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.[Keywords: Telomerase; HPV; transcriptional repressor; ubiquitin; E6; E6-AP] Supplemental material is available at http://www.genesdev.org.

Human papillomavirus type 16 (HPV-16) E6 activates telomerase specifically in epithelial cells. The oncogene c-myc has also been shown to activate telomerase in several cell types. Here we show that while both HPV-16 E6 and c-myc require intact E boxes to transactivate the hTERT promoter, E6 does not induce hTERT transcription simply by inducing expression of c-myc. Moreover, hTERT transactivation by HPV-16 E6 correlates with its ability to bind the cellular E6-associated protein (E6AP), suggesting that E6 and E6AP may target a regulator of hTERT expression.Activation of telomerase is a critical step in cellular transformation (7, 12). Telomerase activity is primarily regulated at the level of expression of the hTERT gene, encoding the catalytic subunit of telomerase (4,24,26,33). Ectopic expression of hTERT in a number of different telomerase-negative cell types has been shown to confer immortality (2,4,18,30). Therefore, much research is now focused on determining the transcriptional regulators of hTERT.The hTERT promoter contains a number of putative transcription factor binding sites. Several studies have defined the minimal core promoter as the proximal 200 bp upstream of the transcription start site (15,29). This core promoter contains numerous SP1 binding sites and two canonical E boxes (MycMax binding sites) (3,15,29,34). Previous in vitro studies have shown that Myc-Max heterodimers can bind these E boxes in the context of the hTERT promoter and can activate hTERT reporter constructs (15,29,35). Myc expression has also been shown to induce telomerase activity in post M 0 human mammary epithelial cells (HMECs), the fibroblast lines IMR90 and WI38 (32), and Epstein-Barr virus-immortalized B cells (35). These studies implicate c-Myc as an important transactivator of hTERT.The human papillomavirus type 16 (HPV-16) E6 oncoprotein can also induce telomerase expression, specifically in epithelial cell types (20). Expression of HPV-16 E6 in either human foreskin keratinocytes (HFKs) or HMECs induces telomerase activity. Another well-established function of HPV-16 E6 is its association with the cellular E6-associated protein (E6AP) to form a ubiquitin protein ligase that specifically targets p53 for degradation (16,17,28). The HPV-16 E6-8S/9A/10T mutant is defective in p53 degradation yet retains the ability to activate telomerase, demonstrating that these two functions of E6 are separate and distinct(20). Expression of HPV-16 E6 does not induce telomerase in human foreskin fibroblasts (20) or in IMR90 cells (32). It has been suggested that a cell-type-specific ability of HPV-16 E6 to induce c-myc expression is responsible for this differential telomerase activation (35). In this study, we show that upregulation of c-myc does not directly correlate with telomerase activation, indicating that other regulators of hTERT expression are also involved. We also demonstrate that activation of telomerase by HPV-16 E6 does not require upregulation of c-myc, yet intact E boxes in the hTERT promoter are required for HPV-16 ...

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 ubiquitinmediated 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.Normally, somatic cells undergo a finite series of population doublings before entering cellular senescence (24,25). A critical marker of a cell's age is the length of its telomeric DNA (1); with each cellular division, up to 200 nucleotides of DNA are lost at the ends of chromosomes (23,41). Cells that require infinite replicative potential, such as stem cells, protect their telomeric DNA from erosion by constitutively expressing telomerase, a ribonucleoprotein complex that extends telomeric DNA, and thus, these cells avoid senescence. Tumors also overcome cellular senescence in order to continue their growth (22), and many activate telomerase through up-regulation of hTERT, the catalytic subunit of telomerase (63). Thus, hTERT expression and telomerase activity are critical in cellular immortalization and carcinogenesis.Various proteins have been shown to be important regulators of hTERT. They include those that act as transcriptional repressors, including p53, p73, AP-1, and Menin (45, 59, 62, 67), as well as transcriptional activators, such as N-terminally truncated p73, c-Myc, and Sp1 (5,56,57,68,70,74). c-Myc and Sp1 have been shown to bind to the core hTERT promoter and increase hTERT mRNA levels (56,57,70,74), although Sp1 and Sp3 can also recruit histone deactylase to the hTERT promoter to repress expression (73). c-Myc and Sp1 have been found to affect hTERT, but their relative protein levels do not always correlate with the downstream hTERT mRNA and protein expression levels (17,57,69). Other important facto...

While p53 activity is critical for a DNA damage-induced G(1) checkpoint, its role in the G(2) checkpoint has not been compelling because cells lacking p53 retain the ability to arrest in G(2) following DNA damage. Comparison between normal human foreskin fibroblasts (HFFs) and HFFs in which p53 was eliminated by transduction with human papillomavirus type 16 E6 showed that treatment with adriamycin initiated arrest in G(2) with active cyclin B/CDC2 kinase, regardless of p53 status. Both E6-transduced HFFs and control (LXSN)-transduced cells maintained a prolonged arrest in G(2); however cells with functional p53 extinguished cyclin B-associated kinase activity. Down regulation was mediated by p53-dependent transcriptional repression of the CDC2 and cyclin B promoters. In contrast, cells lacking p53 showed a prolonged G(2) arrest despite high levels of cyclin B/CDC2 kinase activity, at least some of which translocated into the nucleus. Furthermore, the G(2) checkpoint became attenuated as p53-deficient cells aged in culture. Thus, at late passage, E6-transduced HFFs entered mitosis following DNA damage, whereas the age-matched parental HFFs sustained a G(2) arrest. These results indicate that normal cells have p53-independent pathways to maintain DNA damage-induced G(2) arrest, which may be augmented by p53-dependent functions, and that cells lacking p53 are at greater risk of losing the pathway that protects against aneuploidy.

Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D. radiodurans were developed. These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D. radiodurans and carry homologous sequences for replacement recombination in the D. radiodurans chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D. radiodurans R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the ؊10 and ؊35 regions of these promoters resembled the 70 consensus sequence of E. coli.Its extraordinary tolerance to extremely high doses of ionizing radiation has made Deinococcus radiodurans the focus of growing scientific interest. This non-spore-forming bacterium is able to survive up to 4,000 times the lethal radiation dose for humans without mutation or loss of viability (2, 9). D. radiodurans is also of interest as a representative of a deeply branching family within the domain Bacteria (10). The sequence of the D. radiodurans R1 genome was recently published and shown to consist of two chromosomes, a megaplasmid, and one plasmid (17).Despite the interest in D. radiodurans, little is known concerning basic gene expression and promoters. Earlier studies showed that Deinococcus promoter regions are poorly recognized in Escherichia coli, and E. coli promoters that were tested were not recognized in D. radiodurans (7,14), suggesting that deinococcal promoters might be different from the classical E. coli 70 type. However, no transcriptional analysis of deinococcal promoters has been carried out. Analysis of the recently published genome sequence revealed only three putative sigma factors, one classing with vegetative 70 (rpoD) sequences, and two classing with extracytoplasmic alternative transcription factors (annotated as rpoE and DR0804 [17]). Surprisingly, orthologs of the nitrogen-starvation, general starvation, and heat shock sigma factors (rpoN, rpoS, and rpoH, respectively) were not found.One reason for the lack of information on promoters in deinococci is the lack of convenient genetic tools for studying promoters. A promoter cloning vector has been described (7), but it involves an antibiotic resistance reporter and is a large plasmid with limited cloning sites. Therefore, we developed a suite of integrative promoter-screening vectors that allow the screening and assessment of promoter regions in D. radiodurans based on lacZ and xylE as reporters. These vectors were used to isolate and analyze promoter regions, and promoter regions were further defined by transcripti...

Nearly 20% of cancers worldwide have a component of their etiology that is due to infectious agents. In some cases, infection has an indirect effect, such as the immunosuppression caused by HIV or the inflammation caused by Helicobacter pylori, but in other cases, such as human papillomaviruses (HPVs), viral gene products persist in the cancer and directly promote neoplasia. Understanding the mechanisms by which the viral genes disrupt the checkpoints that normally protect cells from cancer will likely provide insights into cancers in which the underlying critical abnormalities are more difficult to discern.Both epidemiologic observations and molecular data firmly support a causal role for a group of HPVs in the etiology of virtually 100% of cervical carcinomas, as well as the majority of other anogenital cancers and a subset of head and neck cancers (Cogliano et al. 2005). Of these HPV types, HPV-16 DNA is found in more than 50% of tumors (Walboomers et al. 1999). Two viral genes, E6 and E7, are invariably retained and expressed in cervical cancers, and together E6 and E7 efficiently immortalize human epithelial cells. The E7 protein associates with the retinoblastoma (Rb) family of proteins through a LXCXE motif and promotes the ubiquitin-mediated degradation of Rb, p107, and p130 (Munger et al. 2001). Degradation of Rb is necessary but not sufficient for E7's role in cellular immortalization, which also requires sequences in the carboxy-terminal zinc-like finger (Helt and Galloway 2001). HPV-16 E6 associates with a cellular protein, E6AP, and together the complex functions as a ubiquitin ligase (Huibregtse et al. 1993). The p53 tumor suppressor is the best-studied target of E6/E6AP, and its degradation eliminates several checkpoints that normally maintain genetic stability (Kessis et al. 1993;Demers et al. 1994).Disruption of the Rb and p53 pathways is critical for transformation of many human cell types, but it is also essential to prevent telomere shortening (Hahn et al. 1999). In some strains of human fibroblasts, the introduction of hTERT, the catalytic subunit of telomerase, is sufficient for immortalization (Bodnar et al. 1998;Kiyono et al. 1998;Vaziri and Benchimol 1998;Wang et al. 1998). Telomerase may play additional roles in tumorigenic transformation, because hTERT was necessary for transformation of cells that maintained long telomeres by the ALT pathway (Stewart et al. 2002). Nearly all tumors and cells transformed in culture express hTERT at levels that provide sufficient telomerase activity to keep telomeres above a critically short level (Kim et al. 1994). Multiple mechanisms are likely responsible for regulation of telomerase activity, including changes in transcription factors (Xu et al. 2001), loss of transcriptional repressors (Horikawa et al. 1998;Ducrest et al. 2001;Lin and Elledge 2003), changes in chromatin structure (Takakura et al. 2001;Hou et al. 2002), and altered levels of telomere-binding proteins (van Steensel et al. 1998). Additionally, cancers may arise in stem cells in which ...

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