Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

RNF168 Is Expressed at High Levels in HPV⁺ Cancers.

As HPV⁺ tumors exhibit large nuclear foci of RNF168 and 53BP1, we first sought to determine if the levels of these proteins are specifically increased in these tumors. Strikingly, analysis of RNF168 mRNA expression in cancer samples from the cBioPortal database (33, 34) revealed that cervical and head and neck are among the few types of cancers in which the E3 ligase is overexpressed (SI Appendix, Fig. S1A). Next, we analyzed the Illumina HiSeq RNA-seq data from The Cancer Genome Atlas (TCGA) for 297 cervical carcinoma (CESC; 278 HPV⁺ and 19 HPV⁻) and 558 head and neck squamous cell carcinoma (HNSC; 73 HPV⁺, 442 HPV⁻, and 43 normal-adjacent tissues) cohorts for expression of RNF168 and 53BP1 (Fig. 1A). For comparison, the expression levels of 2 other ubiquitin ligases that also accumulate on chromatin following DSBs, RNF8 and RNF169, were analyzed. Strikingly, both types of HPV-related cancers were found to express high levels of RNF168 mRNA, as shown by the normalized RNA-seq absolute read count values. This increase is highly significant when comparing HPV⁺ and HPV⁻ samples from the HNSC cohort (P ≤ 0.0001) and is observed independently of the HR-HPV type detected in these samples (Fig. 1A and SI Appendix, Fig. S1B). In samples from the HNSC cohort, expression levels of 53BP1 were also significantly increased, although this was not the case in the CESC cohort. In contrast to RNF168 and 53BP1, the expression levels for RNF8 and RNF169 were not increased in HPV⁺ samples. In fact, RNF8 expression was markedly reduced in HPV⁺ samples compared with HPV⁻ tumors, indicating that the increased expression of RNF168 does not reflect a general up-regulation of DNA repair genes in these cancers. Consistent with these results, analysis of a panel of HPV⁺ cell lines isolated from cervical (SiHa, HeLa, CaSki) and head and neck carcinomas (UM-SCC-47; hereafter referred to as SCC47) revealed that the levels of RNF168 are markedly increased in HPV⁺ cell lines compared with HPV⁻ CESC (C33-A) and HNSC (UM-SCC-1; referred to hereafter as SCC1) cell lines (Fig. 1B and SI Appendix, Fig. S1 C and D). The later observation suggested that viral proteins encoded by these integrated genomes lead to increased expression of RNF168 in HPV⁺ cells.

When analyzed by immunochemistry, parafilm-embedded HPV⁺ tumor tissues were found to accumulate large foci of RNF168 and 53BP1 (32), a phenotype that is reminiscent of the formation of 53BP1-NBs, a marker of genomic instability induced by replication stress. These nuclear structures shield DNA ends from further processing until they reach the next late S phase, in which they will be repaired by RAD52-mediated repair (35–38). To determine whether the appearance of these foci is related to the HPV status of these cells, we analyzed the localization of RNF168 and 53BP1 in the panel of HPV⁺ and HPV⁻ cells described here earlier by immunofluorescence microscopy. In these experiments, we found that the number of large 53BP1 bodies was significantly increased in HPV⁺ CESC and HNSC cell lines and directly correlated to the number of integrated viral genomes (Fig. 1 C and D and SI Appendix, Fig. S1E). These foci were present only during the G1 phase of the cell cycle (marked by the absence of cyclin A staining; Fig. 1E and SI Appendix, Fig. S1F) and were abolished by depletion of RNF168 (SI Appendix, Fig. S1 G–I), indicating that they correspond to 53BP1-NBs. Consistently, we found that BRCA1, but not the DNA repair factor RAD51, colocalizes with 53BP1 in these bodies (Fig. 1E and SI Appendix, Fig. S1J). Taken together, these results indicate that increased expression of RNF168 and accumulation of genomic instability are associated with the presence of HPV in human cancer cells.

HPV E7 Proteins Directly Interact with RNF168.

Many DSB signaling and repair proteins accumulate at viral replication centers during the viral replication cycle (10). Combined with the results described here earlier, this raises the possibility that RNF168-mediated signaling is directly subverted by one or more viral gene products. To explore this possibility, we used a LacO/LacR system that recapitulates the recruitment of DSB repair factors downstream of γ-H2AX in the absence of DNA damage (Fig. 2A) (24, 39). Specifically, we investigated if the viral replication proteins E1 and E2, E4, or the oncoproteins E6 and E7 from the prevalent HR-HPV31 could promote the recruitment of DSB-signaling proteins at the LacO-array, visible as a single nuclear focus in this cell-based assay. E5 and L1/L2 proteins were not tested, as they are, respectively, a transmembrane protein and capsid structural proteins only expressed in terminally differentiated keratinocytes (4). In this assay, mCherry-LacR fused to the C-terminal DNA cleavage domain of the endonuclease FOKI was used as positive control to monitor the recruitment of DSB-signaling factor at the LacO-array. Surprisingly, we found that mCherry-LacR-E6 and -E7 promoted a low but significant accumulation of 53BP1 at the LacO-array (Fig. 2 B and C and SI Appendix, Fig. S2 A and G). The absence of γ-H2AX at the array 6 h posttransfection shows that E6 and E7 recruit 53BP1 independently of DSB signaling in these conditions, unlike E2, which triggers the accumulation of the histone mark at the array, potentially through its ability to interact with TOPBP1, a factor that promotes ATR activation (40) (Fig. 2D and SI Appendix, Fig. S2B). Consistent with an interaction between the viral proteins and RNF168, we found that FLAG-RNF168, but not FLAG-RNF8, colocalized with the viral oncoproteins at the array in as many as 80% of the cells (Fig. 2 E and F and SI Appendix, Figs. S2 C, D, and H). Furthermore, while depletion of RNF8 drastically reduced the recruitment of 53BP1 to DSBs induced by targeting the nuclease FOKI at the LacO-array, it increased the amount of 53BP1 colocalizing with mCherry-LacR-E6 and -E7 (Fig. 2G and SI Appendix, Fig. S2 E and J). As RNF168 is a limiting factor during DSB signaling (32), it is possible that the depletion of RNF8 increases the amount of RNF168 available for recruitment to the array by E6 and E7.

HPV31 E7 and E6 interact with histone modifiers of the histone acetyl transferase (HAT) (41, 42) and deacetylase (HDAC) (43, 44) families. To exclude that E6 and E7 indirectly promote the recruitment of RNF168 to the array by inducing local epigenetic changes in chromatin, we investigated if mCherry-LacR-RNF168 also promoted the accumulation of the viral oncoproteins at the LacO-array. Under these conditions, only E7 was efficiently recruited to the array (Fig. 2H and SI Appendix, Fig. S2 F and I). Therefore, we decided to focus further analyses on the E7-mediated interaction. Using mCherry-LacR-E7, we also detected the accumulation of ubiquitin conjugates, as well as BRCA1, at the LacO-array (SI Appendix, Fig. S2 K–M), consistent with RNF168 being recruited to the array by HPV31 E7. However, although RNF169 is a paralog of RNF168, it is not recruited directly to the array by E7, as depletion of RNF168 prevents its recruitment (SI Appendix, Fig. S2N).

Finally, the interactions detected using the LacO-LacR tethering system could be indirect. To rule out this possibility, we showed that recombinant GST-HPV16 E7 protein efficiently pulled down recombinant His-RNF168 in vitro (Fig. 2I and SI Appendix, Fig. S2O), indicating that the interaction is direct and independent of posttranslational modifications. Taken together, these findings revealed that the HPV E7 protein specifically interacts with the E3 ligase RNF168 independently of the activation of the cellular response to DNA damage.

RNF168 Interacts with E7 from HR-HPVs and Is Required for Viral Replication.

Human papillomavirus E7 is essential for viral replication and HPV-induced carcinogenic transformation. E7 is a small protein of approximately 100 amino acids that is composed of 3 conserved regions (CR1–3; Fig. 3A). Mutations in any of these regions affect different biological activities of E7 (45). To gain insight into the role of the interaction between E7 and RNF168, we first investigated if this interaction is conserved among the E7s from different types of virus and which domain of E7 contributes to the interaction. According to their oncogenic potential, HPVs of the alpha genus are subdivided as low- and high-risk (4). HPV6 and 11 are classified as low-risk, as they are mainly associated with the emergence of benign genital warts. HPV16, 18, and 31 are part of a group of 12 HPV types classified as high-risk because of their association with cancer development (4). Interestingly, we observed that only the HPV E7 proteins from high-risk viruses promoted efficient recruitment of RNF168 to the LacO-array (Fig. 3B and SI Appendix, Fig. S3 A and B). These findings show that the interaction of E7 with RNF168 is conserved only among the alpha HR-HPV types. Surprisingly, we did not detect an interaction between RNF168 and E7 from HPV5 and HPV8, 2 types of virus that have been associated with the development of nonmelanoma skin cancers (46). In the LacO/LacR system, neither CR1 nor CR2, which comprises the pRb-binding motif (LXCXE), were sufficient to recapitulate the interaction with RNF168 (Fig. 3C and SI Appendix, Fig. S3 C and D). Accordingly, a point mutation or a small deletion that abolishes the interaction of E7 with the tumor suppressor pRb (C24G and DLYC) did not severely impact the recruitment of RNF168 by full-length E7 to the array (Fig. 3D and SI Appendix, Fig. S3 E and F). In contrast to CR1 and CR2, CR3 by itself was able to recruit RNF168 to the array, similarly to full-length E7 (Fig. 3C and SI Appendix, Fig. S3 C and D). This domain was also required for the interaction of E7 with RNF168 in immunoprecipitation experiments using cell extracts (Fig. 3E). CR3 is an atypical zinc finger-like domain that mediates the dimerization of E7 and is needed for its transforming activity (45). Using a panel of well described HPV16 E7 CR3 mutants (45, 47), we found that the recruitment of RNF168 to mCherry-LacR-E7 foci was only slightly impaired by a mutant that abolishes the interaction of E7 with HDACs (L67R; Fig. 3D and SI Appendix, Fig. S3 E and F). Interestingly, mutants of E7 that are transformation-defective and fail to interact with the cullin 2 ubiquitin ligase complex (CVQ, LEDLL) also significantly impair its interaction with RNF168.

As E7 is required for viral replication, we next investigated whether RNF168 is required for the replication of viral genomes. In CIN612 9E cells, which are derived from a biopsy of a low-grade cervical intraepithelial neoplasia (CIN1) and stably maintain multiple copies of the HPV31b genome in episomal form, depletion of RNF168 for 3 d had no impact on the amount of viral genome that is maintained in undifferentiated cells (shRNF168 vs. shScramble at T0; Fig. 4 A and B). Interestingly, Southern blot analysis of viral genome amplification, which occurs when CIN612 9E cells are induced to differentiate in high-calcium medium for 72 h, showed that RNF168 depletion substantially decreased the number of viral episomes compared with shScram (Fig. 4 A and B). In these experiments, the knockdown of RNF168 did not interfere with the ability of CIN612 9E cells to differentiate, as indicated by the increased levels of involucrin and keratin-10, 2 specific markers for differentiation. Consistent with the increased levels of RNF168 detected in HPV⁺ tumors, we also detected higher level of RNF168 in CIN612 9E compared with uninfected keratinocytes (Fig. 4C). Altogether, these results indicate that the expression of RNF168 E3 ligase is increased in HPV-infected cells and is required for productive viral replication upon differentiation. This phenotype is reminiscent of results obtained with knockdown or inhibition of ATM, MRE11, NBS1, RAD51, and BRCA1 (11, 12, 15, 16).

E7 Favors DNA Repair by HDR by Limiting the Function of RNF168 at DSBs.

Although the interaction of E7 with RNF168 can be easily detected at the LacO-array, the pan-nuclear localization of the viral protein observed in untreated cells remained unchanged in cells in which DNA lesions were induced by irradiation (IR), neocarzinostatin, or hydroxyurea, which induces lesions specifically in S-phase by blocking replication fork progression (SI Appendix, Fig. S4A). Similarly, E7 is not recruited to sites of intense DNA damage induced by targeting the FOKI nuclease to the LacO-array (Fig. 2H and SI Appendix, Fig. S2F). However, HPV16 E7 was recently reported to colocalize with 53BP1 using proximity-ligation assays, both in differentiated HPV⁺ keratinocytes and in CaSki (48), arguing that both proteins are near each other during viral replication and cellular transformation. Consistently, we found that E7 and the CR3 domain alone are recruited to foci of RNF168 that have been described as cellular replication factories in unperturbed conditions (Fig. 5A) (49). Thus, these results support a model in which E7 interacts with a subpopulation of RNF168 that localizes at replication forks.

RNF168 is a limiting factor during DSB signaling, as it quickly becomes saturated upon damage induction (32). This led us to investigate if E7 could reduce the amount of RNF168 at DSBs. Although the antibody against RNF168 detects high levels of DNA damage that accumulate in 53BP1-NBs (Fig. 1 C and D), it is less efficient at detecting the recruitment of RNF168 at irradiation-induced foci (SI Appendix, Fig. S1E; only 30% of γ-H2AX foci are RNF168-positive vs. 75% and 90% for conjugated-ubiquitin [FK2] and 53BP1 [Fig. 5B], respectively). Therefore, we quantified the formation of 53BP1 and FK2 foci as markers of RNF168 activity. To do so, U2OS cells stably expressing GFP-HPV31 E7 and E7 CR1-CR2 (referred to here as CR1/2) as a negative control were exposed to increasing doses of IR (Fig. 5B and SI Appendix, Fig. S4 B–F). As the accumulation of 53BP1 to DSBs is regulated in a cell cycle manner (20), we performed the experiment in U2OS cells in which the impact of E7 on DSB signaling can be uncoupled from its impact on cell cycle progression (pRb degradation). Indeed, pRB is always phosphorylated (inactive) in U2OS as a result of the absence of pRB dephosphorylation after mitosis (50). Interestingly, we observed that the total number of radiation-induced 53BP1 and FK2 foci were significantly reduced in cells expressing the viral oncoprotein compared with cells expressing a negative control only (Fig. 5B and SI Appendix, Fig. S4 B, E, and F). Importantly, the levels of γ-H2AX quantified in cells expressing HPV31 E7 were similar to those in the control cell lines (Fig. 5B and SI Appendix, Fig. S4 C, E and F). The same results were obtained when GFP-HPV31 E7 was expressed in human foreskin keratinocytes expressing the catalytic component of telomerase (hTERT-HFK; Fig. 5C and SI Appendix, Fig. S4 G and H) (51). Inhibition of RNF168 or 53BP1 leads to increased repair by HDR (20). Consistent with the reduced accumulation of RNF168 substrates at DSBs in E7-expressing cells, we also found that stable expression of E6/E7 in C33-A cell (HPV⁻ cervical carcinoma) increases DNA repair by HDR (Fig. 5 D and E and SI Appendix, Fig. S4 I–M). These experiments were performed by measuring gene targeting efficiency of an mClover donor sequence at the LMNA locus in C33-A, C33-A stably expressing E6 and E7, and C33-A KO 53BP1 as positive control. Altogether, these observations support a model in which E7 directly hinders the function of RNF168 at DSBs.

E7 Interacts with RNF168 without Affecting Its E3 Ubiquitin Ligase Activity.

RNF168 is an E3 ubiquitin ligase that is composed of a RING domain (catalytic activity) and 2 ubiquitin recognition modules, which enable the recognition of RNF8’s substrate (UDM1) as well as its own substrate (UDM2; Fig. 6A). The latter activity is important for the autoamplification of RNF168 recruitment at DSBs. RNF168 also contains a PALB2-interacting domain (PID), which promotes its interaction with the complex PALB2/BRCA2 (52, 53). In vitro, RNF168 promotes ubiquitylation of histone H2A (23). As E7 reduces the accumulation of conjugated ubiquitin at DSBs (Fig. 5B), we investigated if E7 inhibits the E3 ligase activity of RNF168. Using in vitro ubiquitylation assay on mononucleosomes, we found that GST-HPV16 E7 does not interfere with H2A ubiquitylation by RNF168 (Fig. 6B). In this assay, along with a ubiquitylation assay that was performed without mononucleosomes, we found that GST-HPV16 E7 is not ubiquitylated (Fig. 6C and SI Appendix, Fig. S5A), suggesting that the viral protein is not a direct target of RNF168. We thus turned our attention to the interaction between RNF168 and E7 to gain insight into how the viral oncoprotein subverts the function of this E3 ubiquitin ligase. We assessed the ability of various mCherry-LacR-RNF168 fusion proteins to promote the recruitment of GFP-tagged E7 to the LacO-array. These experiments were done in RNF168-depleted cells to eliminate the endogenous protein as a potential confounding factor (24). Consistent with the results obtained in GST pull-down, we found that the catalytic activity of RNF168 was dispensable for its interaction with E7 (Fig. 6 D and E and SI Appendix, Fig. S5 B–D), but necessary for the recruitment of 53BP1 at the mCherry-LacR locus. Deletion of either one or both motifs of RNF168 that bind ubiquitin (MIUs; Fig. 6A) had little to no effect on the accumulation of GFP-HPV31 E7 at the LacO-array. Surprisingly, deletion of RNF168 amino acids 251 to 383 abolished its interaction with E7 both at the LacO-array and at RNF168-positive foci observed in unperturbed cells, but had no impact on its ability to recruit 53BP1 to the array (Fig. 6 D and E and SI Appendix, Fig. S5E). Furthermore, when residues 251 to 383 of RNF168 were fused to mCherry-LacR, this region was sufficient to recruit GFP-HPV31 E7 to the LacO-array. These results indicate that the ability of RNF168 to recruit 53BP1 and to interact with E7 resides in separate domains of the protein that can be uncoupled genetically. The necessity of the residues 251 to 383 in the interaction between RNF168 and HPV E7 was further confirmed by immunoprecipitation experiments in a U2OS RNF168 KO cell line (SI Appendix, Fig. S6 A–F). Altogether, our study has revealed a previously uncharacterized domain of RNF168, the E7-binding domain (E7BD), that is targeted by the oncoprotein E7 to hinder its signaling function at DSBs.

Cell Culture and Transfections.

Cell lines were maintained at 37 °C and 5% CO₂. All culture media were supplemented with 10% fetal bovine serum (FBS). U-2-OS (U2OS) and U2OS 2-6-5, gift from Roger Greenberg, University of Pennsylvania, Philadelphia, PA (72), cell lines were cultured in McCoy’s medium (Life Technologies). C33-A, CaSki, SiHa, HeLa, SCC1, and SCC47 cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies). The media of SCC1 and SCC47 was also supplemented with 1% minimum essential media (MEM) nonessential amino acid (Life Technologies). U2OS 2-6-5, U2OS, and U2OS RNF168 KO inducible cell lines were cultured in McCoy’s medium containing 40 mg/mL G418 and induced with 5 µg/mL doxycycline when indicated. U2OS Flp-in were cultured in McCoy’s medium containing 200 µg/mL hygromycin and 5 µg/mL blasticidin. hTERT-HFK cells were provided by Aloysius J. Klingelhutz (University of Iowa, Iowa City, IA) (51) and cultured in K-SFM media (Life Technologies) containing 0.16 ng/mL of epithelial growth factor and 25 µg/mL of bovine pituitary extract. Plasmid transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. All cell lines were tested negative for mycoplasma contamination. CIN612 9E cells and HFK-31 cells were grown in E medium supplemented with 5 ng/mL mouse epidermal growth factor (BD Biosciences) in the presence of mitomycin C-treated J2 3T3 fibroblast feeder cells (73). When necessary, J2 feeders were removed from HPV⁺ cells by incubation with 1 mM EDTA in PBS. For differentiation, subconfluent cells were harvested at T0, and the remaining plates of cells were serum-starved overnight in basal keratinocyte growth medium (KGM; Lonza) with supplements for 16 h. Cells were then incubated in keratinocyte basal medium (KBM; Lonza) without supplements but with 1.8 mM CaCl₂. Cells were allowed to differentiate for 48 to 96 h after addition of high-calcium medium. DNA and protein were harvested, and viral genome amplification was measured by Southern blotting as for each experiment to ensure activation of the productive phase of the viral life cycle.

Southern Blot Analysis.

DNA was isolated and Southern blot analysis was performed as described previously (74). Briefly, cells were harvested in buffer containing 400 mM NaCl, 10 mM Tris (pH 7.5), and 10 mM EDTA. Cells were lysed by the addition of 30 µL of 20% SDS and subsequently treated with 15 µL of 10 mg/mL proteinase K overnight at 37 °C. DNA was then extracted with phenol chloroform and precipitated using sodium acetate and ethanol. Five micrograms of resultant DNA were digested with BamHI (which does not cut the HPV31 genome) or HindIII (which linearizes the HPV31 genome), separated on 0.8% agarose gel for 15 h at 40 V, and subsequently transferred to a positively charged nylon membrane (Immobilon-Ny+; Millipore). The ³²P-labeled linearized HPV31 genome was used as a probe.

RNA Interference.

Single siRNA duplexes targeting either RNF168 (D-007152-04) or a nontargeting sequence (D-001210-02-02) were purchased from Dharmacon. To knock down RNF8, a SMARTpools siRNA against RNF8 (D-006900-01-0020) was purchased from Dharmacon. Unless stated otherwise, all siRNAs were transfected in a forward transfection mode 24 h prior to cell processing using RNAimax (Invitrogen) according to the manufacturer’s protocol.

Generation of Lentiviruses.

Lentiviruses were produced as previously described (15). Briefly, plasmids expressing shRNAs or the indicated GFP-tagged proteins were cotransfected with vesicular stomatitis virus G (VSV-G) plasmid DNA and Gag-Pol-Tet-Rev plasmid DNA into HEK-293T cells using polyethyleneimine (PEI). At 48 to 72 h posttransfection, supernatants containing lentivirus were harvested, and CIN612 9E, U2OS, or hTERT-HFKs were transduced in the presence of 8 µg/mL hexadimethrine bromide (Polybrene; Sigma). Three days posttransduction, CIN612 9E were either harvested at T0 (undifferentiated sample) or differentiated in high-calcium medium for 72 h as described here earlier. Knockdown of RNF168 and the expression of the GFP constructs was confirmed for each experiment by Western blotting.

CRISPR-Cas9 Genome Editing of 53BP1 and RNF168.

C33-A and U2OS cells were transiently transfected with sgRNAs targeting 53BP1 or RNF168, respectively (SI Appendix, Table S1), and expressed from the pX459 vector containing Cas9 followed by the 2A-Puromycin cassette. The next day, cells were subcloned to form single colonies. Clones were screened by immunoblot and immunofluorescence to verify the loss of 53BP1 and RNF168. The genomic regions targeted by the CRISPR-Cas9 was subsequently characterized by PCR amplification, DNA sequencing, and TIDE analysis (75).

Immunofluorescence Microscopy.

Immunofluorescence was done as previously described (24) with the indicated antibodies (SI Appendix, Table S2). Micrograph images were taken using a Zeiss LSM700 laser-scanning microscope equipped with a 63× oil lens. In all micrographs, dashed lines indicated nucleus outlines. Insets represent a 10-fold magnification of the indicated region. Quantification was done on 3 biological replicates, and at least 50 cells were counted in each experiment. Unless stated otherwise, t test with Welch correction was realized to assess statistical significance.

Clover Assay.

C33-A stable cell lines were plated at 100,000 cells per well on 6-well plates 24 h post transfection with pX330-LMNAgRNA1 and pCR2.1-CloverLMNAdonor plasmids as previously described (76). At 72 h posttransfection, percentage of mClover-positive cells were analyzed by flow cytometry using an Accuri C6 (BD Biosciences).

Immunoprecipitation.

Cells were transfected with mCherry-LacR, mCherryLacR-HPV31 E7 full length, or mCherryLacR-HPV31 E7 CR1-CR2–expressing plasmids. At 15 h posttransfection, GFP, GFP-RNF168, GFP-RNF168 WT, and ∆E7BD expression were induced. Cells were lysed 9 h later in lysis buffer (50 mM Tris, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100) followed by Micrococcal nuclease (Sigma) treatment for 30 min. Cleared cell lysate (1 mg) was subjected to immunoprecipitation using 20 µL GFP Trap beads for 3 h at 4 °C. After 3 washing steps, beads were eluted in 2× Laemmli buffer for immunoblotting analyses.

Purification of Recombinant Protein.

GST-HPV16 E7, RNF168 WT, and ∆E7BD recombinant proteins were purified with technical help from Marie-Christine Caron from the laboratory of Jean-Yves Masson (Université Laval, Québec, QC, Canada) as previously described (53). For MBP-His recombinant protein purification, E. coli strain BL-21 was induced with 1 mM IPTG when they reached an OD₆₀₀ of 0.6. Next, fusion proteins were purified on Ni-NTA agarose (Qiagen) according to the batch method described in the manufacturer’s manual. All recombinant proteins were stored in 20 mM Tris acetate, pH 8.0, 200 mM potassium acetate, 10% glycerol, 1 mM EDTA, 0.5 mM DTT.

Ubiquitylation Reaction.

Mononucleosomes were isolated from the HeLa cells and ubiquitylated as described previously (25). Briefly, 2.5 µg mononucleosomes were incubated with 30 nM Uba1, 1.5 μM UbcH5a, 4 μM HIS-RNF168, 22 μM ubiquitin (Boston Biochem), and 3.33 mM ATP in a buffer containing 50 mM Tris⋅HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 μM ZnCl₂, and 1 mM DTT at 30 °C for 4 h. His-GST (4.44 µM) or GST-E7 (2.2 to 4.4 µM) proteins were added to the mixture. The reaction was quenched by adding 15 mM EDTA, pH 8.0. Denatured samples were separated on an SDS/PAGE and analyzed by Western blotting experiment.

GST Pull-Down.

For GST pull-down, 1 µg of purified GST-tagged protein was incubated with 4 µg of purified His-tagged protein in 1.5 mL of GSTB buffer (20 mM Hepes, pH 7.4, 150 mM KCl, 10% glycerol, 0.02% Triton X-100, 0.2 mM EDTA, 0.002 µg/mL aprotinin, 0.001 µg/mL pepstatin A, 0.002 µg/mL leupeptin, 1 mM AEBSF, 1 mM DTT, 1 mg/mL bovine serum albumin [BSA]) for 3 h at room temperature. Input was collected before the addition of glutathione Sepharose beads for 90 min at room temperature. Beads were then washed with GSTB buffer without BSA and directly eluted in 2× Laemmli buffer for immunoblotting analyses.

RNA-Seq Analyses.

Level 3 RNA-Seq by Expectation Maximization (RSEM) normalized Illumina HiSeq RNA expression data for the TCGA head and neck cancer (HNSC) and cervical carcinoma (CESC) cohorts were downloaded from the Broad Genome Data Analysis Center’s Firehose server (https://gdac.broadinstitute.org/). For all genes, the gene-level Firehose dataset was used. RSEM-normalized expression data were extracted, and the HPV status was manually curated based on published datasets as previously described (77). Primary patient samples with known HPV status were grouped as HPV-positive, HPV-negative, or normal-adjacent control tissue. For the boxplots, center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by GraphPad Prism, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. P values were assigned using a 2-tailed nonparametric Mann–Whitney U test.