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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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Analysis of p53 and p130 degradation in wild-type and ΔCTCF HPV18 organotypic raft sections. (A) Sections were stained with p53-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (B) The percentage of cells positive for nuclear p53 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p53-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.0005). (C) Sections were stained with p130-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (D) The percentage of cells positive for nuclear p130 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p130-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.001).
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Figure 8: Analysis of p53 and p130 degradation in wild-type and ΔCTCF HPV18 organotypic raft sections. (A) Sections were stained with p53-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (B) The percentage of cells positive for nuclear p53 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p53-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.0005). (C) Sections were stained with p130-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (D) The percentage of cells positive for nuclear p130 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p130-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.001).

Mentions: The increased cell cycle entry and hyperproliferation observed in the organotypic raft cultures derived from HFK lines maintaining ΔCTCF HPV18 genomes could be due to an increase in the expression of E6 and E7 viral oncoproteins. Detection of these proteins by immunostaining currently is not possible; therefore, raft sections were stained with surrogate markers, p53 as a marker for E6 expression and pRb family member p130 for E7 expression (8, 41). Cells stained positive for p53 in WT HPV18 raft sections were apparent throughout the epithelia as previously reported (42), albeit at a noticeably decreased level compared to that of rafts derived from untransfected HFKs (Fig. 8A). In contrast, p53-positive cells were undetectable in rafts derived from ΔCTCF HPV18 lines (Fig. 8A and B). This observation is consistent with an increase in E6 protein levels in ΔCTCF HPV18 compared to that of the WT, resulting in a decrease in detectable p53 protein. Similarly, immunostaining with p130-specific antibodies revealed significant differences between WT and ΔCTCF HPV18 rafts (Fig. 8C and D). In wild-type HPV18 rafts, p130-positive cells were confined to the upper layers, as previously shown (42) and in contrast to HPV-negative HFK raft sections, where cells stained positive for p130 in the parabasal and lower and upper suprabasal layers. However, immunostaining of p130 in the ΔCTCF HPV18 raft sections revealed an almost complete loss of p130-positive cells in the upper layers, suggesting increased and prolonged expression of E7 protein in the ΔCTCF HPV18 rafts compared to that of the WT.


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)

Analysis of p53 and p130 degradation in wild-type and ΔCTCF HPV18 organotypic raft sections. (A) Sections were stained with p53-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (B) The percentage of cells positive for nuclear p53 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p53-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.0005). (C) Sections were stained with p130-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (D) The percentage of cells positive for nuclear p130 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p130-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.001).
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Figure 8: Analysis of p53 and p130 degradation in wild-type and ΔCTCF HPV18 organotypic raft sections. (A) Sections were stained with p53-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (B) The percentage of cells positive for nuclear p53 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p53-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.0005). (C) Sections were stained with p130-specific antibody (green), and DNA was stained with Hoechst (blue). The white arrows indicate the basal layer, and the lower suprabasal/upper suprabasal boundary is highlighted with a dashed line. Scale bar, 5 μm. (D) The percentage of cells positive for nuclear p130 staining in the basal, lower suprabasal (parabasal and lower spinous), and upper suprabasal (upper spinous and granular) layers of 15 fields of view of 3 independent rafts (n = 45) from each donor was determined. The data represent the means and standard errors. A significant reduction in p130-positive cells is observed in all layers of rafts derived from the ΔCTCF HPV18 lines (***, P < 0.001).
Mentions: The increased cell cycle entry and hyperproliferation observed in the organotypic raft cultures derived from HFK lines maintaining ΔCTCF HPV18 genomes could be due to an increase in the expression of E6 and E7 viral oncoproteins. Detection of these proteins by immunostaining currently is not possible; therefore, raft sections were stained with surrogate markers, p53 as a marker for E6 expression and pRb family member p130 for E7 expression (8, 41). Cells stained positive for p53 in WT HPV18 raft sections were apparent throughout the epithelia as previously reported (42), albeit at a noticeably decreased level compared to that of rafts derived from untransfected HFKs (Fig. 8A). In contrast, p53-positive cells were undetectable in rafts derived from ΔCTCF HPV18 lines (Fig. 8A and B). This observation is consistent with an increase in E6 protein levels in ΔCTCF HPV18 compared to that of the WT, resulting in a decrease in detectable p53 protein. Similarly, immunostaining with p130-specific antibodies revealed significant differences between WT and ΔCTCF HPV18 rafts (Fig. 8C and D). In wild-type HPV18 rafts, p130-positive cells were confined to the upper layers, as previously shown (42) and in contrast to HPV-negative HFK raft sections, where cells stained positive for p130 in the parabasal and lower and upper suprabasal layers. However, immunostaining of p130 in the ΔCTCF HPV18 raft sections revealed an almost complete loss of p130-positive cells in the upper layers, suggesting increased and prolonged expression of E7 protein in the ΔCTCF HPV18 rafts compared to that of the WT.

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