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CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression.

Paris C, Pentland I, Groves I, Roberts DC, Powis SJ, Coleman N, Roberts S, Parish JL - J. Virol. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: University of St. Andrews, School of Medicine, St. Andrews, Fife, United Kingdom.

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Creation of HPV18 wild-type and ΔCTCF mutant human foreskin keratinocyte lines. HFKs established from two independent donors were transfected with WT or ΔCTCF HPV18 genomes. (A) Analysis of growth kinetics using a CCK-8 metabolic assay. Cells were seeded at equal density at day 0, and the growth of each line was measured at days 1, 3, 5, and 7. The data show the means and standard errors from two independent experiments performed in triplicate. (B) HPV18 genome copy number was determined by qPCR analysis of DpnI-digested DNA extracted from each line using the Pfaffl comparative CT method and normalized against the TLR2 locus (37). Data show the means and standard errors from three independent repeats (donor 1, P = 0.9; donor 2, P = 0.2). (C) HPV18 genome status was determined by Southern blotting from extracted DNA from donor 1 and donor 2 transfected with either wild-type (WT) or ΔCTCF mutant (ΔC) HPV18 genomes (OC, open circle; L, linear; SC, supercoiled). DNA was linearized with EcoRI, producing a single band of similar intensity running at approximately 8 kbp, demonstrating the maintenance of viral episomes at a similar copy number in each line. Digestion with BglII shows minimal multimeric/integrated HPV genomes in all lines. (D) Abrogation of CTCF binding by mutation of the CTCF binding site was determined by ChIP. Chromatin was either immunoprecipitated with FLAG (negative control) or CTCF antibody, and the percentage of bound HPV18 genome was determined by qPCR with primers that flank the CTCF binding site at position 2989. A significant decrease in CTCF binding was observed in ΔCTCF HPV18 compared to that of the wild type (**, P = 0.01). The data shown represent the means and standard errors from two independent repeats performed in duplicate (donor 1; donor 2 showed a similar decrease in CTCF binding).
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Figure 5: Creation of HPV18 wild-type and ΔCTCF mutant human foreskin keratinocyte lines. HFKs established from two independent donors were transfected with WT or ΔCTCF HPV18 genomes. (A) Analysis of growth kinetics using a CCK-8 metabolic assay. Cells were seeded at equal density at day 0, and the growth of each line was measured at days 1, 3, 5, and 7. The data show the means and standard errors from two independent experiments performed in triplicate. (B) HPV18 genome copy number was determined by qPCR analysis of DpnI-digested DNA extracted from each line using the Pfaffl comparative CT method and normalized against the TLR2 locus (37). Data show the means and standard errors from three independent repeats (donor 1, P = 0.9; donor 2, P = 0.2). (C) HPV18 genome status was determined by Southern blotting from extracted DNA from donor 1 and donor 2 transfected with either wild-type (WT) or ΔCTCF mutant (ΔC) HPV18 genomes (OC, open circle; L, linear; SC, supercoiled). DNA was linearized with EcoRI, producing a single band of similar intensity running at approximately 8 kbp, demonstrating the maintenance of viral episomes at a similar copy number in each line. Digestion with BglII shows minimal multimeric/integrated HPV genomes in all lines. (D) Abrogation of CTCF binding by mutation of the CTCF binding site was determined by ChIP. Chromatin was either immunoprecipitated with FLAG (negative control) or CTCF antibody, and the percentage of bound HPV18 genome was determined by qPCR with primers that flank the CTCF binding site at position 2989. A significant decrease in CTCF binding was observed in ΔCTCF HPV18 compared to that of the wild type (**, P = 0.01). The data shown represent the means and standard errors from two independent repeats performed in duplicate (donor 1; donor 2 showed a similar decrease in CTCF binding).

Mentions: We next used HPV16 and HPV18 genome-containing cells to ascertain whether CTCF associates with the viral genome in cells. W12 cells, derived from a low-grade cervical squamous epithelial lesion, contain ∼100 episomal HPV16 genome copies/cell (39, 40), and HPV18-transfected HFKs contain ∼200 episomal HPV18 copies/cell (see Fig. 5B). CTCF association with the HPV genomes was determined by ChIP followed by qPCR. In both HPV16 and HPV18 genome-containing cells grown in monolayer, we noted a significant enrichment of CTCF binding within the E2 ORF, coinciding with the CTCF binding site conserved in high-risk HPV types but not in low-risk types (Fig. 3). In contrast, we failed to detect CTCF binding to the late gene region in either HPV16 or HPV18 genome-containing model systems.


CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression.

Paris C, Pentland I, Groves I, Roberts DC, Powis SJ, Coleman N, Roberts S, Parish JL - J. Virol. (2015)

Creation of HPV18 wild-type and ΔCTCF mutant human foreskin keratinocyte lines. HFKs established from two independent donors were transfected with WT or ΔCTCF HPV18 genomes. (A) Analysis of growth kinetics using a CCK-8 metabolic assay. Cells were seeded at equal density at day 0, and the growth of each line was measured at days 1, 3, 5, and 7. The data show the means and standard errors from two independent experiments performed in triplicate. (B) HPV18 genome copy number was determined by qPCR analysis of DpnI-digested DNA extracted from each line using the Pfaffl comparative CT method and normalized against the TLR2 locus (37). Data show the means and standard errors from three independent repeats (donor 1, P = 0.9; donor 2, P = 0.2). (C) HPV18 genome status was determined by Southern blotting from extracted DNA from donor 1 and donor 2 transfected with either wild-type (WT) or ΔCTCF mutant (ΔC) HPV18 genomes (OC, open circle; L, linear; SC, supercoiled). DNA was linearized with EcoRI, producing a single band of similar intensity running at approximately 8 kbp, demonstrating the maintenance of viral episomes at a similar copy number in each line. Digestion with BglII shows minimal multimeric/integrated HPV genomes in all lines. (D) Abrogation of CTCF binding by mutation of the CTCF binding site was determined by ChIP. Chromatin was either immunoprecipitated with FLAG (negative control) or CTCF antibody, and the percentage of bound HPV18 genome was determined by qPCR with primers that flank the CTCF binding site at position 2989. A significant decrease in CTCF binding was observed in ΔCTCF HPV18 compared to that of the wild type (**, P = 0.01). The data shown represent the means and standard errors from two independent repeats performed in duplicate (donor 1; donor 2 showed a similar decrease in CTCF binding).
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Figure 5: Creation of HPV18 wild-type and ΔCTCF mutant human foreskin keratinocyte lines. HFKs established from two independent donors were transfected with WT or ΔCTCF HPV18 genomes. (A) Analysis of growth kinetics using a CCK-8 metabolic assay. Cells were seeded at equal density at day 0, and the growth of each line was measured at days 1, 3, 5, and 7. The data show the means and standard errors from two independent experiments performed in triplicate. (B) HPV18 genome copy number was determined by qPCR analysis of DpnI-digested DNA extracted from each line using the Pfaffl comparative CT method and normalized against the TLR2 locus (37). Data show the means and standard errors from three independent repeats (donor 1, P = 0.9; donor 2, P = 0.2). (C) HPV18 genome status was determined by Southern blotting from extracted DNA from donor 1 and donor 2 transfected with either wild-type (WT) or ΔCTCF mutant (ΔC) HPV18 genomes (OC, open circle; L, linear; SC, supercoiled). DNA was linearized with EcoRI, producing a single band of similar intensity running at approximately 8 kbp, demonstrating the maintenance of viral episomes at a similar copy number in each line. Digestion with BglII shows minimal multimeric/integrated HPV genomes in all lines. (D) Abrogation of CTCF binding by mutation of the CTCF binding site was determined by ChIP. Chromatin was either immunoprecipitated with FLAG (negative control) or CTCF antibody, and the percentage of bound HPV18 genome was determined by qPCR with primers that flank the CTCF binding site at position 2989. A significant decrease in CTCF binding was observed in ΔCTCF HPV18 compared to that of the wild type (**, P = 0.01). The data shown represent the means and standard errors from two independent repeats performed in duplicate (donor 1; donor 2 showed a similar decrease in CTCF binding).
Mentions: We next used HPV16 and HPV18 genome-containing cells to ascertain whether CTCF associates with the viral genome in cells. W12 cells, derived from a low-grade cervical squamous epithelial lesion, contain ∼100 episomal HPV16 genome copies/cell (39, 40), and HPV18-transfected HFKs contain ∼200 episomal HPV18 copies/cell (see Fig. 5B). CTCF association with the HPV genomes was determined by ChIP followed by qPCR. In both HPV16 and HPV18 genome-containing cells grown in monolayer, we noted a significant enrichment of CTCF binding within the E2 ORF, coinciding with the CTCF binding site conserved in high-risk HPV types but not in low-risk types (Fig. 3). In contrast, we failed to detect CTCF binding to the late gene region in either HPV16 or HPV18 genome-containing model systems.

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.

View Article: PubMed Central - PubMed

Affiliation: University of St. Andrews, School of Medicine, St. Andrews, Fife, United Kingdom.

Show MeSH
Related in: MedlinePlus