The exposure of mouse embryos in utero and primary cortical neurons to ionizing radiation results in the P53-dependent activation of a subset of genes that is highly induced during brain development and neuronal maturation, a feature that these genes reportedly share with circular RNAs (circRNAs). Interestingly, some of these genes are predicted to express circular transcripts. In this study, we validated the abundance of the circular transcript variants of four P53 target genes (Pvt1, Ano3, Sec14l5, and Rnf169). These circular variants were overall more stable than their linear counterparts. They were furthermore highly enriched in the brain and their transcript levels continuously increase during subsequent developmental stages (from embryonic day 12 until adulthood), while no further increase could be observed for linear mRNAs beyond post-natal day 30. Finally, whereas radiation-induced expression of P53 target mRNAs peaks early after exposure, several of the circRNAs showed prolonged induction in irradiated embryonic mouse brain, primary mouse cortical neurons, and mouse blood. Together, our results indicate that the circRNAs from these P53 target genes are induced in response to radiation and they corroborate the findings that circRNAs may represent biomarkers of brain age. We also propose that they may be superior to mRNA as long-term biomarkers for radiation exposure.

Gallid herpesvirus type 2 (GaHV-2) is an oncogenic alphaherpesvirus that induces malignant T-cell lymphoma in chicken. GaHV-2 encodes a viral telomerase RNA subunit (vTR) that plays a crucial role in virus-induced tumorigenesis, enhances telomerase activity and possesses function independent of the telomerase complex. vTR is driven by a robust viral promoter, highly expressed in virus-infected cells and regulated by two c-Myc response elements (c-Myc REs). The regulatory mechanisms involved in controlling vTR and other genes during viral replication and latency remain poorly understood but are crucial to understand this oncogenic herpesvirus. Therefore, we investigated DNA methylation patterns of CpG dinucleotides found in the vTR promoter and measured the impact of methylation on the telomerase activity. We demonstrated that telomerase activity was considerably increased following viral reactivation. Furthermore, CpG sites within c-Myc REs showed specific changes in methylation after in vitro reactivation and in infected animals over time. Promoter reporter assays indicated that one of the c-Myc RE is involved in regulating vTR transcription, and that methylation strongly influenced vTR promoter activity. To study the importance of the CpG sites found in c-Myc REs in virus-induced tumorigenesis, we generated a recombinant virus containing mutations in both CpG sites of c-Myc REs as well as revertant, by two-step Red-mediated mutagenesis. Introduced mutation in vTR promoter did not affect the replication properties of the recombinant viruses in vitro. In contrast, replication of the mutant virus in infected chickens was severely impaired, and tumour formation completely abrogated. Our data provided a more in-depth characterisation of c-Myc oncoprotein REs and DNA methylation involvement in transcriptional regulation of vTR. IMPORTANCE Previous studies demonstrated that telomerase RNAs possess functions that promote tumour development independent of the telomerase complex. vTR is a herpesvirus-encoded telomerase RNA subunit that plays a crucial role in virus-induced tumorigenesis and is expressed by a robust viral promoter that is highly regulated by the c-Myc oncoprotein binding to the E-boxes. Here we demonstrated that the DNA methylation patterns in the functional c-Myc response elements of the vTR promoter change upon reactivation from latency and that demethylation strongly increases telomerase activity in virus-infected cells. Moreover, the introduction of mutation in the CpG dinucleotides of the c-Myc binding sites resulted in decreased vTR expression and complete abrogation of tumour formation. Our study provides further confirmation of the involvement of specific DNA methylation patterns in the regulation of vTR expression and vTR importance for virus-induced tumorigenesis.