NFX1-123 and Poly(A) Binding Proteins Synergistically Augment Activation of Telomerase in Human Papillomavirus Type 16 E6-Expressing Cells

Abstract

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...

The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins

“…This position as been shown to be very important for binding and very sensitive to mutation (Lim et al 2006;Kozlov et al 2010). However, in two cases (for the human NF-X1 and TOB2 proteins), a PAM2 also presenting a leucine-to-phenylalanine substitution at the same position was found to be involved in binding to PABP (Okochi et al 2005;Lim et al 2006;Katzenellenbogen et al 2007;Kozlov et al 2010). …”

Human papillomavirus and molecular considerations for cancer risk

“…19, 20 The RNA, noncatalytic component of telomerase, hTERC, has not been demonstrated to interact directly with HPV oncoproteins; however, increased copy number has been reported with a gain of chromosome 3q. 21 Epigenetic regulation of hTERT through histone modification has been reported after the interaction between E6, E6AP, and p300, a coactivator/acetyl transferase.…”

Characterization of a cluster of oncogenic mutations in E6 of a human papillomavirus 83 variant isolated from a high-grade squamous intraepithelial lesion

“…Transcriptional control of the catalytic subunit of human telomerase reverse transcriptase (hTERT) plays a major role in the regulation of telomerase activity in many human cancers (Ducrest et al, 2002;Horikawa & Barrett, 2003). In normal somatic cells, E6 of HR-HPV induces the hTERT promoter (Liu et al, 2009;Veldman et al, 2001) through degradation of the transcriptional repressor NFX1-91 (Gewin et al, 2004;Katzenellenbogen et al, 2007Katzenellenbogen et al, , 2009 or through its binding to Myc (Liu et al, 2008;Veldman et al, 2003). Indeed, E6 from HR-HPV can increase cell proliferation through the upregulation of telomerase activity (Veldman et al, 2001).…”