Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

Abstract

Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G 1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.KEYWORDS Epstein-Barr virus, immediate-early protein Rta, lytic cycle reactivation, CTCF, DNA methylation, cell cycle arrest, genome topology, immediate-early protein Rta

Transcriptome analysis of Burkitt lymphoma cells treated with anti-convulsant drugs that are inhibitors of Epstein–Barr virus lytic reactivation

“…Entry of EBV into cells does not use xCT. A small upregulation of SLC7A11 occurred in EBV-negative 293 cells exogenously expressing the EBV lytic transactivator protein RTA [ 39 ]. Changes in SLC7A11 gene expression occur during primary EBV infection when B cells are reprogrammed and thousands of genes are regulated [ 40 , 41 ].…”

Sodium-coupled monocarboxylate transporter is a target of epigenetic repression in cervical cancer

“…Consequently, it is speculated that the absence of SLC5A8 expression is largely influenced by dense methylation in this block of 20 CpG dinucleotides. Along these CpGs, a computational prediction using the Genomatix software (https://genomatix.de/) identified the consensus binding sites for the methylation-sensitive transcription factor E2F and transcriptional repressor CTCF (34,35); however, further experiments are required to determine the role of these transcription factors in the regulation of SLC5A8 expression.…”

Three-Dimensional Chromatin Structure of the EBV Genome: A Crucial Factor in Viral Infection

“…Furthermore, several studies have revealed that CTCF can regulate viral gene expression in other double-stranded DNA viruses. For instance, in Kaposi’s sarcoma herpesvirus (KSHV) [ 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ], herpes simplex virus (HSV) [ 83 , 84 , 85 , 86 , 87 , 88 , 89 ] and human cytomegalovirus (CMV) [ 90 ], CTCF has been shown to bind to the viral genome and regulate viral gene expression by modulating the accessibility of the viral genome to transcription factors. Finally, it has also been suggested that viruses can modulate CTCF activity to promote viral replication.…”