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CTCF prevents the epigenetic drift of EBV latency promoter Qp.

Tempera I, Wiedmer A, Dheekollu J, Lieberman PM - PLoS Pathog. (2010)

Bottom Line: We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome.Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells.EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound approximately 40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection.

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Mutagenesis of CTCF binding site in Qp.A) Schematic of mutations introduced into the Qp region of EBV bacmid. B) Purified bacmid DNA for EBV Wt, GAL K, Wt rescue, and ΔCTCF was analyzed by Sal I restriction digest and 0.7% agarose gel electrophoresis. DNA was visualized by ethidium bromide staining. C) PCR amplification of the region encompassing Qp for EBV Wt, GAL K, Wt rescue and ΔCTCF. D and E) ChIP assay of Wt rescue, or ΔCTCF bacmids in stable 293 cell pools after 8 weeks of hygromycin selection with antibody for CTCF (top panel), or EBNA1 (lower panel). CTCF ChIP was analyzed at Qp or a region −5 kb to Qp. EBNA1 ChIP was analyzed at Qp, or at OriLyt control region. E) Western blot analysis of CTCF, GFP, EBNA1, and PCNA protein levels for Wt rescue or ΔCTCF 293 cell pools.
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ppat-1001048-g004: Mutagenesis of CTCF binding site in Qp.A) Schematic of mutations introduced into the Qp region of EBV bacmid. B) Purified bacmid DNA for EBV Wt, GAL K, Wt rescue, and ΔCTCF was analyzed by Sal I restriction digest and 0.7% agarose gel electrophoresis. DNA was visualized by ethidium bromide staining. C) PCR amplification of the region encompassing Qp for EBV Wt, GAL K, Wt rescue and ΔCTCF. D and E) ChIP assay of Wt rescue, or ΔCTCF bacmids in stable 293 cell pools after 8 weeks of hygromycin selection with antibody for CTCF (top panel), or EBNA1 (lower panel). CTCF ChIP was analyzed at Qp or a region −5 kb to Qp. EBNA1 ChIP was analyzed at Qp, or at OriLyt control region. E) Western blot analysis of CTCF, GFP, EBNA1, and PCNA protein levels for Wt rescue or ΔCTCF 293 cell pools.

Mentions: To investigate the functional significance of CTCF binding at Qp in cell-based assays, we engineered a substitution mutation in the CTCF binding site at Qp in EBV bacmids using recombineering with GALK gene insertion and gene replacement [56] (http://recombineering.ncifcrf.gov) (Fig. 4A). GALK insertion, CTCF substitution mutation (ΔCTCF), and Wild-type (Wt) rescue mutants in EBV bacmids were validated by restriction enzyme digestion (Fig. 4B), PCR across the junctions (Fig. 4C) and sequencing of the insertions (data not shown). Bacmid DNA for ΔCTCF and Wt rescue control was introduced into 293 cells and stable transformants were selected for hygromycin and GFP expression. After 8 weeks of selection, stable cell pools were assayed by ChIP assay to validate that the substitution mutation disrupted CTCF binding in living cells (Fig. 4D). As expected, CTCF failed to bind to Qp in the ΔCTCF mutant (Fig. 4D, top panel). We also found that EBNA1 binding to Qp was reduced in the ΔCTCF mutant (Fig. 4D, lower panel), suggesting that CTCF facilitates EBNA1 binding at Qp in living cells. Stable cell pools were also assayed at 8 weeks for their relative expression GFP, EBNA1, CTCF and PCNA (Fig. 4E). We found no apparent differences in the expression of these proteins after 8 weeks of selection in 293 cell pools.


CTCF prevents the epigenetic drift of EBV latency promoter Qp.

Tempera I, Wiedmer A, Dheekollu J, Lieberman PM - PLoS Pathog. (2010)

Mutagenesis of CTCF binding site in Qp.A) Schematic of mutations introduced into the Qp region of EBV bacmid. B) Purified bacmid DNA for EBV Wt, GAL K, Wt rescue, and ΔCTCF was analyzed by Sal I restriction digest and 0.7% agarose gel electrophoresis. DNA was visualized by ethidium bromide staining. C) PCR amplification of the region encompassing Qp for EBV Wt, GAL K, Wt rescue and ΔCTCF. D and E) ChIP assay of Wt rescue, or ΔCTCF bacmids in stable 293 cell pools after 8 weeks of hygromycin selection with antibody for CTCF (top panel), or EBNA1 (lower panel). CTCF ChIP was analyzed at Qp or a region −5 kb to Qp. EBNA1 ChIP was analyzed at Qp, or at OriLyt control region. E) Western blot analysis of CTCF, GFP, EBNA1, and PCNA protein levels for Wt rescue or ΔCTCF 293 cell pools.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921154&req=5

ppat-1001048-g004: Mutagenesis of CTCF binding site in Qp.A) Schematic of mutations introduced into the Qp region of EBV bacmid. B) Purified bacmid DNA for EBV Wt, GAL K, Wt rescue, and ΔCTCF was analyzed by Sal I restriction digest and 0.7% agarose gel electrophoresis. DNA was visualized by ethidium bromide staining. C) PCR amplification of the region encompassing Qp for EBV Wt, GAL K, Wt rescue and ΔCTCF. D and E) ChIP assay of Wt rescue, or ΔCTCF bacmids in stable 293 cell pools after 8 weeks of hygromycin selection with antibody for CTCF (top panel), or EBNA1 (lower panel). CTCF ChIP was analyzed at Qp or a region −5 kb to Qp. EBNA1 ChIP was analyzed at Qp, or at OriLyt control region. E) Western blot analysis of CTCF, GFP, EBNA1, and PCNA protein levels for Wt rescue or ΔCTCF 293 cell pools.
Mentions: To investigate the functional significance of CTCF binding at Qp in cell-based assays, we engineered a substitution mutation in the CTCF binding site at Qp in EBV bacmids using recombineering with GALK gene insertion and gene replacement [56] (http://recombineering.ncifcrf.gov) (Fig. 4A). GALK insertion, CTCF substitution mutation (ΔCTCF), and Wild-type (Wt) rescue mutants in EBV bacmids were validated by restriction enzyme digestion (Fig. 4B), PCR across the junctions (Fig. 4C) and sequencing of the insertions (data not shown). Bacmid DNA for ΔCTCF and Wt rescue control was introduced into 293 cells and stable transformants were selected for hygromycin and GFP expression. After 8 weeks of selection, stable cell pools were assayed by ChIP assay to validate that the substitution mutation disrupted CTCF binding in living cells (Fig. 4D). As expected, CTCF failed to bind to Qp in the ΔCTCF mutant (Fig. 4D, top panel). We also found that EBNA1 binding to Qp was reduced in the ΔCTCF mutant (Fig. 4D, lower panel), suggesting that CTCF facilitates EBNA1 binding at Qp in living cells. Stable cell pools were also assayed at 8 weeks for their relative expression GFP, EBNA1, CTCF and PCNA (Fig. 4E). We found no apparent differences in the expression of these proteins after 8 weeks of selection in 293 cell pools.

Bottom Line: We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome.Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells.EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound approximately 40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection.

Show MeSH
Related in: MedlinePlus