NFX1-123 and poly(A) binding proteins synergistically augment activation of telomerase in human papillomavirus type 16 E6-expressing cells.

Overcoming senescence signals in somatic cells is critical to cellular immortalization and carcinogenesis. High-risk human papillomavirus (HPV) can immortalize epithelial cells in culture through degradation of the retinoblastoma protein by HPV E7 and activation of hTERT transcription, the catalytic subunit of telomerase, by the heterodimer HPV E6/E6-associated protein (E6AP). Recent work in our laboratory identified a novel repressor of hTERT transcription, NFX1-91, which is targeted for ubiquitin-mediated degradation by HPV type 16 (HPV16) E6/E6AP. In contrast, NFX1-123, a splice variant NFX1, increased expression from an hTERT promoter that was activated by HPV16 E6/E6AP. Here, we show that HPV16 E6 bound both NFX1-91 and NFX1-123 through the common central domain of NFX1 in the absence of E6AP. NFX1-123 positively regulated hTERT expression, as its knockdown decreased hTERT mRNA levels and telomerase activity and its overexpression increased telomerase activity. We identified new protein partners of NFX1-123, including several cytoplasmic poly(A) binding proteins (PABPCs) that interacted with NFX1-123 through its N-terminal PAM2 motif, a protein domain characteristic of other PABPC protein partners. Furthermore, NFX1-123 and PABPCs together had a synergistic stimulatory effect on hTERT-regulated reporter assays. The data suggest that NFX1-123 is integral to hTERT regulation in HPV16 E6-expressing epithelial cells and that the interaction between NFX1-123 and PABPCs is critical to hTERT activity.

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

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.