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Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Karlsson QH, Schelcher C, Verrall E, Petosa C, Sinclair AJ - PLoS Pathog. (2008)

Bottom Line: Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma.The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3.Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton, United Kingdom.

ABSTRACT
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

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ZtaC189S binding to meZREs in Rp is compromised in vivo.A. Schematic representation of the BRLF1 gene. The black arrow indicates the primary transcript. The location of primer sets used to detect sub-regions of Rp and upstream and downstream regions are indicated relative to the transcription start site. B. HisZta and HisZtaC189S were introduced into 293-BZLF1-KO cells and chromatin prepared. Chromatin affinity purification was undertaken and binding to Rp detected with the indicated primer sets by real-time PCR. The signal was set relative to the “empty vector”, pcDNA3 (striped box), and the signal for Zta (filled box) and ZtaC189S (open box) are shown together with the standard error from duplicate experiments. C. Expression of HisZta, HisZtaC189S and a loading control, PARP, were assessed by western blot analysis.
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ppat-1000005-g004: ZtaC189S binding to meZREs in Rp is compromised in vivo.A. Schematic representation of the BRLF1 gene. The black arrow indicates the primary transcript. The location of primer sets used to detect sub-regions of Rp and upstream and downstream regions are indicated relative to the transcription start site. B. HisZta and HisZtaC189S were introduced into 293-BZLF1-KO cells and chromatin prepared. Chromatin affinity purification was undertaken and binding to Rp detected with the indicated primer sets by real-time PCR. The signal was set relative to the “empty vector”, pcDNA3 (striped box), and the signal for Zta (filled box) and ZtaC189S (open box) are shown together with the standard error from duplicate experiments. C. Expression of HisZta, HisZtaC189S and a loading control, PARP, were assessed by western blot analysis.

Mentions: To substantiate the above findings, we used chromatin precipitation to evaluate the binding of Zta and ZtaC189S to the BRLF1 promoter in vivo. Cells (293-BZLF1-KO) that contain an episomal EBV genome [11] were transfected with vectors encoding either His-tagged Zta or ZtaC189S and equivalence of expression was confirmed by immunoblotting (Figure 4). Zta- and ZtaC189S-associated chromatin complexes were isolated and the co-precipitated DNA was amplified using quantitative PCR with primers spanning the ZREs within Rp (Figure 4). As expected [12], ZRE sequences in the precipitated chromatin were clearly enriched, whereas regions lying 5′ and 3′ of Rp were not. Fine mapping of the chromatin complexes allowed us to differentiate between binding to ZRE1, ZRE2 and ZRE3. Whereas Zta and ZtaC189S bound equally well to ZRE1, the interaction between ZtaC189S and ZRE2 was partly compromised (relative to wild-type Zta) and its interaction with ZRE3 was completely eliminated. As Rp is fully methylated in cells harboring EBV [3], the in vivo chromatin association and the in vitro DNA-binding analyses correlate well. Taken together, these data demonstrate a critical role for C189 in the interaction of Zta with meZRE3 and, to a lesser extent, meZRE2.


Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Karlsson QH, Schelcher C, Verrall E, Petosa C, Sinclair AJ - PLoS Pathog. (2008)

ZtaC189S binding to meZREs in Rp is compromised in vivo.A. Schematic representation of the BRLF1 gene. The black arrow indicates the primary transcript. The location of primer sets used to detect sub-regions of Rp and upstream and downstream regions are indicated relative to the transcription start site. B. HisZta and HisZtaC189S were introduced into 293-BZLF1-KO cells and chromatin prepared. Chromatin affinity purification was undertaken and binding to Rp detected with the indicated primer sets by real-time PCR. The signal was set relative to the “empty vector”, pcDNA3 (striped box), and the signal for Zta (filled box) and ZtaC189S (open box) are shown together with the standard error from duplicate experiments. C. Expression of HisZta, HisZtaC189S and a loading control, PARP, were assessed by western blot analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2267006&req=5

ppat-1000005-g004: ZtaC189S binding to meZREs in Rp is compromised in vivo.A. Schematic representation of the BRLF1 gene. The black arrow indicates the primary transcript. The location of primer sets used to detect sub-regions of Rp and upstream and downstream regions are indicated relative to the transcription start site. B. HisZta and HisZtaC189S were introduced into 293-BZLF1-KO cells and chromatin prepared. Chromatin affinity purification was undertaken and binding to Rp detected with the indicated primer sets by real-time PCR. The signal was set relative to the “empty vector”, pcDNA3 (striped box), and the signal for Zta (filled box) and ZtaC189S (open box) are shown together with the standard error from duplicate experiments. C. Expression of HisZta, HisZtaC189S and a loading control, PARP, were assessed by western blot analysis.
Mentions: To substantiate the above findings, we used chromatin precipitation to evaluate the binding of Zta and ZtaC189S to the BRLF1 promoter in vivo. Cells (293-BZLF1-KO) that contain an episomal EBV genome [11] were transfected with vectors encoding either His-tagged Zta or ZtaC189S and equivalence of expression was confirmed by immunoblotting (Figure 4). Zta- and ZtaC189S-associated chromatin complexes were isolated and the co-precipitated DNA was amplified using quantitative PCR with primers spanning the ZREs within Rp (Figure 4). As expected [12], ZRE sequences in the precipitated chromatin were clearly enriched, whereas regions lying 5′ and 3′ of Rp were not. Fine mapping of the chromatin complexes allowed us to differentiate between binding to ZRE1, ZRE2 and ZRE3. Whereas Zta and ZtaC189S bound equally well to ZRE1, the interaction between ZtaC189S and ZRE2 was partly compromised (relative to wild-type Zta) and its interaction with ZRE3 was completely eliminated. As Rp is fully methylated in cells harboring EBV [3], the in vivo chromatin association and the in vitro DNA-binding analyses correlate well. Taken together, these data demonstrate a critical role for C189 in the interaction of Zta with meZRE3 and, to a lesser extent, meZRE2.

Bottom Line: Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma.The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3.Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton, United Kingdom.

ABSTRACT
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

Show MeSH
Related in: MedlinePlus