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Exploration of the global manipulation of transcriptional networks by human papillomavirus

Principal Supervisor: Dr. Joanna Parish, Institute of Cancer and Genomic Sciences

Co-supervisor: Dr. Sally Roberts, Institute of Cancer and Genomic Sciences

PhD project title: Exploration of the global manipulation of transcriptional networks by human papillomavirus

University of Registration: University of Birmingham

Project outline:

Human papillomaviruses (HPV) are a major human pathogen and the cause of persistent genital and skin warts as well as 610,000 cancers per year of the anogenital and oropharyngeal tracts. HPV replication and life cycle completion is dependent on the virus’s ability to manipulate the host environment and on the terminal differentiation of infected epithelial cells. Infection is established in the undifferentiated basal keratinocytes where the chromatinised viral DNA is established as a persistent episome and viral transcripts that encode the E6 and E7 viral proteins are expressed, which modulate the cell cycle to support productive infection. Differentiation and migration of cells to the upper epithelium coincides with activation of the late virus promoter and expression of late capsid proteins. To co-ordinate these complex transcriptional events, HPV utilises a plethora of host transcription factors. For example, we demonstrated that CCCTC-binding factor (CTCF) is recruited to HPV genomes to co-ordinate differentiation-dependent repression of viral genes. As well as controlling expression of their own genes, viruses create a host environment that is supportive of infection. We hypothesise that HPV epigenetically reprograms the host to create an environment supportive of viral persistence and these changes contribute to HPV-induced disease.

The student will use state-of-the-art models of HPV infected tissue and advanced technological methods to explore the complexity of virus transcription control and manipulation of the host.

Objectives:

1. Analyse viral transcripts in undifferentiated and differentiating tissue. RNA harvested from primary keratinocyte donor cells containing episomal HPV18 genomes, grown either in monolayer (undifferentiated) or three-dimensional organotypic rafts that recapitulate the fully differentiated epithelium will be analysed by RNA-Seq. In addition, the function of CTCF in the control of virus transcript splicing will be determined by comparing transcripts present in cultures containing wild type HPV18 genomes to those containing genomes with the CTCF binding site mutated (Paris et al., 2015); ΔCTCF HPV18.

2. Analysis of host cell transcription changes following HPV establishment: To produce high-quality transcriptome analysis pre- and post-HPV establishment,

isogenic donor cells will be included in the RNA-Seq experiments described in Aim 1. Significantly altered host pathways will be identified by Gene Ontology analysis (www.broadinstitute.org) and pathways of interest will be further validated by qRT-PCR and western blotting.

3. Analysis of the mechanistic underpinnings of HPV mediated host transcriptional reprogramming. Preliminary data suggests that HPV induces transcriptional reprogramming of the host by redistributing important transcriptional regulators. The student will explore host cell reprogramming further by ChIP-Seq analysis of these factors and epigenetic marks of actively transcribed chromatin (H3K4Me3) and repressed chromatin (H3K27Me3). Identified changes in transcriptional hubs within the host will be mapped onto the changes in gene transcription identified in the RNA-Seq experiments (aim 2) to give a complete overview of epigenetic host cell reprogramming by HPV.

4. Analysis of global remodelling of host cell chromatin structure and topologically associating domains (TADs) by HPV. Chromatin is compartmentalised by elaborate three-dimensional folding that brings distant functional elements in close physical proximity. To analyse these chromosomal interactions pre- and post-HPV establishment, we will use chromosome conformation capture coupled to high-throughput sequencing (Hi-C) in collaboration with Prof. S. Jha, National University of Singapore.

Completion of these aims will provide a unique overview of host manipulation by HPV to gain novel insight into how the virus alters the cellular milieu to maintain persistent infection.

BBSRC Strategic Research Priority: Molecules, Cells and Systems

Techniques that will be undertaken during the project:

  • Primary cell culture
  • Three dimensional organotypic raft culture of epithelium
  • Chromatin immunoprecipitation
  • Chromatin, conformation, capture (3C) and HiC
  • RNA-Seq
Contact: Dr Joanna Parish, Institute of Cancer and Genomic Sciences