET2-binding defective K30A/P100A/K108A mutant SNIP1 was overexpressed in SNIP1-knockdown U2OS cells, and mRNA expression of the indicated c-MYC direct target genes was determined using real-time qRT-PCR.(E–G) Enrichment of ectopically expressed TET2, SNIP1, and c-MYC in the genome of HEK293T cells. Previous ChIP-seq data (GSM897576 and GSM1493021) were re-analyzed using Bowtie 1.2.1.1 (Langmead et al., 2009) and MACS 2.1.1 (Zhang et al., 2008). ChIP-seq densities of Halo-TET2 and Flag-c-MYC were profiled according to the indicated levels of HA-SNIP1 ChIP-seq signals around TSS regions (E). Heatmap analysis of total 27,569 TSSs showed that HA-SNIP1, Halo-TET2, and Flag-c-MYC had similar binding patterns across all TSSs. All TSSs were ranked by HA-SNIP1 signal intensities (F). The overlap of TET2 and c-MYC-binding promoters and SNIP1 upregulating genes in U2OS identified by RNA-seq is displayed by Venn diagram (G) (p < 10-10).(H) TET2 binds to c-MYC target gene promoters in a manner dependent on SNIP1. In SNIP1-knockdown and the control U2OS cells, the occupancy of endogenous TET2 on the promoters of indicated c-MYC target genes was determined using ChIP-qPCR as described in STAR Methods. Rabbit IgG was used as a negative control.(I and J) SNIP1 knockdown decreases 5hmC and increases 5mC at the promoters of c-MYC target genes. In SNIP1-knockdown and the control U2OS cells, 5hmC and 5mC levels at the promoter regions of indicated c-MYC direct target genes were determined using hMeDIP-qPCR (I) and MeDIP-qPCR (J), respectively, as described in STAR Methods.Shown are average values of triplicated experiments with SD. *p < 0.05, **p < 0.01, and ***p < 0.001 for the indicated comparisons. N.S., not significant.

Fig 3: Identification of SNIP1 as One of the TET2 Potential Interacting Transcription Regulators(A and B) Schematic representation of the mammalian two-hybrid system and dual-luciferase reporter system used to search for TET2-interacting proteins. Human full-length TET2 fused toVP16transactivation domain (AD), preys (1,126 human open reading frames [ORFs]) fused to Gal4 DNA-binding domain (DBD) (A), UAS-luciferase reporter plasmid, and CMV-Renilla control plasmid were co-transfected in HEK293T cells. At 30 hr after transfection, the luciferase reporter activity was measured as an indicator for protein-protein interaction (B). Moredetailed information is provided in STAR Methods.(C) Optimization of the mammalian two-hybrid screening system. One or two copies of VP16 were fused to either the N or C terminus of human full-length or truncated TET2, and OGT as a positive control was fused to C terminus of Gal4(DBD). Gal4(DBD)-OGT and the 9 × UAS-luciferase reporter, which was driven by nine Gal4-binding elements, were co-transfected into HEK293 cells with various TET2 fusion as indicated. At 30 hr after transfection, the luciferase reporter activity was measured.(D) The reporter-based screen identifies SNIP1 as the strongest positive. Candidates with relative luciferase activation higher than OGT were shown, and their protein interaction with TET2 was verified by co-immunoprecipitation (Figure S1).(E and F) Endogenous TET2 interacts with endogenous SNIP1 in U2OS cells. TET2 protein in U2OS cells (E) or TET2 deficient U2OS cells (F) was purified by immunoprecipitation with an antibody against TET2, followed by western blot to detect endogenous SNIP1 using a SNIP1 antibody. Rabbit IgG was used as negative control.(G) Endogenous SNIP1 interacts with endogenous TET2 in U2OS cells. SNIP1 protein in U2OS cells was purified by immunoprecipitation with an antibody against SNIP1, followed by western blot to detect endogenous TET2 using a TET2 antibody. Rabbit IgG was used as negative control.

Fig 4: Depletion of SNIP1 and TET2 Has No Additive Effect on Increasing DNA Double-Strand Breaks and Cell Apoptosis(A–C) SNIP1 knockdown and TET2 knockout have no additive effect on increasing internal DNA double-strand breaks. In the indicated U2OS stable cells, ?H2AX foci was determined using immunofluorescence (A), and the percentage of ?H2AX-positive cells (more than eight foci per cell) was counted from randomly selected cells of three independent experiments (B). Meanwhile, the number of ?H2AX foci per cell was counted from randomly selected cells (C).(D–F) SNIP1 knockdown and TET2 knockout have no additive effect on regulating cisplatin-induced DNA damage response. The indicated U2OS stable cells were treated with cisplatin (20 µM for 6 hr), ?H2AX foci was determined using immunofluorescence (D), and the percentage of ?H2AX-positive cells (more than eight foci per cell) was counted from randomly selected cells of three independent experiments (E). Additionally, ?H2AX level was also determined using western blotting (F) and normalized by ß-ACTIN in these U2OS stable cells.(G and H) SNIP1 knockdown and TET2 knockout has no additive effect on regulating cisplatin-induced apoptosis. The indicated U2OS stable cells were treated with cisplatin (20 µM) as indicated, and the protein level of cleaved PARP was determined using western blotting (G), normalized by ß-ACTIN. The percentage of viable cells (both PI and annexin V negative) was determined using flow cytometry (H).Shown are average values of triplicated experiments with SD. *p < 0.05 and **p < 0.01 for the indicated comparisons. N.S., not significant.

Fig 5: SNIP1 Mediates the Interaction between TET2 and Several Transcription Factors, Including c-MYC(A) SNIP1 bridges TET2 binding with c-MYC. Plasmids expressing the indicated proteins were transiently transfected in HEK293T cells. TET2 protein was purified by immunoprecipitation with Flag beads, followed by western blot to detect the co-precipitated Gal4(DBD)-SNIP1 and HA-c-MYC with antibodies against Gal4-DBD and HA, respectively.(B) K30/P100/K108 residues in the N-terminal domain of SNIP1 are crucial for TET2-SNIP1-c-MYC complex formation. Plasmids expressing the indicated proteins were transiently transfected in HEK293T cells. TET2 protein was purified by immunoprecipitation with Flag beads, followed by western blotto detect the co-precipitated Gal4(DBD)-SNIP1 and HA-c-MYC with antibodies against Gal4-DBD and HA, respectively.(C) Endogenous TET2 interacts with SNIP1 and c-MYC in MCF-7 and U2OS cells. TET2 protein in the indicated cells was immunoprecipitated with a TET2 antibody and followed by western blot to detect the co-precipitated endogenous SNIP1 and c-MYC. Rabbit IgG was used as a negative control.(D) SNIP1 is essential for TET2 and c-MYC interaction. SNIP1 knockout U2OS cells were generated by three different sgRNAs (#1, #2, and #3). CTRL, control sgRNA. TET2 protein was immunoprecipitated and followed by western blot to detect the co-precipitated endogenous SNIP1 and c-MYC.(E) Schematic representation of the mass spectrometry assay to identify SNIP1-binding transcription factors. In MCF-7 stable cells with knockdown of endogenous SNIP1 and put-back of Flag-tagged empty vector or SNIP1, chromatin-bound nuclear proteins were isolated, followed by immunoprecipitation with Flag beads. The immunoprecipitated proteins were then proceeded with isobaric tags for relative and absolute quantification by mass spectrometry.(F and G) SNIP1 bridges TET2 binding with other transcription factors. Plasmids expressing the indicated proteins were transiently transfected in HEK293T cells. TET2 protein was purified by immunoprecipitation with Flag beads, followed by western blot to detect the co-precipitated Gal4(DBD)-SNIP1 and Myc-CDC5L (F)or Myc-BCLAF1 (G) with antibodies against Gal4-DBD and Myc tag, respectively.