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. 2022 Jan 25;25(2):103813. doi: 10.1016/j.isci.2022.103813

Figure 7.

Figure 7

DTX2 mediates K63-linked ubiquitination of NFIC to enhance hTERT transcription

(A) GST-NFIC was transiently co-expressed in HEK293T cells with DTX2-SFB and HA-tagged Ub or Ub K48/K63 mutants as indicated. EGFP-SFB was used as a control. Cells were harvested for GST pull-down (PD) and western blotting as shown.

(B) GST-NFIC was transiently co-expressed in HEK293T cells with SFB-tagged DTX2 or the I/M∗ mutant as well as HA-tagged Ub or Ub K63 mutant as indicated. I/M∗, DTX2 with I414R and I457R mutations. EGFP-SFB was used as a control. Cells were harvested for GST pull-down (PD) and western blotting as shown.

(C) HA-Ub was transiently co-expressed in HEK293T cells with DTX2-SFB and GST-tagged WT or K/R point mutants of NFIC. EGFP-SFB was used as a control. Cells were harvested for GST pull-down (PD) and western blotting as shown.

(D–F) DTX2/NFIC DKO cells ectopically expressing FH-tagged WT or K/R point mutants of NFIC were examined for protein expression with an anti-Flag antibody (D), relative hTERT mRNA level by qPCR (E), and telomerase activity by qTRAP (F). Data were shown as mean ± SD (N = 3). ∗p< 0.05; ∗∗p< 0.01; n.s., not significant.

(G) ChIP-qPCR analysis was performed with an anti-HA antibody and primers for the indicated DNA regions (relative to hTERT TSS) using cells from (D). Data were shown as mean ± SD (N = 3). ∗∗p< 0.01; n.s., not significant. For panels E–G, p values were calculated using Student’s t test.

(H) In telomerase-positive cancer cells with low DTX2 expression, NFIC binds to the consensus half sites on the hTERT promoter with low affinity. Increased DTX2 expression promotes its interaction with NFIC and K63-linked ubiquitination at K65 and K99 sites, which helps stabilize the binding of the DTX2-NFIC complex to the hTERT core promoter and enhance hTERT transcription. (H) In telomerase-positive cancer cells with low DTX2 expression, NFIC binds to the consensus half sites on the hTERT promoter with low affinity. The hTERT gene remains transcription repressive with hypermethylation on H3K9. Increased DTX2 expression promotes its interaction with NFIC and K63-linked ubiquitination at K65 and K99 sites, which helps stabilize the binding of the DTX2-NFIC complex to the hTERT core promoter. The enrichment of DTX2-NFIC further upregulates H3K4me3 level and helps recruit phosphorylated Pol II-S2, therefore enhancing hTERT transcription. See also Figure S7.