Immediate-early Transcription Research Articles

Reversible Silencing of Cytomegalovirus Genomes by Type I Interferon Governs Virus Latency

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNβ blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNβ is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNβ. Finally, IFNβ prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNβ is consistent with the establishment of CMV latency.

Repression of interferon regulatory factor 3 by the Epstein-Barr virus immediate-early protein Rta is mediated through E2F1 in HeLa cells

Interferon regulatory factor 3 (IRF-3), an essential transcriptional regulator of the interferon (IFN) genes, is important in the host defense against viral and microbial infection. Epstein-Barr virus (EBV) immediate-early protein replication and transcription activator (Rta) and the transcription factor E2F1 are two important inhibitive factors, which repress IRF-3 expression. Numerous studies have identified that Rta can directly bind to the Rta-response element in promoters of its target genes and regulate their expression. In the present study, we demonstrated that Rta represses the expression of IRF-3 by E2F1 rather than through its traditional way. Transient transfection analysis and chromatin immunoprecipitation (ChIP) assays revealed that the overexpression of Rta elevated the expression of E2F1 and increased the binding of E2F1 to the promoter of IRF-3. The mutation of the E2F1‑binding site and the knocking down of E2F1 by small interfering RNA (siRNA) can eradicate the inhibitory effect of Rta. These results suggested that Rta represses IRF-3 expression by increasing E2F1 binding to the IRF-3 promoter.

Enucleation-induced rat adrenal gland regeneration: expression profile of selected genes involved in control of adrenocortical cell proliferation.

Enucleation-induced adrenal regeneration is a highly controlled process; however, only some elements involved in this process have been recognized. Therefore, we performed studies on regenerating rat adrenals. Microarray RNA analysis and QPCR revealed that enucleation resulted in a rapid elevation of expression of genes involved in response to wounding, defense response, and in immunological processes. Factors encoded by these genes obscure possible priming effects of various cytokines on initiation of regeneration. In regenerating adrenals we identified over 100 up- or downregulated genes involved in adrenocortical cell proliferation. The changes were most significant at days 2-3 after enucleation and their number decreased during regeneration. For example, expression analysis revealed a notable upregulation of the growth arrest gene, Gadd45, only 24 hours after surgery while expression of cyclin B1 and Cdk1 genes was notably elevated between days 1–8 of regeneration. These changes were accompanied by changes in expression levels of numerous growth factors and immediate-early transcription factors genes. Despite notable differences in mechanisms of adrenal and liver regeneration, in regenerating adrenals we identified genes, the expression of which is well recognized in regenerating liver. Thus, it seems legitimate to suggest that, in the rat, the general model of liver and adrenal regeneration demonstrate some degree of similarity.

Kaposi's Sarcoma-Associated Herpesvirus Transactivator Rta Induces Cell Cycle Arrest in G 0 /G 1 Phase by Stabilizing and Promoting Nuclear Localization of p27 kip

The Kaposi's sarcoma-associated herpesvirus (KSHV) immediate-early gene, replication, and transcription activator (K-Rta) is a key viral protein that serves as the master regulator for viral lytic replication. In this study, we investigated the role of K-Rta in cell cycle regulation and found that the expression of K-Rta in doxycycline (Dox)-inducible BJAB cells induced cell cycle arrest in G0/G1 phase. Western blot analysis of key cell cycle regulators revealed that K-Rta-mediated cell cycle arrest was associated with a decrease in cyclin A and phosphorylated Rb (pS807/pS811) protein levels, both markers of S phase progression, and an increase in protein levels for p27, a cyclin-dependent kinase inhibitor. Further, we found that K-Rta does not affect the transcription of p27 but regulates p27 at the posttranslational level by inhibiting its proteosomal degradation. Immunofluorescence staining and cell fractionation experiments revealed largely nuclear compartmentalization of p27 in K-Rta-expressing cells, demonstrating that K-Rta not only stabilizes p27 but also modulates its cellular localization. Finally, short hairpin RNA knockdown of p27 significantly abrogates cell cycle arrest in K-Rta-expressing cells, supporting its key role in K-Rta-mediated cell cycle arrest. Our findings are consistent with previous studies which showed that expression of immediate-early genes of several herpesviruses, including herpes simplex virus, Epstein-Barr virus, and cytomegalovirus, results in cell cycle arrest at the G0/G1 phase, possibly to avoid competition for resources needed for host cell replication during the S phase.

Lysophosphatidic acid regulates SPHK1 and GPR68/OGR1 in human gingival fibroblasts

Our laboratory has established that lysophosphatidic acid (LPA) regulates the biology of human oral fibroblasts, and that gingival fibroblasts (GF) express the LPA1–5 receptor subtypes. We have also shown that LPA controls immediate‐early gene transcription of inflammatory cytokines, and their receptors and regulators in GF; therefore, we hypothesized that LPA would modulate transcription of the isozymes SPHK1 and/or SPHK2, which make sphingosine‐1‐phosphate (S1P). S1P is under much investigation as a major regulator in immunity and inflammation. Critically, S1P is a primary controller of human neutrophil responses (such as chemotaxis, degranulation, and oxidative burst), making it highly relevant to the inflammation seen in periodontal disease. After GF treatment with 1 × 10−5 M 18:1 LPA for 2h or 8h, RNA was extracted. Agilent Whole Human Genome Oligonucleotide Microarray analysis revealed that LPA consistently and significantly upregulated SPHK1 versus control (3.9 ± 0.5 and 4.7± 0.7 fold at 2h and 8h, respectively). Of great interest, the proton‐sensing receptor GPR68/OGR1 was highly induced: 7.3 ± 4.0 fold (2h) and 18.8 ± 12.8 fold (8h). LPA1–3 can all dimerize with GPR68 /OGR1, which is linked to COX‐2 and inflammatory cytokine induction in other systems. QRT‐PCR was performed to validate the microarray results, using 3 biologic replicates for four periodontal disease‐relevant up‐regulated genes at the 8h time point. These results (level of significance, p < 0.01) provide preliminary evidence that LPA likely exerts complex actions during periodontal inflammation by inducing S1P production and through LPA1–3 hetero‐dimerizing with GPR68. Support: Health Future Foundation (D.R.C).

Regulation of immediate‐early gene transcription following activation of Gαq‐coupled designer receptors

G-protein coupled designer receptors that are specifically activated by designer drugs have been developed. Here, we have analyzed the regulation of gene transcription following activation of Gα(q)-coupled designer receptor (Rα(q)). Stimulation of human embryonic kidney (HEK) 293 cells expressing Rα(q) with clozapine-N-oxide (CNO), a pharmacologically inert compound, induced the expression of biologically active Egr-1, a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor (TCF) Elk-1, a key transcriptional regulator of serum response element (SRE)-driven gene transcription, prevented Egr-1 expression. Stimulation of Rα(q) with CNO increased the transcriptional activation potential of Elk-1 and enhanced transcription of an SRE regulated reporter gene. In addition, AP-1 transcriptional activity was significantly elevated. AP-1 activity was controlled by TCFs and c-Jun in cells expressing an activated Gα(q)-coupled designer receptor. CNO stimulation did not increase Egr-1 and AP-1 activity in neuroblastoma cells expressing endogenous M3 muscarinic acetylcholine receptors, indicating that CNO did not function as a ligand for these receptors. Rα(q) stimulation also increased the transcriptional activation potential of CREB and cAMP response controlled gene transcription. Pharmacological and genetic experiments revealed that the protein kinases Raf and ERK were essential to connect Rα(q) stimulation with enhanced Egr-1 and AP-1 controlled transcription. In contrast, MAP kinase phosphatase-1 functioned as a nuclear shut-off device of stimulus-transcription coupling. The fact that Rα(q) stimulation activates the transcription factors Egr-1, Elk-1, AP-1, and CREB indicates that regulation of gene transcription is an integral part of Gα(q)-coupled receptor signaling.

Roles of the Nuclear Lamina in Stable Nuclear Association and Assembly of a Herpesviral Transactivator Complex on Viral Immediate-Early Genes

ABSTRACTLittle is known about the mechanisms of gene targeting within the nucleus and its effect on gene expression, but most studies have concluded that genes located near the nuclear periphery are silenced by heterochromatin. In contrast, we found that early herpes simplex virus (HSV) genome complexes localize near the nuclear lamina and that this localization is associated with reduced heterochromatin on the viral genome and increased viral immediate-early (IE) gene transcription. In this study, we examined the mechanism of this effect and found that input virion transactivator protein, virion protein 16 (VP16), targets sites adjacent to the nuclear lamina and is required for targeting of the HSV genome to the nuclear lamina, exclusion of heterochromatin from viral replication compartments, and reduction of heterochromatin on the viral genome. Because cells infected with the VP16 mutant virus in1814 showed a phenotype similar to that of lamin A/C−/− cells infected with wild-type virus, we hypothesized that the nuclear lamina is required for VP16 activator complex formation. In lamin A/C−/− mouse embryo fibroblasts, VP16 and Oct-1 showed reduced association with the viral IE gene promoters, the levels of VP16 and HCF-1 stably associated with the nucleus were lower than in wild-type cells, and the association of VP16 with HCF-1 was also greatly reduced. These results show that the nuclear lamina is required for stable nuclear localization and formation of the VP16 activator complex and provide evidence for the nuclear lamina being the site of assembly of the VP16 activator complex.

Effect of Rta protein of Epstein-Barr virus on the cell cycle in HeLa cells

Epstein-Barr virus (EBV) replication and transcription activator (Rta) is an immediate-early transcription factor that mediates the switch from latent to lytic infection. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of Rta on cell cycle progression. Cell cycle analysis revealed that Rta induced HeLa cells in G0/G1-phase to reenter the S-phase. Analysis of the expression pattern of a key set of cell cycle regulators revealed that expression of Rta inhibited the expression of Rb and p53 and induced the expression of E2F1. These findings suggest that Rta plays an active role in redirecting HeLa cell physiology through an Rta-mediated cell cycle transformation.

Choristoneura fumiferana multiple nucleopolyhedrovirus LEF-3–P143 complex can complement DNA replication and budded virus in an AcMNPV LEF-3–P143 double knockout bacmid

Transient replication assays using Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) genes suggested that the interactions between P143, the viral helicase and LEF-3, a ssDNA-binding protein, may represent virus species specificity determinants. P143 and LEF-3 are essential for DNA replication in these assays and together with IE-1, the major immediate-early transcription factor, may be part of the viral replisome. In the current report, a lef-3/p143 double-knockout AcMNPV bacmid was constructed that was defective for viral DNA replication and late gene expression. When the homologous lef-3/p143 CfMNPV genes were introduced into this double-knockout bacmid, DNA replication was restored but the level of replication was lower, budded virus production was delayed, and the yields were reduced from those in an AcMNPV-rescue bacmid. These results suggest that to maximize virus replication, baculovirus replisome assembly and function requires protein-protein interactions between P143 and LEF-3, and other viral proteins.

Phosphorylation of histone H1 by P‐TEFb is a necessary step in skeletal muscle differentiation

Positive transcription elongation factor b (P-TEFb), the complex of Cyclin T1 and CDK9, activates the transcription of many viral and eukaryotic genes at the point of mRNA elongation. The activity of P-TEFb has been implicated in the differentiation of a number of cell types, including skeletal muscle. In order to promote transcription, P-TEFb hyperphosphorylates RNA Pol II, thereby increasing its processivity. Our previous work identified histone H1 as a P-TEFb substrate during HIV-1 and immediate-early transcription. Here, we examine the role of P-TEFb phosphorylation of histone H1 during differentiation, using the myoblast cell line C2C12 as a model for skeletal muscle differentiation. We found that H1 phosphorylation is elevated in differentiating C2C12, and this phosphorylation is sensitive to P-TEFb inhibition. H1 phosphorylation was also necessary for the induction of three muscle marker genes that require P-TEFb for expression. Additionally, ChIP experiments demonstrate that H1 dissociates from muscle differentiation marker genes in C2C12 cells under active P-TEFb conditions. We determine that both P-TEFb activity and H1 phosphorylation are necessary for the full differentiation of C2C12 myoblasts into myotubes.

Latent and lytic Epstein‐Barr virus gene expression in the peripheral blood of astronauts

Epstein-Barr virus (EBV) latent and replicative gene transcription was analyzed in peripheral blood B-lymphocytes from astronauts who flew on short-duration (∼11 days) Shuttle missions and long-duration (∼180 days) International Space Station (ISS) missions. Latent, immediate-early, and early gene replicative viral transcripts were detected in samples from six astronauts who flew on short-duration Shuttle missions, whereas viral gene transcription was mostly absent in samples from 24 healthy donors. Samples from six astronauts who flew on long-duration ISS missions were characterized by expanded expression of latent, immediate-early, and early gene transcripts and new onset expression of late replicative transcription upon return to Earth. These data indicate that EBV-infected cells are no longer expressing the restricted set of viral genes that characterize latency but are expressing latent and lytic gene transcripts. These data also suggest the possibility of EBV-related complications in future long-duration missions, in particular interplanetary travel.

Varicella-Zoster Virus Transcriptome in Latently Infected Human Ganglia

We recently developed a novel multiplex reverse transcription (RT)-PCR assay that allows rapid and sensitive detection of transcripts corresponding to all 68 unique varicella-zoster virus (VZV) open reading frames (ORFs) in only five amplification reactions (M. A. Nagel, D. Gilden, T. Shade, B. Gao, and R. J. Cohrs, J. Virol. Methods 157:62-68, 2009). Herein, we applied multiplex RT-PCR analysis to mRNA extracted from 26 trigeminal ganglia latently infected with VZV and one control trigeminal ganglion negative for VZV DNA that were removed from 14 men and women, 16 to 84 years of age, within 24 h after death. Analysis identified VZV transcripts mapping to VZV ORFs 29, 62, and 63, previously detected and sequence verified; VZV ORFs 4 and 40, previously detected by in situ hybridization; and VZV ORFs 11, 41, 43, 57, and 68, not previously detected. VZV ORF 63 transcripts were the most prevalent. Comparison of the 10 VZV ORFs transcribed during latency to their herpes simplex virus type 1 homologues reveals that the latently transcribed VZV genes encode immediate-early, early, and late transcripts.

Human Cytomegalovirus Early Protein pUL21a Promotes Efficient Viral DNA Synthesis and the Late Accumulation of Immediate-Early Transcripts

We have previously reported that a newly annotated gene of human cytomegalovirus (HCMV), UL21a, encodes an early viral protein termed pUL21a. Most notably, the virions of a UL21a deletion virus had markedly reduced infectivity, indicating that UL21a is required to establish an efficient productive infection. In this study, we infected fibroblasts with equal numbers of DNA-containing viral particles and identified where in the viral life cycle pUL21a acted. The UL21a deletion virus entered cells and initiated viral gene expression efficiently; however, it synthesized viral DNA poorly and accumulated several immediate-early (IE) transcripts at reduced levels at late times of infection. The defect in viral DNA synthesis preceded that in gene expression, and inhibition of viral DNA synthesis reduced the late accumulation of IE transcripts in both wild-type and mutant virus-infected cells to equivalent levels. This suggests that reduced viral DNA synthesis is the cause of reduced IE gene expression in the absence of UL21a. The growth of UL21a deletion virus was similar to that of recombinant HCMV in which pUL21a expression was abrogated by stop codon mutations, and the defect was rescued in pUL21a-expressing fibroblasts. pUL21a expression in trans was sufficient to restore viral DNA synthesis and gene expression of mutant virus produced from normal fibroblasts, whereas mutant virus produced from complementing cells still exhibited the defect in normal fibroblasts. Thus, pUL21a does not promote the functionality of HCMV virions; rather, its de novo synthesis facilitates viral DNA synthesis, which is necessary for the late accumulation of IE transcripts and establishment of a productive infection.

Involvement of the 3′‐untranslated region of the brain‐derived neurotrophic factor gene in activity‐dependent mRNA stabilization

Although gene transcription is controlled by neuronal activity, little is known about post-transcriptional regulation in neurons. Using cultured neurons, we found that the half-life of immediate-early gene transcripts is prolonged or shortened by membrane depolarization. Focusing on the activity-dependent stabilization of brain-derived neurotrophic factor (BDNF) mRNA, we constructed a series of plasmids, in which the short 3'-untranslated region (3'-UTR) of the BDNF gene was fused to the firefly luciferase gene, and found that the 3'-UTR prevented destabilization of luciferase mRNA through Ca(2+) signals evoked via depolarization. No such prevention was observed with the simian virus 40 late poly(A) site. The pre-mRNA covering the entire short 3'-UTR, where multiple poly(A) sites including novel ones are located, was stabilized. Deletion analyses of 3'-UTR revealed a core region (about 130 bases long) and a complementary region to be responsible for the prevention, well consistent with the formation of an extended stem-loop RNA structure and the production of poly(A) mRNAs. Thus, the mRNA stability is activity-dependently controlled in neurons and distinct regions of the 3'-UTR of BDNF mRNA are involved in stabilizing mRNA in response to Ca(2+) signals, suggesting a primary role of the RNA secondary structure affecting the availability of poly(A) sites in activity-dependent mRNA stabilization.

Coactivation of the CLOCK–BMAL1 complex by CBP mediates resetting of the circadian clock

The transcription factor CLOCK-BMAL1 is a core component of the molecular clock machinery that drives circadian gene expression and physiology in mammals. Recently, we reported that this heterodimeric transcription factor functions as a signaling molecule in response to the resetting stimuli via the Ca²+-dependent protein kinase C pathway. Here, we demonstrate that the CREB-binding protein (CBP) plays a key role in rapid activation of the CLOCK-BMAL1 heterodimer that leads to phase resetting of the circadian clock. Under physiological conditions, a bimolecular fluorescence complementation (BiFC) assay revealed that CLOCK and BMAL1 dimerize in the cytoplasm and subsequently translocate into the nucleus in response to serum stimuli (mean time duration was 29.2 minutes and mean velocity 0.7 μm/minute). Concomitantly, BMAL1 rapidly recruited CBP on Per1 promoter E-box, but not p300 (a functional analog of CBP), in the discrete nuclear foci. However, recruitment of CBP by cAMP/Ca²+ response element-binding (CREB) protein on CRE was not markedly increased upon delivery of the resetting stimuli. Furthermore, overexpression of CBP greatly potentiated the CLOCK-BMAL1-mediated Per1 transcription, and this effect was completely abolished by site-directed mutation of E-box elements, but not by the mutation of CRE in the Per1 promoter. Furthermore, molecular knockdown of CBP severely dampened circadian oscillation of clock gene expression triggered by the resetting stimuli. These findings suggest that CBP recruitment by BMAL1 mediates acute transactivation of CLOCK-BMAL1, thereby inducing immediate-early Per1 transcription and phase resetting of the circadian clock.

MicroRNAs encoded by Kaposi's sarcoma‐associated herpesvirus regulate viral life cycle

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with Kaposi's sarcoma and lymphomas. The pathogenesis of KSHV depends on the balance between two phases of the viral cycle: latency and lytic replication. In this study, we report that KSHV-encoded microRNAs (miRNAs) function as regulators by maintaining viral latency and inhibiting viral lytic replication. MiRNAs are short, noncoding, small RNAs that post-transcriptionally regulate the expression of messenger RNAs. Of the 12 viral miRNAs expressed in latent KSHV-infected cells, we observed that expression of miR-K3 can suppress both viral lytic replication and gene expression. Further experiments indicate that miR-K3 can regulate viral latency by targeting nuclear factor I/B. Nuclear factor I/B can activate the promoter of the viral immediate-early transactivator replication and transcription activator (RTA), and depletion of nuclear factor I/B by short hairpin RNAs had similar effects on the viral life cycle to those of miR-K3. Our results suggest a role for KSHV miRNAs in regulating the viral life cycle.

Human Cytomegalovirus pUL83 Stimulates Activity of the Viral Immediate-Early Promoter through Its Interaction with the Cellular IFI16 Protein

The human cytomegalovirus (HCMV) virion protein pUL83 (also termed pp65) inhibits the expression of interferon-inducible cellular genes. In this work we demonstrate that pUL83 is also important for efficient induction of transcription from the viral major immediate-early promoter. Infection with a mutant virus containing a premature translation termination codon in the UL83 open reading frame (ORF) (UL83Stop) resulted in decreased transcription from the major immediate-early promoter in a time- and multiplicity-dependent manner. Expression of pUL83 alone is capable of transactivating the promoter in a reporter assay, and pUL83 associates with the promoter in infected cells. To investigate the mechanism by which the protein regulates the major immediate-early promoter, we utilized a mutant virus expressing an epitope-tagged pUL83 from its own promoter to identify protein binding partners for pUL83 during infection. We identified and confirmed the interaction of pUL83 with cellular IFI16 family members throughout the course of HCMV infection. pUL83 recruits IFI16 to the major immediate-early promoter, and IFI16 binding at the promoter is dependent upon the presence of pUL83. Consistent with the results obtained with the UL83Stop virus, infection of IFI16 knockdown cells with wild-type virus resulted in decreased levels of immediate-early transcripts compared to those of control cells. These data identify a previously unknown role for pUL83 in the initiation of the human cytomegalovirus gene expression cascade.

Promoter specificity and interactions between early and late Arabidopsis heat shock factors

The class A heat shock factors HsfA1a and HsfA1b are highly conserved, interacting regulators, responsible for the immediate-early transcription of a subset of heat shock genes in Arabidopsis. In order to determine functional cooperation between them, we used a reporter assay based on transient over-expression in Arabidopsis protoplasts. Reporter plasmids containing promoters of Hsf target genes fused with the GFP coding region were co-transformed with Hsf effector plasmids. The GFP reporter gene activity was quantified using flow cytometry. Three of the tested target gene promoters (Hsp25.3, Hsp18.1-CI, Hsp26.5) resulted in a strong reporter gene activity, with HsfA1a or HsfA1b alone, and significantly enhanced GFP fluorescence when both effectors were co-transformed. A second set of heat shock promoters (HsfA2, Hsp17.6CII, Hsp17.6C-CI) was activated to much lower levels. These data suggest that HsfA1a/1b cooperate synergistically at a number of target gene promoters. These targets are also regulated via the late HsfA2, which is the most strongly heat-induced class A-Hsf in Arabidopsis. HsfA2 has also the capacity to interact with HsfA1a and HsfA1b as determined by bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts and yeast-two-hybrid assay. However, there was no synergistic effect on Hsp18.1-CI promoter-GFP reporter gene expression when HsfA2 was co-expressed with either HsfA1a or HsfA1b. These data provide evidence that interaction between early and late HSF is possible, but only interaction between the early Hsfs results in a synergistic enhancement of expression of certain target genes. The interaction of HsfA1a/A1b with the major-late HsfA2 may possibly support recruitment of HsfA2 and replacement of HsfA1a/A1b at the same target gene promoters.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-010-9643-2) contains supplementary material, which is available to authorized users.

The De Novo Methyltransferases DNMT3a and DNMT3b Target the Murine Gammaherpesvirus Immediate-Early Gene 50 Promoter during Establishment of Latency

The role of epigenetic modifications in the regulation of gammaherpesvirus latency has been a subject of active study for more than 20 years. DNA methylation, associated with transcriptional silencing in mammalian genomes, has been shown to be an important mechanism in the transcriptional control of several key gammaherpesvirus genes. In particular, DNA methylation of the functionally conserved immediate-early replication and transcription activator (RTA) has been shown to regulate Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus Rta expression. Here we demonstrate that the murine gammaherpesvirus (MHV68) homolog, encoded by gene 50, is also subject to direct repression by DNA methylation, both in vitro and in vivo. We observed that the treatment of latently MHV68-infected B-cell lines with a methyltransferase inhibitor induced virus reactivation. In addition, we show that the methylation of the recently characterized distal gene 50 promoter represses activity in a murine macrophage cell line. To evaluate the role of de novo methyltransferases (DNMTs) in the establishment of these methylation marks, we infected mice in which conditional DNMT3a and DNMT3b alleles were selectively deleted in B lymphocytes. DNMT3a/DNMT3b-deficient B cells were phenotypically normal, displaying no obvious compromise in cell surface marker expression or antibody production either in naïve mice or in the context of nonviral and viral immunogens. However, mice lacking functional DNMT3a and DNMT3b in B cells exhibited hallmarks of deregulated MHV68 lytic replication, including increased splenomegaly and the presence of infectious virus in the spleen at day 18 following infection. In addition, total gene 50 transcript levels were elevated in the spleens of these mice at day 18, which correlated with the hypomethylation of the distal gene 50 promoter. However, by day 42 postinfection, aberrant virus replication was resolved, and we observed wild-type frequencies of viral genome-positive splenocytes in mice lacking functional DNMT3a and DNMT3b in B lymphocytes. The latter correlated with increased CpG methylation in the distal gene 50 promoter, which was restored to levels similar to those of littermate controls harboring functional DNMT3a and DNMT3b alleles in B lymphocytes, suggesting the existence of an alternative mechanism for the de novo methylation of the MHV68 genome. Importantly, this DNMT3a/DNMT3b-independent methylation appeared to be targeted specifically to the gene 50 promoter, as we observed that the promoters for MHV68 gene 72 (v-cyclin) and M11 (v-bcl2) remained hypomethylated at day 42 postinfection. Taken together, these data provide the first evidence of the importance of DNA methylation in regulating gammaherpesvirus RTA/gene 50 transcription during virus infection in vivo and provide insight into the hierarchy of host machinery required to establish this modification.

Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex

Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and sequential chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 the ATPase subunit of the SWI/SNF remodeler, is recruited to the promoter of target genes by transcription factors such as Elk-1 or NF-κB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of IE genes enabling the binding of transcription factors like JUN and the onset of transcription.