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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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RNA expression and promoter utilization in Qp mutated bacmids.A) Schematic representation of the EBV latency genes and promoters. Promoters are indicated by arrows. The position of the six EBNAs ORFs are indicated. B) Schematic representation of different EBNA1 transcripts. Exons present at 5′ end of EBNA1 mRNA are indicated in red. C) Quantitative RT-PCR was used to measure the abundance of EBNA2, EBNA3A and EBNA3C mRNA relative to bacmid GFP for Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection, as indicated. D) Same as in C, except EBNA1-transcripts initiating from either Cp/Wp, Qp, or Fp were measured relative to GFP in Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection. E) RT-PCR was measured for Wt rescue or ΔCTCF bacmids in 293 cell pools at 8 weeks post-transfection, as well as for type I (Mutu I) or type III (Mutu-LCL) controls. RNA was analyzed for the junction specific transcripts QUK (Qp initiation), C1C2W1W2 (Cp initiation), W0W1W2 (Wp initiation), BFLF1 (lytic gene adjacent to Qp), UK (EBNA1 mRNA in both type I and type III), and control cellular GAPDH.
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ppat-1001048-g006: RNA expression and promoter utilization in Qp mutated bacmids.A) Schematic representation of the EBV latency genes and promoters. Promoters are indicated by arrows. The position of the six EBNAs ORFs are indicated. B) Schematic representation of different EBNA1 transcripts. Exons present at 5′ end of EBNA1 mRNA are indicated in red. C) Quantitative RT-PCR was used to measure the abundance of EBNA2, EBNA3A and EBNA3C mRNA relative to bacmid GFP for Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection, as indicated. D) Same as in C, except EBNA1-transcripts initiating from either Cp/Wp, Qp, or Fp were measured relative to GFP in Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection. E) RT-PCR was measured for Wt rescue or ΔCTCF bacmids in 293 cell pools at 8 weeks post-transfection, as well as for type I (Mutu I) or type III (Mutu-LCL) controls. RNA was analyzed for the junction specific transcripts QUK (Qp initiation), C1C2W1W2 (Cp initiation), W0W1W2 (Wp initiation), BFLF1 (lytic gene adjacent to Qp), UK (EBNA1 mRNA in both type I and type III), and control cellular GAPDH.

Mentions: The loss of GFP expression and episome stability in 293 cell pools could be due to a deregulation of viral gene expression. Others have shown that EBV establishes a restricted pattern of latency gene expression in 293 cells, resembling a type I program with stable Qp utilization for EBNA1 expression [57]. To assess viral gene expression patterns in transfected 293 cells, we first assay mRNA expression of EBNA1, EBNA2, EBNA3A, and EBNA3C in 293 cell pools after 4, 8, and 16 weeks of selection (Fig. 6). RNA expression was measured by quantitative RT-PCR and normalized to bacmid expression of GFP mRNA. At 4 weeks, EBNA1 and EBNA2 mRNA levels were expressed at lower levels in ΔCTCF compared to Wt rescue cell pools (Fig. 6C, top panel). At 8 weeks, EBNA1 levels were similar, while EBNA2, EBNA3A, and EBNA3C levels were higher in ΔCTCF relative to Wt rescue cell pools (Fig. 6C, middle panel). By 16 weeks, EBNA1 mRNA levels were maintained in the Wt rescue, but almost undetectable in ΔCTCF cell pools (Fig. 6C, lower panel). EBNA2, EBNA3A, and EBNA3C were expressed at very low levels in both Wt rescue and ΔCTCF cell pools at these later passages in culture. These observations are consistent with a previous study showing that EBV initially expresses EBNA2, but eventually adopts an EBNA1 only, type I latency in 293 cells [57].


CTCF prevents the epigenetic drift of EBV latency promoter Qp.

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

RNA expression and promoter utilization in Qp mutated bacmids.A) Schematic representation of the EBV latency genes and promoters. Promoters are indicated by arrows. The position of the six EBNAs ORFs are indicated. B) Schematic representation of different EBNA1 transcripts. Exons present at 5′ end of EBNA1 mRNA are indicated in red. C) Quantitative RT-PCR was used to measure the abundance of EBNA2, EBNA3A and EBNA3C mRNA relative to bacmid GFP for Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection, as indicated. D) Same as in C, except EBNA1-transcripts initiating from either Cp/Wp, Qp, or Fp were measured relative to GFP in Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection. E) RT-PCR was measured for Wt rescue or ΔCTCF bacmids in 293 cell pools at 8 weeks post-transfection, as well as for type I (Mutu I) or type III (Mutu-LCL) controls. RNA was analyzed for the junction specific transcripts QUK (Qp initiation), C1C2W1W2 (Cp initiation), W0W1W2 (Wp initiation), BFLF1 (lytic gene adjacent to Qp), UK (EBNA1 mRNA in both type I and type III), and control cellular GAPDH.
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Related In: Results  -  Collection

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ppat-1001048-g006: RNA expression and promoter utilization in Qp mutated bacmids.A) Schematic representation of the EBV latency genes and promoters. Promoters are indicated by arrows. The position of the six EBNAs ORFs are indicated. B) Schematic representation of different EBNA1 transcripts. Exons present at 5′ end of EBNA1 mRNA are indicated in red. C) Quantitative RT-PCR was used to measure the abundance of EBNA2, EBNA3A and EBNA3C mRNA relative to bacmid GFP for Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection, as indicated. D) Same as in C, except EBNA1-transcripts initiating from either Cp/Wp, Qp, or Fp were measured relative to GFP in Wt rescue or ΔCTCF bacmids in 293 cell pools at 4, 8, and 16 weeks after transfection. E) RT-PCR was measured for Wt rescue or ΔCTCF bacmids in 293 cell pools at 8 weeks post-transfection, as well as for type I (Mutu I) or type III (Mutu-LCL) controls. RNA was analyzed for the junction specific transcripts QUK (Qp initiation), C1C2W1W2 (Cp initiation), W0W1W2 (Wp initiation), BFLF1 (lytic gene adjacent to Qp), UK (EBNA1 mRNA in both type I and type III), and control cellular GAPDH.
Mentions: The loss of GFP expression and episome stability in 293 cell pools could be due to a deregulation of viral gene expression. Others have shown that EBV establishes a restricted pattern of latency gene expression in 293 cells, resembling a type I program with stable Qp utilization for EBNA1 expression [57]. To assess viral gene expression patterns in transfected 293 cells, we first assay mRNA expression of EBNA1, EBNA2, EBNA3A, and EBNA3C in 293 cell pools after 4, 8, and 16 weeks of selection (Fig. 6). RNA expression was measured by quantitative RT-PCR and normalized to bacmid expression of GFP mRNA. At 4 weeks, EBNA1 and EBNA2 mRNA levels were expressed at lower levels in ΔCTCF compared to Wt rescue cell pools (Fig. 6C, top panel). At 8 weeks, EBNA1 levels were similar, while EBNA2, EBNA3A, and EBNA3C levels were higher in ΔCTCF relative to Wt rescue cell pools (Fig. 6C, middle panel). By 16 weeks, EBNA1 mRNA levels were maintained in the Wt rescue, but almost undetectable in ΔCTCF cell pools (Fig. 6C, lower panel). EBNA2, EBNA3A, and EBNA3C were expressed at very low levels in both Wt rescue and ΔCTCF cell pools at these later passages in culture. These observations are consistent with a previous study showing that EBV initially expresses EBNA2, but eventually adopts an EBNA1 only, type I latency in 293 cells [57].

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