HPV 5 and 8 E6 expression reduces ATM protein levels and attenuates LINE-1 retrotransposition

Abstract

The expression of the E6 protein from certain members of the HPV genus β (β HPV 5 and 8 E6) can disrupt p53 signaling by diminishing the steady state levels of two p53 modifying enzymes, ATR and p300. Here, we show that β-HPV 5 and 8 E6 are also capable of reducing the steady state levels of another p53 modifying enzyme, ATM, and as a result restrict LINE-1 retrotransposition. Furthermore, we show that the reduction of both ATM and LINE-1 retrotransposition is dependent upon the ability of β-HPV 8 E6 to bind and degrade p300. We use inhibitors and dominant negative mutants to confirm that ATM is needed for efficient LINE-1 retrotransposition. Furthermore, neither sensitivity to LINE-1 expression nor LINE-1 induced DSB formation is altered in an ATM deficient background. Together, these data illustrate the broad impact some β-HPVs have on DNA damage signaling by promoting p300 degradation.

Figure 1. Beta HPV E6 expression decreases steady state ATM levels

A. Representative immunoblot showing ATM protein levels in HFK cells. p53 is shown as a control for HPV 16 E6 expression. Unlike β HPV E6 expression, HPV 16 E6 expression promotes constitutive p53 degradation. Nucleolin (Nuc) was included as a loading control. B. Densitometry of immunoblots (n=3) of ATM protein level in HPV E6 expressing HFK cells. Levels were normalized to nucleolin for each experiment and graphed with ATM protein levels in LXSN cells arbitrarily set to 100. Error bars represent standard errors of the mean. * denotes a statistically significant difference from LXSN cells (p ≤ 0.05, Student T test and 2 way ANOVA with Bonferroni correction). C. Representative immunoblot showing ATM levels in HFK cells. D. Densitometer of immunoblots (n=3) of ATM protein levels in HPV E6 HFK cells. Levels were normalized to β-actin for each experiment and graphed with ATM protein levels in LXSN cells arbitrarily set to 100. Error bars represent standard errors of the mean. * denotes a statistically significant difference from LXSN cells (p ≤ 0.05, Student T test and 2 way ANOVA with Bonferroni correction).

Figure 2. Beta HPV E6 expression reduces steady state ATM levels by promoting p300 degradation

A. Representative immunoblot showing ATM levels in HFK cells cotransfected with either p300wt or p300 S1834E and vector control or HPV 8 E6. β-Actin was used as a loading control. B. Densitometry of immunoblots (n=3) of ATM protein levels in HPV E6 expressing HFK cells cotransfected with either p300wt or p300 S1834E and vector control or HPV 8 E6. Levels were normalized to β-actin for each experiment and graphed with p300 wt arbitrarily set to 100. Error bars represent standard errors of the mean. * denotes a statistically significant difference from HFK cells cotransfected with vector control and p300wt (p ≤ 0.05, Student T test). C. p300 was knocked down in HFK cells by transfection with a pool of 4 siRNAs targeting p300 or a pool of 4 non-targeting siRNAs. Representative immunoblots showing ATM and p300 levels in these HFK cells 72 hours post transfection. β-actin was used as a loading control. D. Densitometry of immunoblots (n=3) of ATM and p300 protein levels in HFK cells transfected with a pool of four p300 targeting siRNAs or a pool of four non targeting siRNAs. Levels were normalized to β-actin for each experiment and graphed with control siRNA arbitrarily set to 100. Error bars represent standard errors of the mean. * denotes a statistically significant difference from HFK cells transfected with control siRNAs (p ≤ 0.05, Student T test).

Figure 3. LINE-1 retrotransposition requires ATM kinases activity

A. LINE-1 retrotransposition assay. A tagged full Length LINE-1 retrotransposon is transfected into cells. This 3′ tag consists of a reporter (Blastocidin/Neomicin resistance) construct including promoter in the opposite orientation of the retrotransposon. A self-splicing intron in the same orientation as the retrotransposon disrupts the reporter gene. Following transcription from the LINE-1 promoter, this intron is removed resulting in LINE-1 mRNA that contains a reporter gene that is no longer disrupted by the intron. This mRNA is then reverse transcribed and inserted into the genome by target primed reverse transcription (TPRT). The new LINE-1 insert also contains a now functional reporter gene that is capable of providing Neomycin/Blasticidin resistance to the cell containing the new insertion. Resistant colonies resulting from the cells containing these inserts are scored as evidence of at least one LINE-1 retrotransposition event. B. NeoR/BlastR colony formation assay. In order to assess the impact of inhibitor treatment or exogenous gene expression on colony formation, cells transfected with a reporter plasmid are either co-transfected with the exogenous gene of interest (an untagged LINE-1 or ATM) or treated with inhibitor. These cells are then grown in selection media and relative colony formation is used as an indication of the impact inhibitor treatment/exogenous gene expression has on colony formation. C. Caffeine, DMSO, wortmannin, and vanillin were analyzed for effects on colony formation following transfection with a tagged LINE-1 (L1) expression vector or Neomycin resistance (NeoR) vector as a control. HeLa were transfected with either LINE-1 NeoR or NeoR vectors and treated with the indicated final concentrations of inhibitors or DMSO as a control. Selection media was added 48 hours post transfection. Charts show average number of colonies relative to untreated control that was arbitrarily set to 100. Error bars represent standard errors of the mean. * Denotes significant lower than NeoR control (p ≤ 0.05 student T-test). D. Tagged LINE-1 expression vector or Neomycin resistance vector were cotransfected with ATM cDNA or ATM kinase defective (ATMkd) and colonies were quantified after growth for 2 weeks in selection media. Charts show mean (triplicate in four independent experiments) number of colonies relative to ATM cDNA control that was arbitrarily set to 100. Error bars represent standard errors of the mean. * Denotes significant lower than NeoR control (p ≤ 0.05 student T-test).

Figure 4. HPV E6 expression attenuates LINE-1 retrotransposition

A. and B. Retrotransposition of a tagged LINE-1 (LINE-1 BlastR) was measured in HPV E6 expressing HT1080 cells. The chart shows mean colony numbers (n=4) relative to LXSN control HT1080 cells. Error Bars represent standard errors of the mean. * denotes statistically significant difference from LXSN (p ≤ 0.05, Student T test and 2 way ANOVA with Bonferroni correction).

Figure 5. Decreased ATM levels do not sensitize cells to LINE-1 expression

A. The AT derived cell lines YZ-5 (ATM complemented) and EBS-7 (ATM−) were transfected with LINE-1 expression vector (L1) or control vector (pCEP4) in combination with a blasticidin resistance plasmid. Cells underwent selection for blasticidin for two weeks and colonies were counted to measure LINE-1 toxicity. Error bars represent standard errors of the mean. * denotes significant difference from empty vector control (p ≤ 0.05, Student T test). 1 denotes significantly different from YZ-5 cells. B. HPV E6 expressing HT1080 cells were transfected with either an empty vector or one expressing LINE-1 ORF2. 24 hours after transfection, growth media was replaced with selection media and cells were grown for an additional three days. Chart shows mean (n=3) proliferation in LINE-1 ORF2 transfected cells relative to control transfected cells. Error bars represent standard errors of the mean. * denote a statistical difference from LINE-1 ORF2 transfected LXSN cells (p ≤ 0.05, Student T test and 2 way ANOVA with Bonferroni correction). 1 denotes a statistically significant difference from control transfected cells (p ≤ 0.05, Student T test and 2-way ANOVA with Bonferroni correction).

Figure 6. LINE-1 induced DSB formation is not altered in ATM−/− cells

A.YZ-5 (ATM complemented) and EBS (ATM−) cells were transfected with LINE-1 expression vectors, either wild type or an endonuclease mutant (L1 EN205A), or a control empty vector. 24 hours later the cells were harvested and subjected to neutral COMET assay. The relative tail moment is shown for each cell. B. Means of two replicates of neutral COMET assays. Tail length was set relative to empty vector control. Error Bars represent standard errors of the mean. * denote statistical difference from vector control (p ≤ 0.05, Student T test).