Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers.

CTCF is an essential epigenetic regulator mediating chromatin insulation, long-range regulatory interactions, and the organization of large topological domains in the nucleus. Phenotypes of CTCF haploinsufficient mutations in humans, knockout in mice, and depletion in cells are often consistent with impaired genome stability, but a role of CTCF in genome maintenance has not been fully investigated. Here, we report that CTCF maintains genome stability, is recruited to sites of DNA damage, and promotes homologous recombination repair of DNA double-strand breaks (DSBs). CTCF depletion increased chromosomal instability, marked by chromosome breakage and end fusions, elevated genotoxic stress-induced genomic DNA fragmentation, and activated the ataxia telangiectasia mutated (ATM) kinase. We show that CTCF could be recruited to drug-induced 53BP1 foci and known fragile sites, as well as to I-SceI endonuclease-induced DSBs. Laser irradiation analysis revealed that this recruitment depends on ATM, Nijmegen breakage syndrome (NBS), and the zinc finger DNA-binding domain of CTCF. We demonstrate that CTCF knockdown impaired homologous recombination (HR) repair of DSBs. Consistent with this, CTCF knockdown reduced the formation of γ-radiation-induced Rad51 foci, as well as the recruitment of Rad51 to laser-irradiated sites of DNA lesions and to I-SceI-induced DSBs. We further show that CTCF is associated with DNA HR repair factors MDC1 and AGO2, and directly interacts with Rad51 via its C terminus. These analyses establish a direct, functional role of CTCF in DNA repair and provide a potential link between genome organization and genome stability.CTCF | chromatin | genome stability | DNA repair | homologous recombination

Kaposi’s sarcoma associated herpesvirus (KSHV) infection stabilizes hypoxia inducible factors (HIFs). The interaction between KSHV encoded factors and HIFs plays a critical role in KSHV latency, reactivation and associated disease phenotypes. Besides modulation of large-scale signaling, KSHV infection also reprograms the metabolic activity of infected cells. However, the mechanism and cellular pathways modulated during these changes are poorly understood. We performed comparative RNA sequencing analysis on cells with stabilized hypoxia inducible factor 1 alpha (HIF1α) of KSHV negative or positive background to identify changes in global and metabolic gene expression. Our results show that hypoxia induces glucose dependency of KSHV positive cells with high glucose uptake and high lactate release. We identified the KSHV-encoded vGPCR, as a novel target of HIF1α and one of the main viral antigens of this metabolic reprogramming. Bioinformatics analysis of vGPCR promoter identified 9 distinct hypoxia responsive elements which were activated by HIF1α in-vitro. Expression of vGPCR alone was sufficient for induction of changes in the metabolic phenotype similar to those induced by KSHV under hypoxic conditions. Silencing of HIF1α rescued the hypoxia associated phenotype of KSHV positive cells. Analysis of the host transcriptome identified several common targets of hypoxia as well as KSHV encoded factors and other synergistically activated genes belonging to cellular pathways. These include those involved in carbohydrate, lipid and amino acids metabolism. Further DNA methyltranferases, DNMT3A and DNMT3B were found to be regulated by either KSHV, hypoxia, or both synergistically at the transcript and protein levels. This study showed distinct and common, as well as synergistic effects of HIF1α and KSHV-encoded proteins on metabolic reprogramming of KSHV-infected cells in the hypoxia.