SUMMARY Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication nor did the DDR marks co-localize with latent episomes. Rather, a transient period of EBV-induced hyper-proliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoproteins EBNA3C was required to attenuate the EBV-induced DNA damage response We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR which is attenuated by viral latency products to induce cell immortalization.

b Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-B activity. It was previously thought that the major viral protein responsible for NF-B activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-B activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-B activation during the first week after infection increased the transformation efficiency, while early NF-B inhibition had no effect on transformation. Rather, inhibition of NF-B was not toxic to EBV-infected cells until LMP1 levels and NF-B activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.

Mediator is a modular coactivator complex involved in the transcription of the majority of RNA polymerase II-regulated genes. However, the degrees to which individual core subunits of Mediator contribute to its activity have been unclear. Here, we investigate the contribution of two essential architectural subunits of Mediator to transcription in Saccharomyces cerevisiae. We show that acute depletion of the main complex scaffold Med14 or the head module nucleator Med17 is lethal and results in global transcriptional downregulation, though Med17 removal has a markedly greater negative effect. Consistent with this, Med17 depletion impairs pre-initiation complex (PIC) assembly to a greater extent than Med14 removal. Co-depletion of Med14 and Med17 reduced transcription and TFIIB promoter occupancy similarly to Med17 ablation alone, indicating that the contributions of Med14 and Med17 to Mediator function are not additive. We propose that, while the structural integrity of complete Mediator and the head module are both important for PIC assembly and transcription, the head module plays a greater role in this process and is thus the key functional module of Mediator in this regard.

Eukaryotic RNA polymerase II (RNAPII) transcribes mRNA genes and non-protein-coding RNA (ncRNA) genes, including those encoding small nuclear and nucleolar RNAs (sn/snoRNAs). In metazoans, RNAPII transcription of sn/snoRNAs is facilitated by a number of specialized complexes, but no such complexes have been discovered in yeast. It has been proposed that yeast sn/snoRNA and mRNA expression relies on a set of common factors, but the extent to which regulators of mRNA genes function at yeast sn/snoRNA genes is unclear. Here, we investigated a potential role for the Mediator complex, essential for mRNA gene transcription, in sn/ snoRNA gene transcription. We found that Mediator maps to sn/snoRNA gene regulatory regions and that rapid depletion of the essential structural subunit Med14 strongly reduces RNAPII and TFIIB occupancy as well as nascent transcription of sn/ snoRNA genes. Deletion of Med3 and Med15, subunits of the activator-interacting Mediator tail module, does not affect Mediator recruitment to or RNAPII and TFIIB occupancy of sn/snoRNA genes. Our analyses suggest that Mediator promotes PIC formation and transcription at sn/snoRNA genes, expanding the role of this critical regulator beyond its known functions in mRNA gene transcription and demonstrating further mechanistic similarity between the transcription of mRNA and sn/snoRNA genes. E ukaryotic RNA polymerase II (RNAPII), responsible for the transcription of mRNA genes, also transcribes several classes of non-protein-coding RNA (ncRNA) genes. Two prominent classes of RNAPII-transcribed eukaryotic ncRNA genes are small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). snRNAs are involved in pre-mRNA splicing (1), while snoRNAs primarily function in rRNA maturation via guiding methylation and pseudouridylation of specific rRNA bases (2).Most sn/snoRNA transcription in eukaryotes is carried out by RNAPII, with a few notable RNAPIII-transcribed exceptions, including the U6 snRNA gene (3). While transcription of both mRNA and the majority of sn/snoRNA genes is carried out by RNAPII, notable differences between their regulation have been described, particularly in metazoans. In addition to the documented dependence of a few sn/snoRNAs on several general transcription factors (GTFs) that are components of the preinitiation complex and generally essential for RNAPII transcription (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) (4-6), metazoan sn/snoRNA gene transcription relies on a number of specialized factors. One such factor is the small nuclear RNA activating complex (SNAPc), which contains the TATA-binding protein (TBP) as well as five specific subunits and is essential for