PhD Studentship: Exploration of the global manipulation of transcriptional networks by human papill

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:

• 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.
• 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.
• Analysis of the mechanistic underpinnings of HPV mediated host transcriptional reprogramming. Preliminary data suggests that HPV induces transcriptional reprogramming of the host. The student will explore host cell reprogramming further by ChIP-Seq analysis of key factors and epigenetic marks. 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.
• 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).

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

Person Specification

Applicants should have a strong background in molecular and cellular biology, with experience of one or more of the following laboratory techniques: mammalian tissue culture; immunofluorescence; immunoblotting or chromatin immunoprecipitation. Previous experience of a research lab environment is essential. They should be ambitious, enthusiastic and self-motivated, and should meet the eligibility criteria outlined below.

Funding Notes

Funding:

This studentship is competition funded by the BBSRC MIBTP scheme.

Deadline:

January 8, 2018

Number of Studentships available:

30

Stipend:

RCUK standard rate (plus travel allowance in Year 1 and a laptop). The Midlands Integrative Biosciences Training Partnership (MIBTP) is a BBSRC-funded doctoral training partnership between the universities of Warwick, Birmingham and Leicester.


 

References

1. Pentland. I., Parish, J.L. Targetting CTCF to control virus gene expression: A common theme amongst diverse DNA viruses. (2015) Viruses. 7(7):3574-85. doi: 10.3390/v7072791
2. Paris, C., Pentland, I., Groves, I., Roberts, D.C., Coleman, N., Roberts, S. and Parish J.L. CCCTC-Binding Factor Recruitment to the Early Region of the Human Papillomavirus Type 18 Genome Regulates Viral Oncogene Expression. (2015) Journal of Virology. 89(9):4770-85.
3. Harris, L., McFarlane, L., Campos Leon, K., Roberts, S. and Parish, J.L. The cellular DNA helicase ChlR1 regulates chromatin and nuclear matrix attachment of the human papillomavirus type 16 E2 protein and high copy viral genome establishment: ChlR1 regulates the chromatin association of HPV16 E2. (2017) Journal of Virology. 91(1):1-16. doi: 10.1128/JVI.01853-16.