Exploration of the global manipulation of transcriptional networks by oncogenic

Background
HPV causes 610,000 cancers per year of the anogenital and oropharyngeal tracts. HPV life cycle completion is dependent on the 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 oncoproteins are expressed. Differentiation and migration of cells to the upper epithelium coincides with activation of the late virus promoter and expression of late capsid proteins. This differentiation-dependent model of the HPV life cycle is well accepted but the molecular mechanisms controlling gene expression remain largely unknown. To co-ordinate these complex transcriptional events, HPV utilises a plethora of host transcription factors. For example, we demonstrated that the host chromatin regulator CTCF (CCCTC-binding factor) is recruited to oncogenic HPV genomes to co-ordinate differentiation-dependent repression of viral oncogenes.

As well as controlling expression of their own genes, viruses create a host environment that supports infection and replication. Studies have shown that expression of isolated HPV proteins alters cellular gene expression to evade immune activation and increase cellular growth and motility. However, these studies have not been extended to examine changes that occur during infection and the mechanisms of transcriptional reprogramming are not understood.

Hypothesis
HPV epigenetically reprograms the host to create a cellular milieu supportive of viral persistence and these changes contribute to HPV-driven carcinogenesis.

Aim
The student will use state-of-the-art models of HPV infected tissue and advanced technological methods to explore the complexity of genome-wide manipulation of the host and resulting transcriptional changes that contribute to HPV-induced disease.

Objectives
1. Analyse host transcription changes following HPV establishment: To produce high-quality transcriptome analysis pre- and post-HPV establishment RNA-Seq will be carried out on isogenic primary keratinocytes harvested from different disease-relevant body sites before and after establishment of HPV16 or HPV18 episomes. Differential gene expression patterns following HPV infection in differentiating epithelia will also be determined by RNA-Seq. 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, western blotting and phenotypic studies.
2. Analyse 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, such as CTCF. 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 to give a complete overview of epigenetic host cell reprogramming by HPV. These changes will also be assessed in differentiating epithelia in organotypic raft cultures and will be complemented by the analysis of specific transcription factor recruitment to host cell enhancers by ATAC-Seq.
3. Analyse the 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.