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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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Related in: MedlinePlus

Analysis of unspliced E6E7 transcript and protein expression in organotypic raft culture. RNA extracted from 14-day-old raft cultures was converted to cDNA and amplified between nucleotides 121 and 295. The products of this PCR are unspliced early transcripts (14). Amplification of GADPH from the same samples is shown as a loading control. Products were separated by electrophoresis (A) and quantified by densitometry using ImageJ (B). An increase in E6E7 transcript was observed in ΔCTCF HPV18 lines established from individual donors (*, P = 0.03 for donor 1 and donor 2). (C) Proteins extracted from raft cultures were analyzed by Western blotting. Fold increase in virus protein expression compared to the wild type (normalized to GAPDH protein) is indicated below each membrane section. The images shown are representative of three technical repeats of lysates extracted from two independent donor lines. (D) E2 protein localization (red in the merged image; DNA is blue) in raft sections of HFK, wild-type, and ΔCTCF HPV18 genome-containing lines. The images shown are representative of two independent raft cultures of each individual donor line. Scale bar, 10 μm.
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Figure 9: Analysis of unspliced E6E7 transcript and protein expression in organotypic raft culture. RNA extracted from 14-day-old raft cultures was converted to cDNA and amplified between nucleotides 121 and 295. The products of this PCR are unspliced early transcripts (14). Amplification of GADPH from the same samples is shown as a loading control. Products were separated by electrophoresis (A) and quantified by densitometry using ImageJ (B). An increase in E6E7 transcript was observed in ΔCTCF HPV18 lines established from individual donors (*, P = 0.03 for donor 1 and donor 2). (C) Proteins extracted from raft cultures were analyzed by Western blotting. Fold increase in virus protein expression compared to the wild type (normalized to GAPDH protein) is indicated below each membrane section. The images shown are representative of three technical repeats of lysates extracted from two independent donor lines. (D) E2 protein localization (red in the merged image; DNA is blue) in raft sections of HFK, wild-type, and ΔCTCF HPV18 genome-containing lines. The images shown are representative of two independent raft cultures of each individual donor line. Scale bar, 10 μm.

Mentions: Since p53 and p130 expression only provide an indication of E6 and E7 activity, we also quantified expression of early transcripts that have the potential to encode E6 and E7 by reverse transcriptase PCR (RT-PCR). As expected, the relative abundance of unspliced E6E7 transcripts in ΔCTCF HPV18 raft cultures was significantly increased compared to that of the WT (Fig. 9A and B). E6E7 transcript levels also were measured by qPCR using the same primer set as that described above and compared to the human RPLPO gene (Life Technologies). A ratio of E6E7 transcript to RPLPO transcript in HPV18 wild-type and ΔCTCF rafts was calculated using the Livak 2ΔΔCT method. Donor 1 was shown to have a 21.19-fold increase (±10.48-fold standard errors [SE]) and donor 2 had a 44.08-fold increase (±26.95-fold SE) in E6E7 transcript in the HPV18 ΔCTCF rafts compared to wild-type levels. In addition, Western blot analysis of protein extracts from raft cultures harvested at day 14 demonstrated a clear increase in E6 and E7 protein levels (Fig. 9C), which was consistent in both donor lines.


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 unspliced E6E7 transcript and protein expression in organotypic raft culture. RNA extracted from 14-day-old raft cultures was converted to cDNA and amplified between nucleotides 121 and 295. The products of this PCR are unspliced early transcripts (14). Amplification of GADPH from the same samples is shown as a loading control. Products were separated by electrophoresis (A) and quantified by densitometry using ImageJ (B). An increase in E6E7 transcript was observed in ΔCTCF HPV18 lines established from individual donors (*, P = 0.03 for donor 1 and donor 2). (C) Proteins extracted from raft cultures were analyzed by Western blotting. Fold increase in virus protein expression compared to the wild type (normalized to GAPDH protein) is indicated below each membrane section. The images shown are representative of three technical repeats of lysates extracted from two independent donor lines. (D) E2 protein localization (red in the merged image; DNA is blue) in raft sections of HFK, wild-type, and ΔCTCF HPV18 genome-containing lines. The images shown are representative of two independent raft cultures of each individual donor line. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403478&req=5

Figure 9: Analysis of unspliced E6E7 transcript and protein expression in organotypic raft culture. RNA extracted from 14-day-old raft cultures was converted to cDNA and amplified between nucleotides 121 and 295. The products of this PCR are unspliced early transcripts (14). Amplification of GADPH from the same samples is shown as a loading control. Products were separated by electrophoresis (A) and quantified by densitometry using ImageJ (B). An increase in E6E7 transcript was observed in ΔCTCF HPV18 lines established from individual donors (*, P = 0.03 for donor 1 and donor 2). (C) Proteins extracted from raft cultures were analyzed by Western blotting. Fold increase in virus protein expression compared to the wild type (normalized to GAPDH protein) is indicated below each membrane section. The images shown are representative of three technical repeats of lysates extracted from two independent donor lines. (D) E2 protein localization (red in the merged image; DNA is blue) in raft sections of HFK, wild-type, and ΔCTCF HPV18 genome-containing lines. The images shown are representative of two independent raft cultures of each individual donor line. Scale bar, 10 μm.
Mentions: Since p53 and p130 expression only provide an indication of E6 and E7 activity, we also quantified expression of early transcripts that have the potential to encode E6 and E7 by reverse transcriptase PCR (RT-PCR). As expected, the relative abundance of unspliced E6E7 transcripts in ΔCTCF HPV18 raft cultures was significantly increased compared to that of the WT (Fig. 9A and B). E6E7 transcript levels also were measured by qPCR using the same primer set as that described above and compared to the human RPLPO gene (Life Technologies). A ratio of E6E7 transcript to RPLPO transcript in HPV18 wild-type and ΔCTCF rafts was calculated using the Livak 2ΔΔCT method. Donor 1 was shown to have a 21.19-fold increase (±10.48-fold standard errors [SE]) and donor 2 had a 44.08-fold increase (±26.95-fold SE) in E6E7 transcript in the HPV18 ΔCTCF rafts compared to wild-type levels. In addition, Western blot analysis of protein extracts from raft cultures harvested at day 14 demonstrated a clear increase in E6 and E7 protein levels (Fig. 9C), which was consistent in both donor lines.

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