Beta human papillomavirus 8E6 promotes alternative end joining
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Curated by eLife
Evaluation Summary:
This manuscript reports useful data on how human papillomavirus 8E6 protein regulates DSB repair pathways in human cells. The data support the claim that 8E6 promotes alternative end-joining through binding and destabilizing the p300 acetyltransferase, but the study remains relatively descriptive and incomplete as it is not yet clear which alternative end-joining pathway is involved lacking a test of a direct involvement of DNA polymerases theta (POLθ).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. The E6 protein of beta-human papillomavirus (HPV8 E6) impairs two critical DSB repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). However, HPV8 E6 only delays DSB repair. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesize that HPV8 E6 promotes a less commonly used DSB repair pathway, alternative end joining (Alt-EJ). Using CAS9-based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. At least some of this repair occurs through a subset of Alt-EJ known as polymerase theta dependent end joining. Finally, whole genome sequencing analysis showed HPV8 E6 caused an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap of how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.
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Evaluation Summary:
This manuscript reports useful data on how human papillomavirus 8E6 protein regulates DSB repair pathways in human cells. The data support the claim that 8E6 promotes alternative end-joining through binding and destabilizing the p300 acetyltransferase, but the study remains relatively descriptive and incomplete as it is not yet clear which alternative end-joining pathway is involved lacking a test of a direct involvement of DNA polymerases theta (POLθ).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Joint Public Review:
As demonstrated in alpha genus human papillomavirus (HPV) 16 positive head and neck squamous cell carcinoma, Hu et al. report that the E6 protein of beta genus HPV8, which is implicated in the development of skin cancer, promotes genomic instability by increasing use of error-prone alternative end-joining repair. Liu et al. (2018) showed that loss of TGFβ signaling compromised HR in HPV+ head and neck squamous cell carcinomas (HNSCC) and shifted repair to alt-EJ. A follow up paper in 2021 showed that cancers with an alt-EJ gene signature have high frequency of microhomology flanked indels, pathognomonic for alt-EJ repair. Leeman et al. (2019) demonstrated that HPV16 E7 promotes error-prone, alt-EJ and suppresses NHEJ. Hence the finding that E6 from beta HVP8 does so as well is not unexpected but it is important to …
Joint Public Review:
As demonstrated in alpha genus human papillomavirus (HPV) 16 positive head and neck squamous cell carcinoma, Hu et al. report that the E6 protein of beta genus HPV8, which is implicated in the development of skin cancer, promotes genomic instability by increasing use of error-prone alternative end-joining repair. Liu et al. (2018) showed that loss of TGFβ signaling compromised HR in HPV+ head and neck squamous cell carcinomas (HNSCC) and shifted repair to alt-EJ. A follow up paper in 2021 showed that cancers with an alt-EJ gene signature have high frequency of microhomology flanked indels, pathognomonic for alt-EJ repair. Leeman et al. (2019) demonstrated that HPV16 E7 promotes error-prone, alt-EJ and suppresses NHEJ. Hence the finding that E6 from beta HVP8 does so as well is not unexpected but it is important to provide convincing experimental evidence as is done in this manuscript. Here the authors confirm and extend earlier studies (Hu et al., 2020; Hu and Wallace, 2022; Wallace et al., 2015) showing that loss of p300, a transcription factor necessary for robust expression of key DNA repair genes, impairs execution of HR and NHEJ, which is a prerequisite for driving up use of alt-EJ as a salvage path. Both pathways are inhibited past the initiation step. During NHEJ 8E6 allows DNA-PKcs assembly and activation at DSBs but attenuates downstream steps, Likewise, during HR 8E6 allows assembly of RAD51 foci but blocks their processing. As 8E6 does not block but rather delays DSB repair, this manuscript provides convincing evidence that alternative end-joining of DSBs is increased in 8E6 expressing cells. The study employs gamma-H2AX foci as a surrogate for DSB levels and employs appropriate reporter assays to monitor HR and alt-EJ. Genomic DNA sequencing of 8E6 expressing human foreskin keratinocytes and control cells documents the typical genome scars of elevated alt-EJ including small deletions flanked by microhomology and small templated insertions. The results contribute to our understanding of DSB repair pathway control and may explain the association of HPV8 with nonmelanoma skin cancer, although this tumor type specificity remains unclear. A key limitation of the study that it is not established which alt-EJ pathway is active in 8E6 expressing cells, in particular whether DNA polymerase theta (POLθ) is involved. The mechanism by which p300 favors NHEJ and HR and its absence favors alt-EJ remains to be determined.
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