Limits...
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.

Show MeSH

Related in: MedlinePlus

All four methylation sites on ZRE3 contribute to the binding by Zta.A. Schematic representation of the four methyl-cytosine residues in ZRE3. Methylation is indicated by an asterisk and the numbering system is shown. B, C. Competition EMSA reactions were undertaken with a labeled ZRE (ZIIIB) and non-labeled Zta protein. As indicated, increasing amounts (6×, 10×, 20×, 50×, 75× and 100× excess) of unlabelled competitor ZRE3 DNA (methylated or not) was included in the EMSA reaction. D–G. EMSA competition from replicate experiments showing the ability of the indicated excess of each singly methylated ZRE3 site to compete for the binding of Zta. Experiments were undertaken in duplicate and were used to calculate the standard deviation shown in the error bars.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2267006&req=5

ppat-1000005-g005: All four methylation sites on ZRE3 contribute to the binding by Zta.A. Schematic representation of the four methyl-cytosine residues in ZRE3. Methylation is indicated by an asterisk and the numbering system is shown. B, C. Competition EMSA reactions were undertaken with a labeled ZRE (ZIIIB) and non-labeled Zta protein. As indicated, increasing amounts (6×, 10×, 20×, 50×, 75× and 100× excess) of unlabelled competitor ZRE3 DNA (methylated or not) was included in the EMSA reaction. D–G. EMSA competition from replicate experiments showing the ability of the indicated excess of each singly methylated ZRE3 site to compete for the binding of Zta. Experiments were undertaken in duplicate and were used to calculate the standard deviation shown in the error bars.

Mentions: As the interaction of Zta with ZRE3 is methylation-dependent and the site contains four methylated cytosine residues, we explored the relevance of each methylation site (see Figure 5A for numbering convention). A series of double-strand versions of ZRE3 was generated, each containing a single methyl-cytosine, and binding to Zta was assessed in a competition assay. Initial validation of the assay revealed that whereas a 20-fold excess of fully methylated ZRE3 competes efficiently for Zta binding, non-methylated ZRE3 fails to compete, even at a 100-fold excess (Figure 5B,C). Each singly methylated version of ZRE3 competed for binding to some degree (Figure 5D–G) implying that all four methyl groups contribute to the interaction with Zta. The order of competition efficiency was methyl cytosine1′>methyl cytosine−2>methyl cytosine0≥methyl cytosine−1′. Thus, methylation within the CpG motif that is common to both ZRE2 and ZRE3 gave stronger competition than within the motif uniquely present in ZRE3.


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)

All four methylation sites on ZRE3 contribute to the binding by Zta.A. Schematic representation of the four methyl-cytosine residues in ZRE3. Methylation is indicated by an asterisk and the numbering system is shown. B, C. Competition EMSA reactions were undertaken with a labeled ZRE (ZIIIB) and non-labeled Zta protein. As indicated, increasing amounts (6×, 10×, 20×, 50×, 75× and 100× excess) of unlabelled competitor ZRE3 DNA (methylated or not) was included in the EMSA reaction. D–G. EMSA competition from replicate experiments showing the ability of the indicated excess of each singly methylated ZRE3 site to compete for the binding of Zta. Experiments were undertaken in duplicate and were used to calculate the standard deviation shown in the error bars.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000005-g005: All four methylation sites on ZRE3 contribute to the binding by Zta.A. Schematic representation of the four methyl-cytosine residues in ZRE3. Methylation is indicated by an asterisk and the numbering system is shown. B, C. Competition EMSA reactions were undertaken with a labeled ZRE (ZIIIB) and non-labeled Zta protein. As indicated, increasing amounts (6×, 10×, 20×, 50×, 75× and 100× excess) of unlabelled competitor ZRE3 DNA (methylated or not) was included in the EMSA reaction. D–G. EMSA competition from replicate experiments showing the ability of the indicated excess of each singly methylated ZRE3 site to compete for the binding of Zta. Experiments were undertaken in duplicate and were used to calculate the standard deviation shown in the error bars.
Mentions: As the interaction of Zta with ZRE3 is methylation-dependent and the site contains four methylated cytosine residues, we explored the relevance of each methylation site (see Figure 5A for numbering convention). A series of double-strand versions of ZRE3 was generated, each containing a single methyl-cytosine, and binding to Zta was assessed in a competition assay. Initial validation of the assay revealed that whereas a 20-fold excess of fully methylated ZRE3 competes efficiently for Zta binding, non-methylated ZRE3 fails to compete, even at a 100-fold excess (Figure 5B,C). Each singly methylated version of ZRE3 competed for binding to some degree (Figure 5D–G) implying that all four methyl groups contribute to the interaction with Zta. The order of competition efficiency was methyl cytosine1′>methyl cytosine−2>methyl cytosine0≥methyl cytosine−1′. Thus, methylation within the CpG motif that is common to both ZRE2 and ZRE3 gave stronger competition than within the motif uniquely present in ZRE3.

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