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Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.

Ramasubramanyan S, Osborn K, Al-Mohammad R, Naranjo Perez-Fernandez IB, Zuo J, Balan N, Godfrey A, Patel H, Peters G, Rowe M, Jenner RG, Sinclair AJ - Nucleic Acids Res. (2015)

Bottom Line: Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome.Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites.This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK.

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Zta can activate gene expression through binding sites associated with FOSB. (A) The DNA sequences at three Zta binding sites associated with FOSB were cloned upstream from an EBV lytic cycle promoter BHLF1 containing no ZREs (BHLF1mut) to generate BHLF1mut FOSB. A control region from the human genome is also included. (B) Promoter activity of BHLF1mut, BHLF1mut control and BHLF1mut FOSB in the presence and absence of His-Zta expression in the EBV negative cell line (DG75). The relative luciferase activity is shown as fold regulation by Zta with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B).
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Figure 9: Zta can activate gene expression through binding sites associated with FOSB. (A) The DNA sequences at three Zta binding sites associated with FOSB were cloned upstream from an EBV lytic cycle promoter BHLF1 containing no ZREs (BHLF1mut) to generate BHLF1mut FOSB. A control region from the human genome is also included. (B) Promoter activity of BHLF1mut, BHLF1mut control and BHLF1mut FOSB in the presence and absence of His-Zta expression in the EBV negative cell line (DG75). The relative luciferase activity is shown as fold regulation by Zta with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B).

Mentions: We then asked whether authentic Zta binding sequences from the cellular targets genes can be transferred onto a heterologous promoter. We chose the FOSB and RASA3 genes as examples. In FOSB each of the four non-CPG Zta binding sites was ≥2 kb from the FOSB TSS (16 and 9 kb upstream, 2.4 and 10 kb downstream) and the gene is up regulated during the EBV lytic replication cycle (Figure 7E). We cloned the Zta binding sites from −16 and −9 kb upstream from a viral promoter (BHLF1mut) that had been rendered unresponsive to Zta by virtue of mutations in each of its ZREs (Figure 9A). We also included a similar size control region from the cellular genome. The reporter plasmids were transfected into DG75 cells, together with a plasmid encoding Zta or the empty vector control. The presence of the FOSB sequences caused a 2.5-fold increase in the response of the promoter to Zta compared to the controls (Figure 9B). Equivalent expression of Zta protein was seen for all reporters (Figure 9C). We therefore conclude that the distal Zta binding sites from the FOSB gene are able to confer Zta responsiveness. For RASA3, where there are multiple Zta binding sites across a >100 kb region, we focused on elements within seven Zta binding sites from the 3′ end of the RASA3 gene (≥40 kb from the TSS). These conferred Zta-mediated induction of expression of the minP promoter when cloned into the enhancer position of the PGL3 luciferase reporter vector. The average increase in expression was 2.2-fold over three experiments (Figure 10), which is in line with typical ESC enhancers in similar assays (56).


Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.

Ramasubramanyan S, Osborn K, Al-Mohammad R, Naranjo Perez-Fernandez IB, Zuo J, Balan N, Godfrey A, Patel H, Peters G, Rowe M, Jenner RG, Sinclair AJ - Nucleic Acids Res. (2015)

Zta can activate gene expression through binding sites associated with FOSB. (A) The DNA sequences at three Zta binding sites associated with FOSB were cloned upstream from an EBV lytic cycle promoter BHLF1 containing no ZREs (BHLF1mut) to generate BHLF1mut FOSB. A control region from the human genome is also included. (B) Promoter activity of BHLF1mut, BHLF1mut control and BHLF1mut FOSB in the presence and absence of His-Zta expression in the EBV negative cell line (DG75). The relative luciferase activity is shown as fold regulation by Zta with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B).
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Related In: Results  -  Collection

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Figure 9: Zta can activate gene expression through binding sites associated with FOSB. (A) The DNA sequences at three Zta binding sites associated with FOSB were cloned upstream from an EBV lytic cycle promoter BHLF1 containing no ZREs (BHLF1mut) to generate BHLF1mut FOSB. A control region from the human genome is also included. (B) Promoter activity of BHLF1mut, BHLF1mut control and BHLF1mut FOSB in the presence and absence of His-Zta expression in the EBV negative cell line (DG75). The relative luciferase activity is shown as fold regulation by Zta with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B).
Mentions: We then asked whether authentic Zta binding sequences from the cellular targets genes can be transferred onto a heterologous promoter. We chose the FOSB and RASA3 genes as examples. In FOSB each of the four non-CPG Zta binding sites was ≥2 kb from the FOSB TSS (16 and 9 kb upstream, 2.4 and 10 kb downstream) and the gene is up regulated during the EBV lytic replication cycle (Figure 7E). We cloned the Zta binding sites from −16 and −9 kb upstream from a viral promoter (BHLF1mut) that had been rendered unresponsive to Zta by virtue of mutations in each of its ZREs (Figure 9A). We also included a similar size control region from the cellular genome. The reporter plasmids were transfected into DG75 cells, together with a plasmid encoding Zta or the empty vector control. The presence of the FOSB sequences caused a 2.5-fold increase in the response of the promoter to Zta compared to the controls (Figure 9B). Equivalent expression of Zta protein was seen for all reporters (Figure 9C). We therefore conclude that the distal Zta binding sites from the FOSB gene are able to confer Zta responsiveness. For RASA3, where there are multiple Zta binding sites across a >100 kb region, we focused on elements within seven Zta binding sites from the 3′ end of the RASA3 gene (≥40 kb from the TSS). These conferred Zta-mediated induction of expression of the minP promoter when cloned into the enhancer position of the PGL3 luciferase reporter vector. The average increase in expression was 2.2-fold over three experiments (Figure 10), which is in line with typical ESC enhancers in similar assays (56).

Bottom Line: Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome.Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites.This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK.

Show MeSH
Related in: MedlinePlus