Washington University School of Medicine Digital Commons@Becker Open Access Publications 2016 Role of WDHD1 in Human Papillomavirus- Mediated Oncogenesis Identified yb Transcriptional profiling of E7-expressing cells Yunying Zhou Shandong University School of Medicine Qishu Zhang Shandong University School of Medicine Ge Gao Washington University School of Medicine Xiaoli Zhang Shandong University School of Medicine Yafei Liu Shandong University School of Medicine See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Zhou, Yunying; Zhang, Qishu; Gao, Ge; Zhang, Xiaoli; Liu, Yafei; Yuan, Shoudao; Wang, Xiaowei; and Chen, Jason J., ,"Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional profiling of E7-expressing cells." The ourJ nal of Virology.90,13. 6071-84. (2016). https://digitalcommons.wustl.edu/open_access_pubs/5100 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Authors Yunying Zhou, Qishu Zhang, Ge Gao, Xiaoli Zhang, Yafei Liu, Shoudao Yuan, Xiaowei Wang, and Jason J. Chen This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/open_access_pubs/5100 crossmark Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells Yunying Zhou,a Qishu Zhang,a Ge Gao,b Xiaoli Zhang,c Yafei Liu,a Shoudao Yuan,a Xiaowei Wang,b Jason J. Chena The Cancer Research Center, Shandong University School of Medicine, Jinan, Shandong, Chinaa; Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri, USAb; The Qilu Hospital, Shandong University School of Medicine, Jinan, Shandong, Chinac Downloaded from ABSTRACT The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abro- gates the G1 cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differ- entially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the ac- tivities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the http://jvi.asm.org/ DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our stud- ies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregu- lation of WDHD1 reduced E7-induced G1 checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. on July 19, 2016 by Washington University in St. Louis IMPORTANCE The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcino- genesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformat- ics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is up- regulated in E7-expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. uman papillomaviruses (HPVs) are small DNA viruses that out entering mitosis (12). Rereplication may lead to not only Hreplicate in squamous epithelia. Specific types of HPV (high- polyploidy but also gene amplification, DNA fragmentation, DNA risk HPVs) are the causative agents for cervical and several other breaks, and cellular DNA damage response (13–15). We recently cancers (1). The transforming properties of high-risk HPVs such demonstrated that HPV-16 E7 induces rereplication and that the as HPV 16 (HPV-16) primarily depend on E7 as well as E6 onco- cellular DNA replication initiation factor Cdt1 plays a role in this genes (1, 2). HPV E6 and E7 proteins promote the degradation of process (16). p53 and pRb, respectively (3, 4). E7 from the high-risk HPV types DNA replication is regulated by sequential and interactive can abrogate cell cycle checkpoints and induces genomic instabil- mechanisms to ensure that the genome is accurately replicated ity. Although several transcription profiling studies for E7 have only once per cell cycle. The process of replication initiation is been conducted using DNA microarray analysis (3, 5–7), the HPV E7 activities downstream from, or independent of, pRb responsi- ble for deregulation of cell cycle and induction of genomic insta- Received 21 March 2016 Accepted 16 April 2016 bility are not fully understood. Accepted manuscript posted online 20 April 2016 Cell cycle progression is regulated by cyclins and by cyclin- Citation Zhou Y, Zhang Q, Gao G, Zhang X, Liu Y, Yuan S, Wang X, Chen JJ. 2016. dependent kinases (Cdks) and their regulatory proteins at sev- Role of WDHD1 in human papillomavirus-mediated oncogenesis identified by eral checkpoints (8). Once the checkpoint becomes abnormal, transcriptional profiling of E7-expressing cells. J Virol 90:6071–6084. genomic instability may occur (8). Genomic instability is a hall- doi:10.1128/JVI.00513-16. Editor: L. Banks, International Centre for Genetic Engineering and Biotechnology mark of cancer progression (9). Polyploidy is a type of genomic Address correspondence to Xiaowei Wang, [email protected], or instability where cells have more than two sets of chromosomes Jason J. Chen, [email protected]. and has been recognized as a causal factor for tumorigenesis (10). Supplemental material for this article may be found at http://dx.doi.org/10.1128 Significantly, polyploidy can be detected in the early stage of cer- /JVI.00513-16. vical carcinogenesis (11). Polyploidy can be formed via rereplica- Copyright © 2016, American Society for Microbiology. All Rights Reserved. tion, a process of successive rounds of host DNA replication with- July 2016 Volume 90 Number 13 Journal of Virology jvi.asm.org 6071 Zhou et al. divided into two steps, pre-replicative complex (pre-RC) assem- RNA-seq libraries with a NEBNext mRNA library preparation kit (New bly and activation; the latter leads to generation of replication England BioLabs). In this way, double-stranded cDNA was synthesized forks. Pre-RC starts with the association of the origin recognition from rRNA-depleted total RNA. RNA was then end repaired, dA tailed, complex (ORC), which then promotes the recruitment of two and ligated to standard Illumina adaptor oligonucleotides. Adaptor-li- proteins, Cdc6 and Cdt1, onto origins. This is followed by recruit- gated cDNA libraries were amplified with Phusion PCR master mix. The ment of minichromosome maintenance 2-7 (MCM2-7) onto cDNA libraries were then loaded into a HiSeq 2000 system (Illumina) for sequencing at the Washington University Genome Technology Access chromatin as a result of concerted actions of Cdc6 and Cdt1 (9). Center. The resulting raw sequence reads were first analyzed using a cus- Prior to the S phase, origins are licensed by the binding of com- tom bioinformatics pipeline to remove low-quality reads and then clus- ponents of the replicative DNA helicase MCMs in eukaryotes (17). tered before sequentially mapping to the human transcriptome and ge- Afterward, licensing proteins are downregulated or inhibited such nome with Bowtie was performed (25). In this way, about 90% of all Downloaded from that no more origins can be licensed and rereplication of DNA is sequence reads were mapped to known human sequences. Sequence reads prevented. Cells employ a licensing checkpoint to monitor that mapping to the same transcript were combined and normalized on the sufficient origins are licensed, inhibiting S-phase entry until that basis of the length of the transcript and the number of total reads from state is established (18). The G1 arrest observed in cells that have each sample (reads per kilobase per million [RPKM]). Normalized read engaged in the licensing checkpoint is associated with low levels of counts were compared across samples to identify changes in transcript abundance. Transcripts from the same