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

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

A prospective prostate cancer screening programme for men with pathogenic variants in mismatch repair genes (IMPACT): initial results from an international prospective study.

Funder: Victorian Cancer AgencyFunder: NIHR Manchester Biomedical Research CentreFunder: Cancer Research UKFunder: Cancer Council TasmaniaFunder: Instituto de Salud Carlos IIIFunder: Cancer AustraliaFunder: NIHR Oxford Biomedical Research CentreFunder: Fundación Científica de la Asociación Española Contra el CáncerFunder: Cancer Council South AustraliaFunder: Swedish Cancer SocietyFunder: NIHR Cambridge Biomedical Research CentreFunder: Institut Català de la SalutFunder: Cancer Council VictoriaFunder: Prostate Cancer Foundation of AustraliaFunder: National Institutes of HealthBACKGROUND: Lynch syndrome is a rare familial cancer syndrome caused by pathogenic variants in the mismatch repair genes MLH1, MSH2, MSH6, or PMS2, that cause predisposition to various cancers, predominantly colorectal and endometrial cancer. Data are emerging that pathogenic variants in mismatch repair genes increase the risk of early-onset aggressive prostate cancer. The IMPACT study is prospectively assessing prostate-specific antigen (PSA) screening in men with germline mismatch repair pathogenic variants. Here, we report the usefulness of PSA screening, prostate cancer incidence, and tumour characteristics after the first screening round in men with and without these germline pathogenic variants. METHODS: The IMPACT study is an international, prospective study. Men aged 40-69 years without a previous prostate cancer diagnosis and with a known germline pathogenic variant in the MLH1, MSH2, or MSH6 gene, and age-matched male controls who tested negative for a familial pathogenic variant in these genes were recruited from 34 genetic and urology clinics in eight countries, and underwent a baseline PSA screening. Men who had a PSA level higher than 3·0 ng/mL were offered a transrectal, ultrasound-guided, prostate biopsy and a histopathological analysis was done. All participants are undergoing a minimum of 5 years' annual screening. The primary endpoint was to determine the incidence, stage, and pathology of screening-detected prostate cancer in carriers of pathogenic variants compared with non-carrier controls. We used Fisher's exact test to compare the number of cases, cancer incidence, and positive predictive values of the PSA cutoff and biopsy between carriers and non-carriers and the differences between disease types (ie, cancer vs no cancer, clinically significant cancer vs no cancer). We assessed screening outcomes and tumour characteristics by pathogenic variant status. Here we present results from the first round of PSA screening in the IMPACT study. This study is registered with ClinicalTrials.gov, NCT00261456, and is now closed to accrual. FINDINGS: Between Sept 28, 2012, and March 1, 2020, 828 men were recruited (644 carriers of mismatch repair pathogenic variants [204 carriers of MLH1, 305 carriers of MSH2, and 135 carriers of MSH6] and 184 non-carrier controls [65 non-carriers of MLH1, 76 non-carriers of MSH2, and 43 non-carriers of MSH6]), and in order to boost the sample size for the non-carrier control groups, we randomly selected 134 non-carriers from the BRCA1 and BRCA2 cohort of the IMPACT study, who were included in all three non-carrier cohorts. Men were predominantly of European ancestry (899 [93%] of 953 with available data), with a mean age of 52·8 years (SD 8·3). Within the first screening round, 56 (6%) men had a PSA concentration of more than 3·0 ng/mL and 35 (4%) biopsies were done. The overall incidence of prostate cancer was 1·9% (18 of 962; 95% CI 1·1-2·9). The incidence among MSH2 carriers was 4·3% (13 of 305; 95% CI 2·3-7·2), MSH2 non-carrier controls was 0·5% (one of 210; 0·0-2·6), MSH6 carriers was 3·0% (four of 135; 0·8-7·4), and none were detected among the MLH1 carriers, MLH1 non-carrier controls, and MSH6 non-carrier controls. Prostate cancer incidence, using a PSA threshold of higher than 3·0 ng/mL, was higher in MSH2 carriers than in MSH2 non-carrier controls (4·3% vs 0·5%; p=0·011) and MSH6 carriers than MSH6 non-carrier controls (3·0% vs 0%; p=0·034). The overall positive predictive value of biopsy using a PSA threshold of 3·0 ng/mL was 51·4% (95% CI 34·0-68·6), and the overall positive predictive value of a PSA threshold of 3·0 ng/mL was 32·1% (20·3-46·0). INTERPRETATION: After the first screening round, carriers of MSH2 and MSH6 pathogenic variants had a higher incidence of prostate cancer compared with age-matched non-carrier controls. These findings support the use of targeted PSA screening in these men to identify those with clinically significant prostate cancer. Further annual screening rounds will need to confirm these findings. FUNDING: Cancer Research UK, The Ronald and Rita McAulay Foundation, the National Institute for Health Research support to Biomedical Research Centres (The Institute of Cancer Research and Royal Marsden NHS Foundation Trust; Oxford; Manchester and the Cambridge Clinical Research Centre), Mr and Mrs Jack Baker, the Cancer Council of Tasmania, Cancer Australia, Prostate Cancer Foundation of Australia, Cancer Council of Victoria, Cancer Council of South Australia, the Victorian Cancer Agency, Cancer Australia, Prostate Cancer Foundation of Australia, Asociación Española Contra el Cáncer (AECC), the Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional (FEDER), the Institut Català de la Salut, Autonomous Government of Catalonia, Fundação para a Ciência e a Tecnologia, National Institutes of Health National Cancer Institute, Swedish Cancer Society, General Hospital in Malmö Foundation for Combating Cancer

The reversal of epigenetic silencing of the EBV genome is regulated by viral bZIP protein

EBV (Epstein-Barr virus) 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 protein Zta with three ZREs (Zta-response elements) from the BRLF1 promoter (referred to as Rp). Two of these ZREs contain CpG motifs and are methylated in the latent genome. Biochemical analyses and molecular modelling of Zta bound to methylated RpZRE3 indicate the precise contacts made between a serine and a cysteine residue of Zta with methyl cytosines. A single point mutant of Zta, C189S, is defective in binding to the methylated ZREs both in vitro and in vivo. This was used to probe the functional relevance of the interaction. ZtaC189S was not able to activate Rp in a B-cell line, demonstrating the relevance of the interaction with methylated ZREs. This demonstrates that Zta plays a role in overturning the epigenetic control of viral latency. © The Authors Journal compilation © 2008 Biochemical Society

Evidence for C189- and S186- me cytosine⁻² interactions.

<p>A. Contacts between CpG motif 1 and Zta residues. B. EMSA analysis of unprogramed <i>in vitro</i> translation reaction (none), or Zta protein with the indicated probes was undertaken. The protein-DNA complex is shown. C. The ability of Zta and ZtaS186A to interact with a probe that omits the methylation of cytosine⁻² was determined by EMSA. D. The ability of Zta and ZtaC189S to interact with a probe that omits the methylation of cytosine⁻² was determined by EMSA.</p

ZtaC189S binding to meZREs in Rp is compromised <i>in vitro</i>.

<p>A. EMSA analysis was undertaken for meZRE2 with the indicated proteins or an unprogrammed lysate (IVT). B. Zta, ZtaC189A and ZtaC189S were generated <i>in vitro</i> and analyzed by SDS-PAGE. C. EMSA analysis of equivalent amounts of the indicated proteins was undertaken with meZRE3 as described above.</p

Structural model of Zta bound to meZRE3.

<p>A. Alignment of Zta recognition sequences. The numbering convention is shown for base pair positions (bold italics) and individual nucleotides (plain font). Cytosines modified by methylation are indicated by a dot. B. Model of the Zta-meZRE3 complex viewed along the pseudodyad. The methylation sensitive C189 residue (red asterisk) and bidentate hydrogen bond interactions between R190^Left and Guanine^0′ (dotted black lines) are indicated. Cytosine methyl groups are semi-transparent. C. Orthogonal view. The hydrophobic contact between Cytosine^1′ and S186^Left (broken blue line) and hydrogen bond network involving S186^Left, N182^Left and Guanine2′ (dotted black lines) are shown. D. Schematic summary of contacts. van der Waals contacts involving the CpG methyl groups and Zta residues are shown as broken lines.</p

All four methylation sites on ZRE3 contribute to the binding by Zta.

<p>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.</p

Zta and ZtaC189S interact with non-methylated Rp ZREs equivalently.

<p>A. Schematic diagram showing the location of ZREs 1–3 in Rp. Transcription of this gene occurs in a leftwards direction with respect to the viral genome. The numbering relates to the type I EBV genome accession number NC_007605. Asterixes mark the methylated Cytosine residues. B. Zta and Zta C189s were generated in an <i>in vitro</i> translation system and fractionated on SDS-PAGE, together with a non-programmed translation reaction (IVT). C.–E. Equivalent amounts of the indicated proteins were subject to EMSA analysis with the probes indicated above. A reaction with no added protein was also included, indicated probe. F. The three ZREs associated with Rp are aligned and their areas of conservation indicated by boxes. The interactions of Zta and ZtaC189S with each site are summarized.</p

Single amino acid in basic region of Zta blocks the ability to transactivate Rp in BL cells.

<p>A. Schematic diagram showing the relationship between Zta expression and activation of the <i>BRLF1</i> promoter. B. Expression vectors for Zta, ZtaC189S and the relevant “empty” vector (pBABE) were introduced into Raji cells and their ability to activate the endogenous <i>BRLF1</i> gene determined. 24 hours after transfection, RNA was prepared, cDNA was synthesized then amplified using quantitative PCR with specific primers for the <i>BRLF1</i> transcript and a housekeeping gene, L32. Expression of <i>BRLF1</i> mRNA, following normalization for expression of L32 is shown, relative to that seen following Zta transfection (100%). C. Expression of Zta and ZtaC89S were determined by quantitative PCR and expressed relative to expression of Zta (100%).</p