The acetyltransferase Tip60 is a critical regulator of the differentiation-dependent amplification of human papillomaviruses

Abstract

The life cycle of human papillomaviruses (HPVs) is dependent upon differentiation of the infected host epithelial cell as well as activation of the ataxia telangiectasia mutated (ATM) DNA repair pathway that in normal cells acts to repair double-strand DNA breaks. In normal cells, following DNA damage the acetyltransferase Tip60 must acetylate ATM proteins prior to their full activation by autophosphorylation. E6 proteins have been shown to induce the degradation of Tip60, suggesting that Tip60 action may not be required for activation of the ATM pathway in HPV-positive cells. We investigated what role, if any, Tip60 plays in regulating the differentiation-dependent HPV life cycle. Our study indicates that Tip60 levels and activity are increased in cells that stably maintain complete HPV genomes as episomes, while low levels are seen in cells that express only HPV E6 and E7 proteins. Knockdown of Tip60 with short hairpin RNAs in cells that maintain HPV episomes blocked ATM induction and differentiation-dependent genome amplification, demonstrating the critical role of Tip60 in the viral life cycle. The JAK/STAT transcription factor STAT-5 has previously been shown to regulate the phosphorylation of ATM. Our studies demonstrate that STAT-5 regulates Tip60 activation and this occurs in part by targeting glycogen synthase kinase 3β (GSK3β). Inhibition of either STAT-5, Tip60, or GSK3β blocked differentiation-dependent genome amplification. Taken together, our findings identify Tip60 to be an important regulator of HPV genome amplification whose activity during the viral life cycle is controlled by STAT-5 and the kinase GSK3β.

Importance: Human papillomaviruses (HPVs) are the etiological agents of cervical and other anogenital cancers. HPVs regulate their differentiation-dependent life cycle by activation of DNA damage pathways. This study demonstrates that HPVs regulate the ATM DNA damage pathway through the action of the acetyltransferase Tip60. Furthermore, the innate immune regulator STAT-5 and the kinase GSK3β mediate the activation of Tip60 in HPV-positive cells. This study identifies critical regulators of the HPV life cycle.

FIG 1

The levels of Tip60 are increased in HPV-positive cells that stably maintain episomes. (A) Western blot (immunoblot [IB]) analysis of Tip60, p-Tip60, and GAPDH levels in HFKs (lane HFK) and cells positive for HPV31 (lane 31), HPV18 (lane 18), and HPV16 (lane 16) grown in undifferentiated monolayer cultures. (B) Western blot analysis of Tip60 and GAPDH levels in HFKs and cells positive for HPV31 E5 KO (31E5KO), HPV31, HPV31 E6 (31E6), and HPV31 E6E7 (31E6E7) grown in monolayer cultures. (C) Western blot analysis of Tip60, p-Tip60, and GAPDH levels in HFKs, HPV31-positive keratinocytes, and HPV31-positive CIN612 cells differentiated in high-calcium medium for the times (in hours) indicated beneath the blots. All results are representative of observations from 3 independent experiments.

FIG 2

Suppression of Tip60 by shRNA knockdown blocks HPV genome amplification and late gene expression upon keratinocyte differentiation. HPV31-positive CIN612 cells were infected with lentiviruses expressing TRC1 scrambled shRNA or Tip60 shRNA and incubated for 72 h postinfection, followed by an additional 72 h of differentiation in high-calcium medium. (A) Western blot analysis of Tip60 and GAPDH proteins in monolayer CIN612 cells transduced with different shRNAs against Tip60. TRC1, TRC1 plasmid backbone; #1 to #4, shTip60-1 to shTip60-4, respectively. (B) Southern blot analysis for HPV31 episomes in CIN612 cells following infection with shRNA-expressing lentiviruses and differentiation in high-calcium medium for the times (in hours) indicated beneath the blots. (C) Northern blot analysis for HPV31 early and late gene expression in CIN612 cells following infection with shRNA-expressing lentiviruses and differentiation in high-calcium medium for the times (in hours) indicated beneath the blot. All results are representative of observations from 2 or more independent experiments.

FIG 3

Knockdown of Tip60 suppresses activation of the ATM DNA damage response. HPV31-positive CIN612 cells were transduced with lentivirus expressing shRNAs against Tip60 (shTip60-1) as described in legend to Fig. 2. The transduced cells were assayed by Western blotting for Tip60, p-ATM, ATM, p-CHK2, CHK2, acetyl-p53, p53, and GAPDH protein levels upon differentiation in high-calcium medium for the times (in hours) indicated beneath the blots. All results are representative of observations from 2 or more independent experiments.

FIG 4

Inhibition of STAT-5 suppresses Tip60-dependent activation of the ATM DNA damage response. (A) RT-PCR analysis of Tip60 mRNA levels in CIN612 cells in the presence or absence of pimozide (10 μM) for the times (in hours) indicated beneath the bars. (B) Western blot analysis of Tip60, p-Tip60, and GAPDH protein levels of CIN612 cells for the times (in hours) indicated beneath the blots. (C) Western blot analysis for p-STAT-5, STAT-5, p-Tip60, Tip60, and GAPDH levels in cells positive for HPV31, HPV31 E5 KO, HPV31 E6, and HPV31 E6E7 grown in monolayer cultures. (D) Western blot analysis for p-Tip60, Tip60, and GAPDH levels in CIN612 cells treated with erlotinib at different concentrations for the various times (in hours) indicated beneath the blots. All results are representative of observations from 3 independent experiments.

FIG 5

Inhibition of GSK3β activity by CT98014 blocks HPV genome amplification upon keratinocyte differentiation. (A) Western blot analysis of GSK3α, GSK3β, and GAPDH levels in HFKs, HPV31-positive keratinocytes, and HPV31-positive CIN612 cells differentiated in high-calcium medium for the times (in hours) indicated beneath the blots. (B) Relative levels of expression of target proteins normalized to the levels of GAPDH expression from the Western analysis whose results are shown in panel A. The statistical analysis was performed by 2-tailed t test. Data are means ± standard errors. *, P < 0.05. The band intensities were determined by ImageJ (64-bit) software. (C) Southern blot analysis for HPV31 episomes in CIN612 cells untreated or treated with CT98014 (2 μM) following differentiation in high-calcium medium for the times (in hours) indicated beneath the blots. (D) Western blot analysis of the GSK3α, GSK3β, p-Tip60, Tip60, p-ATM, ATM, and GAPDH proteins of CIN612 cells following differentiation induced in high-calcium medium in the presence or absence of CT98014 (2 μM) for the times (in hours) indicated beneath the blots. All results are representative of observations from 3 independent experiments.

FIG 6

Loss of STAT-5 reduces GSK3β activation. (A) Western blot analysis of the GSK3α, GSK3β, and GAPDH proteins of CIN612 cells following differentiation induced in high-calcium medium in the presence or absence of pimozide (10 μM) for the times (in hours) indicated beneath the blots. (B) RT-PCR analysis of GSK3β mRNA levels in CIN612 cells in the presence or absence of pimozide (10 μM). The difference in the results between assays performed in the presence and the absence of pimozide was statistically significant (P < 0.05). All results are representative of observations from 3 independent experiments. DMSO, dimethyl sulfoxide.

FIG 7

Model of how Tip60 activation by HPV could lead to ATM activation and amplification upon differentiation. HPV proteins work in combination to suppress E6 expression and/or function, leading to Tip60 activation. Tip60 activation can be suppressed by pimozide treatment, leading to inhibition of amplification. Activated Tip60 acetylates (Ac) the ATM kinase, resulting in ATM autophosphorylation. The activated ATM then induces the phosphorylation (P) of SMC1, CHK2, and γ-H₂AX, which contribute to HPV genome amplification.