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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 a long-range element. (A) A synthetic element containing tandem ZREs was cloned 2.2 kb from either of two minimal promoters (minC and minP) in the pGL3 control luciferase reporter plasmid. (B) Promoter activities of the pGL3 based plasmids SV40p, minC-Null and minC ZREs in the presence (black) and absence (gray) of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B). (D) Promoter activity of the minP-Null and minP-ZREs reporters in the presence and absence of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (E) Western blot analysis of Zta and actin from samples in (B).
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Figure 8: Zta can activate gene expression through a long-range element. (A) A synthetic element containing tandem ZREs was cloned 2.2 kb from either of two minimal promoters (minC and minP) in the pGL3 control luciferase reporter plasmid. (B) Promoter activities of the pGL3 based plasmids SV40p, minC-Null and minC ZREs in the presence (black) and absence (gray) of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B). (D) Promoter activity of the minP-Null and minP-ZREs reporters in the presence and absence of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (E) Western blot analysis of Zta and actin from samples in (B).

Mentions: The ability of Zta to activate transcription by binding to promoter elements is well documented (53) but we are not aware of any previous reports that Zta can act through distal regulatory elements. To gain support for the idea that Zta can also influence transcription from a distance, we generated luciferase reporter constructs in which a tandem array of non CpG ZREs was cloned into a classical enhancer vector, 2.2 kb upstream of one of two minimal promoters (Figure 8A and Supplementary Figure S2). Tandem arrays of ZREs embedded within minimal promoters have been previously used to measure Zta activity (54,55), but action away from a promoter has not been assessed. In the MinC-ZREs reporter, we used a minimal promoter derived from the CIITA gene, which is not regulated by Zta. An alternative reporter, designated MinP-ZREs, was based on the minimal promoter in the commercially sourced pGL4.23 reporter vector (Promega). In each case, we compared the activity of the promoters with and without the ZREs, with an SV40 promoter construct as an additional control (Figure 8B and D). The reporter plasmids were introduced into EBV negative DG75 BL cells, together with a vector encoding Zta or empty vector control. Immunoblotting confirmed that Zta was expressed at equivalent levels in the transfected cell populations (Figure 8C and E). Whereas Zta had a minimal effect on the activity of the MinC and SV40 promoters, the inclusion of the ZRE array resulted in an 8-fold increase in the activity of the MinC construct. Similarly, although Zta appeared able to activate the MinP promoter on its own, the presence of the ZRE array resulted in more substantial activation (75-fold versus 13-fold). The 6-fold differential between these effects is consistent with the impact of the ZRE array on the MinP promoter. Taken together, the data suggest that Zta can regulate gene expression via ZRE elements that are located at a distance from promoters, in a classical enhancer assay.


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 a long-range element. (A) A synthetic element containing tandem ZREs was cloned 2.2 kb from either of two minimal promoters (minC and minP) in the pGL3 control luciferase reporter plasmid. (B) Promoter activities of the pGL3 based plasmids SV40p, minC-Null and minC ZREs in the presence (black) and absence (gray) of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B). (D) Promoter activity of the minP-Null and minP-ZREs reporters in the presence and absence of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (E) Western blot analysis of Zta and actin from samples in (B).
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Figure 8: Zta can activate gene expression through a long-range element. (A) A synthetic element containing tandem ZREs was cloned 2.2 kb from either of two minimal promoters (minC and minP) in the pGL3 control luciferase reporter plasmid. (B) Promoter activities of the pGL3 based plasmids SV40p, minC-Null and minC ZREs in the presence (black) and absence (gray) of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (C) Western blot analysis of Zta and Actin from samples in (B). (D) Promoter activity of the minP-Null and minP-ZREs reporters in the presence and absence of His-Zta expression in the EBV negative BL cell line (DG75). The relative luciferase activity is shown as promoter activity together with the standard deviation from three replicates. (E) Western blot analysis of Zta and actin from samples in (B).
Mentions: The ability of Zta to activate transcription by binding to promoter elements is well documented (53) but we are not aware of any previous reports that Zta can act through distal regulatory elements. To gain support for the idea that Zta can also influence transcription from a distance, we generated luciferase reporter constructs in which a tandem array of non CpG ZREs was cloned into a classical enhancer vector, 2.2 kb upstream of one of two minimal promoters (Figure 8A and Supplementary Figure S2). Tandem arrays of ZREs embedded within minimal promoters have been previously used to measure Zta activity (54,55), but action away from a promoter has not been assessed. In the MinC-ZREs reporter, we used a minimal promoter derived from the CIITA gene, which is not regulated by Zta. An alternative reporter, designated MinP-ZREs, was based on the minimal promoter in the commercially sourced pGL4.23 reporter vector (Promega). In each case, we compared the activity of the promoters with and without the ZREs, with an SV40 promoter construct as an additional control (Figure 8B and D). The reporter plasmids were introduced into EBV negative DG75 BL cells, together with a vector encoding Zta or empty vector control. Immunoblotting confirmed that Zta was expressed at equivalent levels in the transfected cell populations (Figure 8C and E). Whereas Zta had a minimal effect on the activity of the MinC and SV40 promoters, the inclusion of the ZRE array resulted in an 8-fold increase in the activity of the MinC construct. Similarly, although Zta appeared able to activate the MinP promoter on its own, the presence of the ZRE array resulted in more substantial activation (75-fold versus 13-fold). The 6-fold differential between these effects is consistent with the impact of the ZRE array on the MinP promoter. Taken together, the data suggest that Zta can regulate gene expression via ZRE elements that are located at a distance from promoters, in a classical enhancer assay.

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