Supplementary Materials Supplemental material supp_90_13_6071__index. in E7-expressing cells. WDHD1 can be a component from the replisome that regulates DNA replication. Latest studies claim that WDHD1 could also work as a DNA replication initiation element and a G1 checkpoint regulator. We discovered that in E7-expressing cells, the steady-state degree of WDHD1 proteins was increased combined with the half-life. Furthermore, downregulation 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 Edaravone (MCI-186) instability and have Edaravone (MCI-186) therapeutic implications. IMPORTANCE The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. 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 bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated 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. INTRODUCTION Human papillomaviruses (HPVs) are small DNA viruses that replicate in squamous epithelia. Specific types of HPV (high-risk HPVs) are the causative agents for cervical and several other cancers (1). The transforming properties of high-risk HPVs such as HPV 16 (HPV-16) primarily depend on E7 as well as E6 oncogenes (1, 2). HPV E6 and E7 proteins promote the degradation of p53 and pRb, respectively (3, Rabbit polyclonal to c-Kit 4). E7 from the high-risk HPV types can abrogate cell Edaravone (MCI-186) cycle checkpoints and induces genomic instability. Although several transcription profiling studies for E7 have been conducted using DNA microarray analysis (3, 5,C7), the HPV E7 activities downstream from, or independent of, pRb responsible for deregulation of cell cycle and induction of genomic instability are not fully understood. Cell cycle progression is regulated by cyclins and by cyclin-dependent kinases (Cdks) and their regulatory proteins at several checkpoints (8). Once the checkpoint becomes abnormal, genomic Edaravone (MCI-186) instability may occur (8). Genomic instability is a hallmark of cancer progression (9). Polyploidy is a type of genomic instability where cells have more than two sets of chromosomes and has been recognized as a Edaravone (MCI-186) causal factor for tumorigenesis (10). Significantly, polyploidy can be detected in the early stage of cervical carcinogenesis (11). Polyploidy can be formed via rereplication, a process of successive rounds of host DNA replication without entering mitosis (12). Rereplication may lead to not only polyploidy but also gene amplification, DNA fragmentation, DNA breaks, and cellular DNA damage response (13,C15). We recently demonstrated that HPV-16 E7 induces rereplication and that the cellular DNA replication initiation factor Cdt1 plays a role in this process (16). DNA replication is regulated by sequential and interactive mechanisms to ensure that the genome is accurately replicated only once per cell cycle. The process of replication initiation is divided into two steps, pre-replicative complex (pre-RC) assembly and activation; the latter leads to generation of replication forks. Pre-RC starts with the association of the origin.