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      CCCTC-Binding Factor Recruitment to the Early Region of the Human Papillomavirus 18 Genome Regulates Viral Oncogene Expression

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          Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression.

          IMPORTANCE The establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

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          Most cited references 70

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          The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53.

          The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.
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            CTCF: master weaver of the genome.

            CTCF is a highly conserved zinc finger protein implicated in diverse regulatory functions, including transcriptional activation/repression, insulation, imprinting, and X chromosome inactivation. Here we re-evaluate data supporting these roles in the context of mechanistic insights provided by recent genome-wide studies and highlight evidence for CTCF-mediated intra- and interchromosomal contacts at several developmentally regulated genomic loci. These analyses support a primary role for CTCF in the global organization of chromatin architecture and suggest that CTCF may be a heritable component of an epigenetic system regulating the interplay between DNA methylation, higher-order chromatin structure, and lineage-specific gene expression.
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              CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing.

              Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.

                Author and article information

                [a ]University of Birmingham, School of Cancer Sciences, Birmingham, United Kingdom
                [b ]University of St. Andrews, School of Medicine, St. Andrews, Fife, United Kingdom
                [c ]University of Cambridge, Department of Pathology, Cambridge, United Kingdom
                Author notes
                Address correspondence to Sally Roberts, S.Roberts@ , or Joanna L. Parish, j.l.parish@ .

                Citation Paris C, Pentland I, Groves I, Roberts DC, Powis SJ, Coleman N, Roberts S, Parish JL. 2015. CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression. J Virol 89:4770–4785. doi: 10.1128/JVI.00097-15.

                Role: Editor
                J Virol
                J. Virol
                Journal of Virology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                18 February 2015
                1 May 2015
                18 February 2015
                : 89
                : 9
                : 4770-4785
                Copyright © 2015 Paris et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Figures: 10, Tables: 3, Equations: 0, References: 71, Pages: 16, Words: 11327
                Genome Replication and Regulation of Viral Gene Expression

                Microbiology & Virology


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