Anita Koroknai

Acetylated histone H3 and H4 mark the upregulated LMP2A promoter of Epstein-Barr virus in lymphoid cells.

ABSTRACT We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap.

Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein-Barr virus is unaffected by CpG methylation and does not correlate with Cp activity

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein-Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.

THE IMPORTANCE OF EPIGENETIC ALTERATIONS IN THE DEVELOPMENT OF EPSTEIN-BARR VIRUS-RELATED LYMPHOMAS

Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with a series of malignant tumors. These include lymphomas (Burkitt’s lymphoma, Hodgkin’s disease, T/NK-cell lymphoma, post-transplant lymphoproliferative disease, AIDS-associated lymphoma, X-linked lymphoproliferative syndrome), carcinomas (nasopharyngeal carcinoma, gastric carcinoma, carcinomas of major salivary glands, thymic carcinoma, mammary carcinoma) and a sarcoma (leiomyosarcoma). The latent EBV genomes persist in the tumor cells as circular episomes, co-replicating with the cellular DNA once per cell cycle. The expression of latent EBV genes is cell type specific due to the strict epigenetic control of their promoters. DNA methylation, histone modifications and binding of key cellular regulatory proteins contribute to the regulation of alternative promoters for transcripts encoding the nuclear antigens EBNA1 to 6 and affect the activity of promoters for transcripts encoding transmembrane proteins (LMP1, LMP2A, LMP2B). In addition to genes transcribed by RNA polymerase II, there are also two RNA polymerase III transcribed genes in the EBV genome (EBER 1 and 2). The 5′ and internal regulatory sequences of EBER 1 and 2 transcription units are invariably unmethylated. The highly abundant EBER 1 and 2 RNAs are not translated to protein. Based on the cell type specific epigenetic marks associated with latent EBV genomes one can distinguish between viral epigenotypes that differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Whereas latent EBV genomes are regularly targeted by epigenetic control mechanisms in different cell types, EBV encoded proteins may, in turn, affect the activity of a set of cellular promoters by interacting with the very same epigenetic regulatory machinery. There are EBNA1 binding sites in the human genome. Because high affinity binding of EBNA1 to its recognition sites is known to specify sites of DNA demethylation, we suggest that binding of EBNA1 to its cellular target sites may elicit local demethylation and contribute thereby to the activation of silent cellular promoters. EBNA2 interacts with histone acetyltransferases, and EBNALP (EBNA5) coactivates transcription by displacing histone deacetylase 4 from EBNA2-bound promoter sites. EBNA3C (EBNA6) seems to be associated both with histone acetylases and deacetylases, although in separate complexes. LMP1, a transmembrane protein involved in malignant transformation, can affect both alternative systems of epigenetic memory, DNA methylation and the Polycomb-trithorax group of protein complexes. In epithelial cells LMP1 can up-regulate DNA methyltransferases and, in Hodgkin lymphoma cells, induce the Polycomb group protein Bmi-1. In addition, LMP1 can also modulate cellular gene expression programs by affecting, via the NF-κB pathway, levels of cellular microRNAs miR-146a and miR-155. These interactions may result in epigenetic dysregulation and subsequent cellular dysfunctions that may manifest in or contribute to the development of pathological changes (e.g. initiation and progression of malignant neoplasms, autoimmune phenomena, immunodeficiency). Thus, Epstein-Barr virus, similarly to other viruses and certain bacteria, may induce pathological changes by epigenetic reprogramming of host cells. Elucidation of the epigenetic consequences of EBV-host interactions (within the framework of the emerging new field of patho-epigenetics) may have important implications for therapy and disease prevention, because epigenetic processes are reversible and continuous silencing of EBV genes contributing to patho-epigenetic changes may prevent disease development.

High-resolution analysis of CpG methylation and in vivo protein-DNA interactions at the alternative Epstein-Barr virus latency promoters Qp and Cp in the nasopharyngeal carcinoma cell line C666-1.

Transcripts for the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNAs) are initiated at alternative promoters (Wp, Cp, for EBNA 1–6 transcripts and Qp, for EBNA 1 transcripts only) located in the BamHI W, C or Q fragment of the viral genome. To understand the host-cell dependent expression of EBNAs in EBV-associated tumors (lymphomas and carcinomas) and in vitro transformed cell lines, it is necessary to analyse the regulatory mechanisms governing the activity of the alternative promoters of EBNA transcripts. Such studies focused mainly on lymphoid cell lines carrying latent EBV genomes, due to the lack of EBV-associated carcinoma cell lines maintaining latent EBV genomes during cultivation in tissue culture. We took advantage of the unique nasopharyngeal carcinoma cell line, C666-1, harboring EBV genomes, and undertook a detailed analysis of CpG methylation patterns and in vivo protein-DNA interactions at the latency promoters Qp and Cp. We found that the active, unmethylated Qp was marked with strong footprints of cellular transcription factors and the viral protein EBNA 1. In contrast, we could not detect binding of relevant transcription factors to the methylated, silent Cp. We concluded that the epigenetic marks at Qp and Cp in C666-1 cells of epithelial origin resemble those of group I Burkitt’s lymphoma cell lines.

CpG-methylation silences the activity of the RNA polymerase III transcribed EBER-1 promoter of Epstein-Barr virus

CpG‐methylation blocks the activity of RNA polymerase II transcribed promoters in most cases. In contrast, the role of DNA methylation in the regulation of RNA polymerase III transcribed promoters is less clarified. There are two untranslated viral RNAs (EBER‐1 and EBER‐2) in most malignant cells carrying latent Epstein‐Barr virus (EBV) genomes. We found that in vitro methylation blocked binding of the cellular proteins c‐Myc and ATF to the 5′‐region of the EBER‐1 gene, and silenced the expression of the EBER‐1 and EBER‐2 genes, transcribed by RNA polymerase III, in transfected cells.

Lineage-specific silencing of human IL-10 gene expression by promoter methylation in cervical cancer cells

Epigenetic analysis was performed to demonstrate that the normal and neoplastic epithelial cells do not serve as the source of the locally elevated IL-10 production during cervical carcinogenesis. Bisulfite sequencing was used to correlate promoter CpG methylation with the transcription of the gene. Lack of IL-10 transcription in HeLa, SiHa, Caski, HT-3, C33-A, HaCaT cell lines and in primary human keratinocytes correlated consistently with the methylated state of the proximal CpG residues, particularly with the two most proximal CpGs at positions -185 and -110. These two sites were also highly methylated in normal and malignant cervical cells directly isolated from patient material. On the other hand, IL-10 producing peripheral blood mononuclear cells had unmethylated CpG residues in the proximal promoter associated with acetylated H3 and H4 histones as determined by chromatin immunoprecipitation. In HeLa carrying epigenetically silenced endogeneous IL-10 promoter, the transfected non-CpG methylated 1 kb and 0.6 kb proximal promoter fragments could drive reporter gene expression, which was reversed by cassette methylation of these promoter fragments. In conclusion, the CpG methylation pattern of the proximal promoter is implicated as a major determinant of transcriptional silencing of human IL-10 expression in cells of cervical epithelial origin.

The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.

The role of DNA hypomethylation, histone acetylation and in vivo protein-DNA binding in Epstein-Barr virus-induced CD23 upregulation.

We analyzed epigenetic marks at the CD23 regulatory regions in well characterized Epstein-Barr virus (EBV)-carrying cell lines covering the major latency types. Bisulfite sequencing showed that DNA methylation is not a major regulator of EBV-induced CD23 transcription, although a wide hypomethylated DNA sequence in the regulatory regions is always present in the cell lines with high CD23 expression. Acetylated histone H3 levels at the CD23b promoter showed strong correlation with CD23b expression, while a weaker correlation could be observed at the CD23a core promoter. DMS in vivo footprinting at the intronic EBV-responsive enhancer and the intermediate-affinity CBF1 site at the CD23a core promoter did not reveal any significant sign of in vivo protein-DNA interactions, despite the presence of strong, characteristic footprints in the same DMS-treated DNA samples at the two CBF1 sites of the LMP2A-promoter. Our in vivo results suggest a minor role for DNA methylation, while a more important role for histone acetylation in the regulation of EBV-induced CD23 expression. Furthermore, our in vivo footprinting results support the complex model of CD23 induction by EBV, rather than a simple model with direct transactivation of CD23 by EBNA-2.

Terminal Repeat Analysis of EBV Genomes

Epstein-Barr virus (EBV) was the first human virus associated directly with human malignancies. During EBV infection of various host cells the double-stranded linear EBV DNA carried by the virions undergoes circularization. Since there are variable numbers of terminal repetitions (TRs) at the ends of the linear EBV genome, the resulting circular episomes enclose a variable number of TRs. Thus, in cells carrying viral episomes, the sizes of the terminal restriction enzyme fragments of EBV is affected by the number of TRs (Raab-Traub and Flynn Cell 47:883-889, 1986). Southern blot analysis revealed that in monoclonal proliferations, arising from a single cell, there was only a single band representing the joined EBV termini, whereas multiple terminal restriction enzyme fragments that differ in size were characteristic for oligoclonal or polyclonal proliferations. Using suitable probes, one can distinguish the episomal form from the linear EBV genomes that are formed during lytic EBV replication or during integration into the host genome. TR analysis is a useful tool for the determination of EBV clonality in different clinical samples and in cell lines carrying EBV genomes. A single terminal restriction enzyme fragment may indicate EBV infection at an early phase of clonal cell proliferation, whereas polyclonal EBV genomes may derive from multiple infections of proliferating cells.

Up-Regulation of Lamin A/C Expression in Epstein-Barr Virus Immortalized B Cells and Burkitt Lymphoma Cell Lines of Activated B Cell Phenotype

Lamin A, B and C, the nuclear intermediate-filament proteins, play a role in epigenetic regulation. Lamin B could be detected in all nucleated cells studied, whereas the lamin A and lamin C isoforms (lamin A/C) encoded by the LMNA gene are co-expressed in most somatic cell types except mature B lymphocytes. Since Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with tumorigenic processes and is known to alter the epigenotype of its host cells, we studied the expression of the LMNA gene and its epigenetic marks in EBV-carrying human lymphoid cell lines. We observed a high lamin A/C mRNA expression in EBV-immortalized B lymphoblastoid cell lines (LCLs) and in a subset of Burkitt lymphoma (BL) lines characterized by an activated B cell phenotype and a unique latent EBV gene expression pattern (latency III). In these cells the first exon of LMNA was hypomethylated and associated with activating histone marks. In contrast, we observed a low level of lamin A/C mRNA expression in EBV negative BL lines and BL lines with a restricted expression of latent EBV products (latency I). Low LMNA promoter activity was associated with hypermethylation of the LMNA first exon. These data suggest a role for EBV latency products in switching on or upregulating the LMNA promoter (LMNAp) in EBV-infected activated B cells in vitro. Lamin A/C may contribute to the establishment of the activated B cell phenotype. Our data also imply a role of LMNA first exon methylation in the silencing of LMNAp. *Corresponding author: Janos M, Department of Oral Biology and Experimental Dental Research, University of Szeged, Hungary, Tel: 36703948279; E-mail: minimicrobi@hotmail.com Received April 27, 2017; Accepted May 15, 2017; Published May 22, 2017 Citation: Banati F, Koroknai A, Szenthe K, Tereh T, Hidasi A, et al. (2017) UpRegulation of Lamin A/C Expression in Epstein-Barr Virus Immortalized B Cells and Burkitt Lymphoma Cell Lines of Activated B Cell Phenotype. J Microb Biochem Technol 9:087-094. doi: 10.4172/1948-5948.1000349 Copyright: