Abstract 227: Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) reveals dynamic and temporal changes in transcription in response to UVB exposure

Abstract

Abstract Skin cancer is the most common form of cancer, with over 5 million cases reported in 2021. UVB exposure derived from sunlight is the major carcinogenic driver of skin cancer and poses a major public health risk. In response to UVB exposure, keratinocytes must activate DNA repair pathways to avoid mutagenesis. In addition to DNA repair pathways, cells initiate well-coordinated DNA damage response pathways to facilitate the repair process, including arresting global transcription. Here we report the use of kethoxal-assisted single-strand DNA-Sequencing (KAS-Seq) technology to profile the transcriptional dynamics that underlie the DNA damage response upon UVB exposure. Mechanistically, KAS-Seq involves the addition of an azide-tagged kethoxal group (N3-kethoxal) to DNA samples, which then reacts with guanines on single-stranded DNA (ssDNA). A “KAS signal” is therefore only obtained when DNA is unwound, allowing the N3-kethoxal reaction with accessible guanines to occur. Accordingly, in response to DNA damage, DNA can form ssDNA bubbles in order to facilitate DNA damage recognition and repair processes. KAS-Seq is therefore a suitable method to profile the transcriptional dynamics that occur in response to UVB-induced DNA damage. Here we show that UVB exposure in normal human keratinocytes (NHEK) cells induces a temporal and dynamic reprogramming in transcription using KAS-Seq. In terms of global transcription, our data show that the greatest loss of KAS signal occurs rapidly between 30mins and 3hrs post-UVB exposure, signifying transcriptional arrest. Interestingly, KAS signal is regained between 6-24hrs post-UVB, which is representative of a transition towards transcriptional recovery and induction of DNA damage processes. Furthermore, a more discrete and targeted analysis shows that the gain and loss of KAS signals primarily occur within the gene body, followed by promoter and intergenic regions. Overall, our data shows that transcriptional control in response to UVB-induced DNA damage is both dynamic and temporal. Further analysis is required to identify which transcriptional phenotype (transcriptional arrest or transcriptional recovery) is the most deleterious to the cell and DNA damage repair processes when disrupted. Additionally, we aim to further elucidate the molecular mechanisms that underlie these global changes in transcription, which will provide expanded insights into the wide-ranging oncogenic mechanisms that underlie UVB exposure. Citation Format: Emma A. Wilkinson, Tong Wu, Gayoung Park, Yan-Hong Cui, Chuan He, Yu-Ying He. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) reveals dynamic and temporal changes in transcription in response to UVB exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 227.