CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression.
Bottom Line: 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.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.
Affiliation: University of St. Andrews, School of Medicine, St. Andrews, Fife, United Kingdom.Show MeSH
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Mentions: To assess the biological function of CTCF binding within the E2 ORF, mutations were introduced into the HPV18 genome to prevent CTCF binding (Fig. 4A). Three nucleotide substitutions were introduced into the predicted binding site that did not alter the amino acid coding sequence of E2 (ΔCTCF HPV18). It should be noted that CTCF also has the potential to bind to the cDNA strand within this region (at the sequence 5′ CACCACCTGGTGGT 3′), although the mutations introduced also would affect binding at this site. We observed a near-complete loss of CTCF binding to the ΔCTCF HPV18 sequence in EMSA, confirming that the mutations prevented CTCF binding (Fig. 4B). HFKs were transfected with recircularized wild-type (WT) or ΔCTCF HPV18 genomes, and immortalized lines were established. To account for donor-specific effects, cells from two independent donors were transfected, and all downstream analyses were performed on both lines. No significant differences in cellular morphology (data not shown) or growth were observed between WT and ΔCTCF lines (Fig. 5A). The physical state of the HPV genomes was determined by Southern blotting and qPCR. Both WT and ΔCTCF HPV18 lines were shown to contain episomal HPV genomes at a similar copy number of approximately 200 copies/cell (Fig. 5B and C). Importantly, we demonstrated a 10-fold reduction in CTCF binding to ΔCTCF HPV18 genomes compared to the level for the WT (Fig. 5D).
Affiliation: University of St. Andrews, School of Medicine, St. Andrews, Fife, United Kingdom.