Identification of a novel telomerase repressor that interacts with the human papillomavirus type-16 E6/E6-AP complex

Abstract

The critical immortalizing activity of the human papillomavirus (HPV) type-16 E6 oncoprotein is to induce expression of hTERT, the catalytic and rate-limiting subunit of telomerase. Additionally, E6 binds to a cellular protein called E6-associated protein (E6-AP) to form an E3 ubiquitin ligase that targets p53 for proteasome-dependent degradation. Although telomerase induction and p53 degradation are separable and distinct functions of E6, binding of E6 to E6-AP strongly correlated with the induction of hTERT. Here, we demonstrate using shRNAs to reduce E6-AP expression that E6-AP is required for E6-mediated telomerase induction. A yeast two-hybrid screen to find new targets of the E6/E6-AP E3 ubiquitin ligase complex identified NFX1. Two isoforms of NFX1 were found: NFX1-123, which coactivated with c-Myc at the hTERT promoter, and NFX1-91, which repressed the hTERT promoter. NFX1-91 was highly ubiquitinated and destabilized in epithelial cells expressing E6. Furthermore, knockdown of NFX1-91 by shRNA resulted in derepression of the endogenous hTERT promoter and elevated levels of telomerase activity. We propose that the induction of telomerase by the HPV-16 E6/E6-AP complex involves targeting of NFX1-91, a newly identified repressor of telomerase, for ubiquitination and degradation.

Figure 1.

E6-AP expression is required for telomerase induction by HPV 16E6. (A) Northern blot. Three shRNA constructs targeting E6-AP (esh1,2,3) were transduced into HFKs and RNA was harvested to examine levels of E6-AP message. 36B4 is a loading control. Relative expression levels are presented normalized to 36B4 and the pB empty vector control. (B) Western blot. HFKs expressing the E6-AP shRNA constructs were subsequently transduced with LXSN empty vector (-) or LXSN-16E6 (+). p53 levels were examined by Western blot. Actin is a loading control. (C) TRAP assay. Extracts from the same cells shown in B were assayed for telomerase activity. HeLa cells are a positive control lysate. CHAPs is a lysis buffer negative control. TSR8 is a synthetic template of eight telomeric repeats used as a PCR positive control. (D) RT-PCR. Expression of hTERT RNA was examined by RT-PCR of RNA extracts. 36B4 is a loading control.

Figure 2.

NFX1 isoforms have differential repressive and coactivating functions in hTERT reporter assays. (A) Schematic of NFX1 isoforms. The black box represents a PHD/RING finger domain. Multiple hatched boxes indicate NFX1-type zinc-finger domains. The dotted box represents an R3H domain present only in NFX1-123. The cross-hatched box indicates the unique lysine-rich C-terminal domain of NFX1-91. (B) Schematic of hTERT promoter and the regions included in the luciferase reporter constructs. Several potential SP1-binding sites and E boxes are indicated. Potential NFX1-binding sites are indicated by Xs. (C) hTERT reporter assay using the 710 hTERT construct in transient transfections in HFKS. The indicated genes were cotransfected with CMV, CMV-NFX1-123, or CMV-NFX1-91. The experiment was done in triplicate. (D) hTERT reporter assay with two different regions of the promoter in attempt to map the activity of the two NFX1 isoforms. The indicated DNAs were cotransfected with either the 219 hTERT or 710 hTERT reporter construct in HFKs. The data are a representative experiment of three trials done in duplicate.

Figure 3.

NFX1-91 binds to the proximal putative X box in the hTERT promoter. EMSA assays. (A) His-tagged recombinant NFX1 protein fragments, schematically represented at the top of the figure, were generated and purified from E. coli. Titrations of His-tagged NFX1-91 (His-N91) were able to shift a wild-type (WT) probe of 48 bp surrounding the proximal E box and including the overlapping putative X box. This shift was slightly diminished on mutation of five of the nucleotides within the putative X-box region (MUT). His-tagged NFX1-123 (His-N123) was much less efficient at binding and shifting the same probes. (B) A C-terminal peptide of NFX1-91 was sufficient to induce a small shifting (brackets) of the wild-type (WT) X-box DNA probe that could be dramatically supershifted (arrowhead) with titrations of a rabbit polyclonal antibody (Ab) raised to this peptide. The last three lanes demonstrate that antibody alone is unable to induce a similar shift.

Figure 4.

NFX1-91 rather than NFX1-123 is targeted by the E6/E6-AP complex. (A) Western blot. HFK/LXSN and HFK/E6 cells were treated with 25 μg/mL cycloheximide for the indicated time points. Lysates were assayed for NFX1 expression levels using an affinity-purified NFX1 antibody. c-Myc is shown as a positive control for a short-lived protein. Nucleolin is a stable protein used as a loading control. (B) Half-life of NFX1-91. A pulse-chase experiment to calculate the half-life of NFX1-91 was performed using metabolically labeled HFKs expressing the indicated E6 construct or LXSN vector control. The data presented are the average of two or three independent experiments for each cell line. Error bars represent the standard error of the mean. (C) Decreased expression of NFX1-91 seen in E6-expressing HFKs was proteasome dependent. Proteasome inhibition with MG-132 restored NFX1-91 protein levels. NFX1 protein was detected using an IgG-purified NFX1 antibody. The asterisk indicates a nonspecific background band used as a loading control. p53 expression is shown as a control for proteasome inhibition. (D) Endogenous NFX1-91 coimmunoprecipitated with AU1-tagged 16E6 in transiently transfected 293T cells. A longer exposure of the NFX1 blot shows the presence of a ubiquitin ladder. The reciprocal IP to precipitate AU1-16E6 with NFX1 antibody did not work.

Figure 5.

NFX1-91 is ubiquitinated by E6-independent and E6-dependent means. (A) Western blot. 293Ts were transiently transfected with Flag-tagged forms of NFX1. Cells were treated with MG-132 prior to lysis. Proteins were detected with rabbit anti-Flag antibody. (B) The lysates shown in A were immunoprecipitated with rabbit IgG (rIgG) or a rabbit anti-Flag antibody (α-Flag). Western blots with a mouse anti-ubiquitin antibody indicate that NFX1-91 was highly ubiquitinated, whereas NFX1-123 was not. (C) Endogenous NFX1 isoforms were immunoprecipitated from HFK/LXSN and HFK/E6 cells with antibodies specific for each isoform or with preimmune serum. Anti-NFX1 detects both NFX1 isoforms and anti-ubiquitin detects ubiquitinated proteins. Nucleolin is a loading control for the input lysates.

Figure 6.

Knockdown of NFX1-91 expression using shRNAs derepresses the endogenous hTERT gene in HFKs. (A) Western blot. NFX1-91 protein expression was reduced with expression of n91sh. (B) TRAP assay. Lysates from HFKs transduced with pB, esh1, or n91sh were analyzed for telomerase activity. Two micrograms and 0.5 μg of each lysate were used in the TRAP reactions. HeLa is a positive control lysate (0.2 μg). The percent of TRAP activity is presented relative to that in the HeLa lane. (C) TRAP assay. The cells shown in A and B were subsequently transduced with LXSN or LXSN-16E6. Lysates were examined for telomerase activity. The percent of TRAP activity is presented relative to that in the pB/LXSN-16E6 cells. (D) RT-PCR. Expression of hTERT and NFX1-91 was examined in RNA extracts from the cells in C. (E) Western blots. In vivo ubiquitination assays were performed as in Figure 4 with HFKs transduced with pB/LXSN, pB/E6, or esh1/E6. The blots on the left show protein expression in the lysates used for IP (5% input). The blots on the right show levels of ubiquitinated NFX1-91 immunoprecipitated from the cells. (F) NFX1-91 half-life. A pulse-chase analysis of NFX1-91 protein half-life in two independent lines of HFKs expressing E6 and empty vector (E6) or E6 and the esh1 shRNA (E6 + esh1) found an increase in protein half-life from 1.2 to 1.7 h. Error bars represent the standard deviation of two independent experiments.

Figure 7.

Senescence-associated β-galactosidase activity in late-passage HFKs. (A) Growth curves. HFKs expressing the indicated shRNA and LXSN (closed symbols) or E6 (open symbols) were grown in culture for ∼2 mo. The pBabe vector control is indicated by black squares, the esh1 cells are represented by red circles, and the n91sh cells are indicated by green triangles. The time point at which cells were stained for SA-β-galactosidase is indicated. (B) Micrographs of SA-β-galactosidase staining of late-passage HFKs expressing the indicated shRNA and LXSN empty vector or LXSN-16E6. The blue staining indicates senescent cells. (C) Quantitation of SA-β-galactosidase staining seen in B. The data represent the average percent of blue cells counted in four different fields at 100× magnification. The error bars indicate the standard error of the mean.