CpG-methylation regulates a class of Epstein-Barr virus promoters

Abstract

DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to 'read' DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing.

Figure 1. Zta binds preferentially to CpG-methylated EBV DNA in vitro.

In vitro immunoprecipitation assays with GFP:BZLF1 and subsequent deep sequencing analysis indicate the preferred binding of Zta to CpG-methylated EBV DNA. E.coli-derived genomic EBV DNA free of methylated CpG dinucleotides (blue) and after complete in vitro CpG methylation by the methyltransferase M.SssI (red) was used as probes for the GFP:BZLF1 immunoprecipitations. Reads were mapped to the reference B95.8 EBV genome. Depicted is the read depth at single base pair resolution. In addition, the difference of read depth between the two experiments (Δdepth, black) is calculated and shown at the bottom.

Figure 2. Identification of methylation-dependent Zta binding to selected viral promoter elements in vivo and in vitro.

Deep sequencing data obtained from in vitro immunoprecipitation experiments with GFP:BZLF1 as in Figure 1 are plotted on selected genes and their promoters as indicated. EBV DNA free of CpG-methylation (blue) and after full methylation by the methyltransferase M.SssI (red) were used as probes in the experiments. In vivo ChIP-seq data obtained after immunoprecipitation of chromatin of Raji cells (black) or B95.8 cells (green) with GFP:BZLF1 are plotted. Ten genes, their exon compositions and the location of selected ZREs are shown. (A) The two divergently transcribed genes BHLF1 and BHRF1, which bracket EBV's lytic origin of DNA replication are shown. (B) The two genes BZLF1 and BHRF1 and the intervening gene BRRF1 are shown. (C) The BMRF1 gene is shown, which encodes the viral DNA polymerase accessory protein.

Figure 3. Identification of methylation-dependent Zta binding to selected viral promoter elements in vivo and in vitro.

Deep sequencing data obtained from in vitro immunoprecipitation experiments with GFP:BZLF1 as in Figure 1 are plotted on selected genes and their promoters as indicated. EBV DNA free of CpG-methylation (blue) and after full methylation by the methyltransferase M.SssI (red) were used as probes in the experiments. In vivo ChIP-seq data obtained after immunoprecipitation of chromatin of Raji cells (black) or B95.8 cells (green) with GFP:BZLF1 are plotted. Ten genes, their exon compositions and the location of selected ZREs are shown. (A) The two annotated genes BSLF2 and BMLF1 are shown, which result in the spliced BMLF2/BSLF1 transcript encoding the viral SM protein also called EB2. (B) The BFRF1 gene is shown. (C) The BALF2 gene is shown, which encodes the major DNA binding protein of EBV.

Figure 4. Identification of methylation-dependent Zta binding to selected viral promoter elements in vivo and in vitro.

Deep sequencing data obtained from in vitro immunoprecipitation experiments with GFP:BZLF1 as in Figure 1 are plotted on selected genes and their promoters as indicated. EBV DNA free of CpG-methylation (blue) and after full methylation by the methyltransferase M.SssI (red) were used as probes in the experiments. In vivo ChIP-seq data obtained after immunoprecipitation of chromatin of Raji cells (black) or B95.8 cells (green) with GFP:BZLF1 are plotted. Ten genes, their exon compositions and the location of selected ZREs are shown. (A) The BBLF4 gene encoding the viral DNA helicase and the BKRF4 gene are shown. (B) The two open reading frames BBLF2 and BBLF3 are spliced and encode the primase-associated factor BBLF2/3. (C) The BALF5 and the ECRF4 genes are shown, which encode the viral DNA polymerase and a hypothetical protein, respectively.

Figure 5. Identification of methylation-dependent Zta binding to the viral BSLF1 and BSLF2/BMLF1 promoter elements in vivo and in vitro.

Deep sequencing data obtained from in vitro immunoprecipitation experiments with GFP:BZLF1 as in Figure 1 are plotted on selected genes and their promoters as indicated. EBV DNA free of CpG-methylation (blue) and after full methylation by the methyltransferase M.SssI (red) were used as probes in the experiments. In vivo ChIP-seq data obtained after immunoprecipitation of chromatin of Raji cells (black) or B95.8 cells (green) with GFP:BZLF1 are plotted. Ten genes, their exon compositions and the location of selected ZREs are shown. The loci of the BSRF1 and BSLF2/BMLF1 genes are shown in conjunction with the BSLF1 gene, which encodes the viral primase.

Figure 6. Motif discovery of Zta bound to unmethylated, CpG-methylated EBV DNA or Raji cell chromatin.

ChIP-seq data and deep sequencing data after in vitro immunoprecipitation assays with GFP:BZLF1 were analyzed with the SISSRs (default parameters) or QuEST algorithms (kernel density estimate of 60, threshold value of 2) for putative stretches of DNA to which Zta binds. The outputs of SISSRs or QuEST were used as training sets for MEME, which identifies gapless, local, multiple sequence motifs . (A) A total of 101 motifs were identified in in vitro immunoprecipitation experiments followed deep sequencing with E.coli-derived EBV DNA free of CpG methylation. (B) A total of 167 motifs were identified in in vitro immunoprecipitation experiments followed deep sequencing with fully CpG-methylated E.coli-derived EBV DNA. (C) The identified motifs in (B) were selected at the level of the SISSRs training set data and grouped into ZRE motifs with (bottom panel) and without (top panel) CpG dinucleotides followed by MEME analysis. (D) A total of 46 motifs were identified in ChIP-seq data after chromatin immunoprecipitations from Raji cells stably transfected with an expression plasmid encoding GFP:BZLF1 (Supporting Figure S1). (E) The identified motifs in (D) were selected at the level of the QuEST training set data and grouped into ZRE motifs encompassing no (top panel) or one or more (bottom panel) CpG dinucleotides. Subsequent MEME analysis identified two classes of ZREs.

Figure 7. Zta binds preferentially to CpG-methylated promoters.

In EMSAs, Zta bound preferentially to the CpG methylated promoters of BRLF1, BALF2, BSLF2/BMLF1, BALF5 and BMRF1 and BSLF1 but it bound only the CpG-methylated promoters of BBLF4 and BBLF2/3. CpG methylation did not influence Zta binding to the promoters of BZLF1, BHLF1, and BHRF1. EMSAs were performed with affinity-purified Strep/FLAG-tagged BZLF1 fusion protein transiently expressed in HEK293 cells. Eleven PCR fragments encompassing EBV promoters with BZLF1 binding sites identified in Figures 2 to 5 served as radioactive probes. PCR fragments (Table S3 in Text S1) were used either unmethylated or fully CpG-methylated in vitro with M.SssI. Supershifts with a FLAG-antibody confirmed the identity of the protein-DNA complexes. The signals were scanned and the percent ratios of bound DNAs versus total input DNAs are shown for each sample.

Figure 8. Zta transactivates CpG-methylated promoters with superior efficiency.

Promoters of eleven different genes (Table S3 in Text S1) were inserted into a synthetic luciferase reporter plasmid free of CpG-dinucleotides . Reporter plasmids were either used after isolation from E.coli and thus free of methylated CpG dinucleotides (unmethylated, white) or after complete in vitro CpG-methylation (black) and co-transfected with a BZLF1 expression plasmid or with a negative control DNA as indicated (none). After data normalization to a luciferase control plasmid free of promoter elements, the x-fold differences in the different data sets were calculated as shown.

Figure 9. Functional identification of single ZREs and meZREs.

Pentamers of ten single ZREs present in the promoters of BHLF1, BBLF4, BMRF1, BSLF2/BMLF1, and BALF5 were introduced into a basic luciferase reporter plasmid with a minimal EF1α promoter and free of CpGs . Unmethylated and fully CpG methylated reporter constructs were analyzed in the presence or absence of a co-transfected BZLF1 expression plasmid.