Understanding the role of the chromatin insulator CTCF in human papillomavirus gene expression and disease progression

Viral infections are estimated to cause over 1 in 10 of all cancers, resulting in over 1.3 million deaths a year, worldwide. Human papillomavirus (HPV) infections cause almost half of these cancers, including cancer of the uterine cervix, genitals and in the mouth and throat. While this causal link between HPV and the development of cancer is well established, the specific changes that occur in HPV infections that drive the formation of HPV tumours are not well understood. Thus, it is important to understand these changes so that new therapies to treat HPV infections and cancer, and diagnostic tools for early disease detection, can be developed. Our work has shown that a host cell protein, CTCF, binds to the HPV genome and limits production of viral proteins that cause increased host cell growth and cancer development. Therefore, we hypothesize that in normal HPV infections CTCF is a key regulator of viral gene expression and works to allow the virus to complete the infectious cycle and produce new virus. We predict that the control of HPV-induced cell growth by CTCF is lost in HPV-driven cancers. The experiments described in this proposal are designed to answer long-standing questions about how the virus behaves in a normal infection and how this novel virus-host interaction contributes to virus productivity. Using clinical samples of HPV-associated disease, we will also explore how this virus-host interaction is disrupted in HPV-driven cancer.

Infection with high-risk human papillomavirus (HR-HPV) is the cause of cancers within the anogenital and oropharyngeal tracts. The fundamental link between HR-HPV infection and neoplastic progression is well established, but the molecular changes are poorly defined. The HPV life cycle is dependent on the differentiation of the infected cell. Infection is established in the undifferentiated basal layer of the epithelium where early gene expression is initiated. Terminal differentiation of the cell results in late promoter activation and expression of late transcripts. This switch in gene expression is essential for life cycle completion, but how it is controlled has not been elucidated. We have recently demonstrated that the host cell transcriptional insulator protein CTCF binds to the HPV genome and regulates early gene expression. Our preliminary data provide strong evidence that CTCF function is essential for the transcriptional regulation of HPV gene expression by influencing transcriptional boundary formation and through the control of RNA polymerase II programming and gene splicing. Loss of CTCF binding not only enhances early gene expression but also prevents late gene expression, suggesting that CTCF function is important in the early to late switch in transcription control. We will study the function of CTCF in the differentiation-dependent control of virus gene expression using physiologically relevant models of the HPV life cycle at different body sites (anogenital and oropharyngeal) using innovative and quantitative methods. These experiments will be underpinned by the study of CTCF-HPV association in cervical cancer specimens to determine whether loss of CTCF binding contributes to cancer development. Consolidation of our findings will provide important insight into the function of CTCF in the HPV life cycle and HPV-driven disease progression and will impact on our understanding of CTCF function in gene expression control in healthy and disease states.

Impact Summary 4000 characters

The work in this proposal focuses on fundamental pathways that regulate virus gene expression and their involvement in HPV-driven carcinogenesis. The results will contribute to our knowledge of HPV biology and HPV-driven disease progression, but more broadly to our understanding of the host cell transcription and gene splicing regulation. The main beneficiaries will be:

THE ACADEMIC COMMUNITY
To our knowledge we are the only grouping addressing the specific questions raised in our proposal and, with our combined expertise, we are ideally placed to answer these long-standing and important questions. Therefore, the results obtained will benefit those interested in the HPV life cycle and the molecular control of viral gene expression. Our study will also be beneficial to those interested in CTCF-mediated regulation of the life cycles of other DNA viruses and host cell gene expression. CTCF is mutated and dysregulated in many cancers and human disease and therefore our study will also benefit those interested in CTCF function in healthy and disease states.
Establishment of state-of-the-art methods (e.g. PAT-ChIP) will benefit our local academic community, as these methods will be shared with our colleagues at the University of Birmingham and further afield. The academic community will also benefit from the training of a postdoctoral scientist in the use of such methods to study the HPV life cycle, gene expression and chromatin structure, as well as more general training in project management and communication skills.

CLINICIANS
By identifying molecular events that contribute to HPV-driven carcinogenesis, we will benefit clinicians interested in HPV-associated disease. The proposed work will facilitate immediate engagement with clinicians (Nick Coleman, University of Cambridge and Hisham Mehanna, University of Birmingham) and future studies will take advantage of the highly integrated clinical academic environment at the University of Birmingham and within Birmingham Health Partners (BHP).

STAFF WORKING ON THE PROJECT
The postdoctoral scientist working on this project will develop a skill set highly desirable for future employment. With guidance from Parish and Roberts, the researcher will gain project management skills, obtained from running a research project, which are transferrable to many other disciplines in addition to the scientific employment sector. The researcher will also develop her communication and supervision/teaching skills that will be transferable to any management discipline.

SOCIETY
The immediate impact on society will be in the development of a highly skilled academic researcher. The proposed work could also have an economic impact in the long term through the development of novel biomarkers and therapies for HPV-driven disease.