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Transcriptional Repression of E-Cadherin by Human Papillomavirus Type 16 E6

D'Costa, Zarina J., Jolly, Carol, Androphy, Elliot J., Mercer, Andrew, Matthews, Charles M., Hibma, Merilyn H.·2012·PLoS ONE
DOI 10.1371/journal.pone.0048954PMC PMC3506579
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Figure 1 HPV16-infected epidermis displays lowered and irregular E-cadherin staining HPV16 E6 expressing cells have reduced E-cadherin expression. E-cadherin (green) and DAPI stained nuclei (blue) in (a) normal and (b) HPV16-infected human epidermis. E = epidermis, D = dermis. (c) HCT116 and (d) HCT116 E6 cells grown on glass coverslips, permeabilised and stained for E-cadherin (green) and DAPI to identify nuclei (blue).
Figure 1 HPV16-infected epidermis displays lowered and irregular E-cadherin staining HPV16 E6 expressing cells have reduced E-cadherin expression. E-cadherin (green) and DAPI stained nuclei (blue) in (a) normal and (b) HPV16-infected human epidermis. E = epidermis, D = dermis. (c) HCT116 and (d) HCT116 E6 cells grown on glass coverslips, permeabilised and stained for E-cadherin (green) and DAPI to identify nuclei (blue).CC BY · PubMed Central
Figure 2 E-cadherin expression and E-cadherin mediated adhesion is reduced in cells expressing HPV16 E6 and E7. HCT116 cells were transfected with plasmids encoding GFP-E5, E6 or E7 and were trypsinised, cleaving surface E-cadherin, 48 h later. Single cell suspensions were cultured for 2 h in low adhesion plates for E-cadherin re-expression and cell-to-cell adhesion to occur. Cells were stained for E-cadherin and GFP positive cells with comparable levels of expression between samples were analysed using flow cytometry. (a) Dot plots showing the E-cadherin negative single cell population (lower left), E-cadherin positive single cells (upper left), E-cadherin positive aggregates (upper right), with the percentage of events in each quadrant shown. (b) Cell surface E-cadherin fluorescence and (c) cellular aggregates mediated by E-cadherin on GFP-positive cells. n = 5 independent experiments. Error bars represent ± SEM. * P<0.05, ** P<0.01, *** P<0.001.
Figure 2 E-cadherin expression and E-cadherin mediated adhesion is reduced in cells expressing HPV16 E6 and E7. HCT116 cells were transfected with plasmids encoding GFP-E5, E6 or E7 and were trypsinised, cleaving surface E-cadherin, 48 h later. Single cell suspensions were cultured for 2 h in low adhesion plates for E-cadherin re-expression and cell-to-cell adhesion to occur. Cells were stained for E-cadherin and GFP positive cells with comparable levels of expression between samples were analysed using flow cytometry. (a) Dot plots showing the E-cadherin negative single cell population (lower left), E-cadherin positive single cells (upper left), E-cadherin positive aggregates (upper right), with the percentage of events in each quadrant shown. (b) Cell surface E-cadherin fluorescence and (c) cellular aggregates mediated by E-cadherin on GFP-positive cells. n = 5 independent experiments. Error bars represent ± SEM. * P<0.05, ** P<0.01, *** P<0.001.CC BY · PubMed Central
Figure 3 HPV16 E6 reduces E-cadherin protein expression by reducing E-cadherin promoter and transcript levels, independent of the major Epal repressor element and HDAC. (a) Levels of E-cadherin mRNA were determined by qRT-PCR analysis on RNA extracted from HCT116 control and HCT116 E6 cells. GAPDH mRNA was amplified in the same RNA samples for normalization. n = 5 independent experiments. (b) Luciferase activity of the −178/+92 E-cadherin promoter was measured in HCT116 and HCT116 E6 cells. n = 3 independent experiments. (c) Schematic representation of the luciferase plasmid under the control of the E-cadherin promoter. Negative regulatory elements Epal, E-box and positive elements CAAT, GC-I and GC-II are shown as is the mt Epal, with the two-nucleotide inactivating mutation. (d) Activity of wild type and mt Epal E-cadherin promoters in HCT116 and HCT116 E6 cells. Luciferase activity of co-transfected pRL-SV40 was used as a control for transfection efficiency and for normalization. n = 8 independent experiments. (e) RNA was extracted from HCT116 and HCT116 E6 cells that had been either untreated or treated with HDAC inhibitor, TSA, for 24 h. E-cadherin mRNA levels were measured by qRT-PCR and normalized to GAPDH mRNA levels. n = 3 independent experiments.
Figure 3 HPV16 E6 reduces E-cadherin protein expression by reducing E-cadherin promoter and transcript levels, independent of the major Epal repressor element and HDAC. (a) Levels of E-cadherin mRNA were determined by qRT-PCR analysis on RNA extracted from HCT116 control and HCT116 E6 cells. GAPDH mRNA was amplified in the same RNA samples for normalization. n = 5 independent experiments. (b) Luciferase activity of the −178/+92 E-cadherin promoter was measured in HCT116 and HCT116 E6 cells. n = 3 independent experiments. (c) Schematic representation of the luciferase plasmid under the control of the E-cadherin promoter. Negative regulatory elements Epal, E-box and positive elements CAAT, GC-I and GC-II are shown as is the mt Epal, with the two-nucleotide inactivating mutation. (d) Activity of wild type and mt Epal E-cadherin promoters in HCT116 and HCT116 E6 cells. Luciferase activity of co-transfected pRL-SV40 was used as a control for transfection efficiency and for normalization. n = 8 independent experiments. (e) RNA was extracted from HCT116 and HCT116 E6 cells that had been either untreated or treated with HDAC inhibitor, TSA, for 24 h. E-cadherin mRNA levels were measured by qRT-PCR and normalized to GAPDH mRNA levels. n = 3 independent experiments.CC BY · PubMed Central
Figure 4 DNMT activity is increased in E6 expressing cells and E-cadherin can be restored following treatment with the DNMT inhibitor, 5-Aza-dC. (a) Nuclear extracts from HCT116 and HCT116 E6 cells were assayed for DNMT activity. n = 3 independent experiments. Error bars represent ± SEM. * P<0.05. (b) RNA was harvested from HCT116 and HCT116 E6 cells treated with 2.5 µM and 7.5 µM 5-Aza-dC for 72 h and analysed by qRT-PCR for E-cadherin mRNA levels, normalized to GAPDH. n = 3 independent experiments. Error bars represent ± SEM. * P<0.05; ns = not significant. (c) Lysates from HCT116 and HCT116 E6 cells treated with 5-Aza-dC, as above, were tested for E-cadherin expression by western blot. Data representative of three experiments. (d) Bisulfite-converted DNA from HCT116 and HCT116 E6 cells was prepared and sequenced to determine the methylation state of CpGs within the minimal promoter region (−191 to +94 bp relative to transcription start site). Nucleotide sequences of ten individual clones of bisulfite converted DNA from each cell line were analysed.
Figure 4 DNMT activity is increased in E6 expressing cells and E-cadherin can be restored following treatment with the DNMT inhibitor, 5-Aza-dC. (a) Nuclear extracts from HCT116 and HCT116 E6 cells were assayed for DNMT activity. n = 3 independent experiments. Error bars represent ± SEM. * P<0.05. (b) RNA was harvested from HCT116 and HCT116 E6 cells treated with 2.5 µM and 7.5 µM 5-Aza-dC for 72 h and analysed by qRT-PCR for E-cadherin mRNA levels, normalized to GAPDH. n = 3 independent experiments. Error bars represent ± SEM. * P<0.05; ns = not significant. (c) Lysates from HCT116 and HCT116 E6 cells treated with 5-Aza-dC, as above, were tested for E-cadherin expression by western blot. Data representative of three experiments. (d) Bisulfite-converted DNA from HCT116 and HCT116 E6 cells was prepared and sequenced to determine the methylation state of CpGs within the minimal promoter region (−191 to +94 bp relative to transcription start site). Nucleotide sequences of ten individual clones of bisulfite converted DNA from each cell line were analysed.CC BY · PubMed Central
Figure 5 E6 reduces the luciferase activities of the full length and a series of deletion constructs of the human E-cadherin regulatory region, whilst reducing the occupancy of the transcriptional regulators within this region. (a) The pGL3 Firefly luciferase constructs were transfected into HCT116 (black bars) and HCT116 E6 (white bars) cells and 48 h later luciferase activities were measured. Activity was normalized to pRL-SV40 Renilla luciferase activity. Results are from three independent experiments. (b) HCT116 and HCT116 E6 cells were subjected to ChIP analysis followed by PCR to determine occupancy of transcriptional regulators AML1, Sp1 and Slug within the E-cadherin regulatory region. (c) Three independent experiments were quantified (Image J, NIH) to measure the relative percentage of AML1, Slug or Sp1 bound to the E-cadherin promoter in E6 cells relative to control cells.
Figure 5 E6 reduces the luciferase activities of the full length and a series of deletion constructs of the human E-cadherin regulatory region, whilst reducing the occupancy of the transcriptional regulators within this region. (a) The pGL3 Firefly luciferase constructs were transfected into HCT116 (black bars) and HCT116 E6 (white bars) cells and 48 h later luciferase activities were measured. Activity was normalized to pRL-SV40 Renilla luciferase activity. Results are from three independent experiments. (b) HCT116 and HCT116 E6 cells were subjected to ChIP analysis followed by PCR to determine occupancy of transcriptional regulators AML1, Sp1 and Slug within the E-cadherin regulatory region. (c) Three independent experiments were quantified (Image J, NIH) to measure the relative percentage of AML1, Slug or Sp1 bound to the E-cadherin promoter in E6 cells relative to control cells.CC BY · PubMed Central
Figure 6 Co-expression of E7 in E6-expressing cells further decreases surface E-cadherin levels and function. HCT116 E6 cells were transfected with GFP, GFP-E5, or GFP-E7 and an adhesion assay followed by flow cytometric analysis of the GFP positive cells was carried out. (a) Dot plots showing E-cadherin negative single cell population (lower left), E-cadherin positive single cells (upper left) and E-cadherin positive aggregates (upper right). The percentage of events in each quadrant is shown. (b) Cell surface E-cadherin and (c) E-cadherin-mediated cellular aggregates were measured. n = 5 independent experiments. *** P<0.001.
Figure 6 Co-expression of E7 in E6-expressing cells further decreases surface E-cadherin levels and function. HCT116 E6 cells were transfected with GFP, GFP-E5, or GFP-E7 and an adhesion assay followed by flow cytometric analysis of the GFP positive cells was carried out. (a) Dot plots showing E-cadherin negative single cell population (lower left), E-cadherin positive single cells (upper left) and E-cadherin positive aggregates (upper right). The percentage of events in each quadrant is shown. (b) Cell surface E-cadherin and (c) E-cadherin-mediated cellular aggregates were measured. n = 5 independent experiments. *** P<0.001.CC BY · PubMed Central

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Cited by

Metadata

Authors
D'Costa, Z. J., Jolly, C., Androphy, E. J., Mercer, A., Matthews, C. M., & Hibma, M. H.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-11-26
Volume
7
Issue
11
Pages
e48954
ISSN
1932-6203
DOI
10.1371/journal.pone.0048954
PMC
PMC3506579
Cited by
83
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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