Highlights d A genetic, functional, and structural analysis of mammalian Mediator is provided d Contacts between a conserved core and the tail impact mMED-Pol II interaction d Loss of non-essential mMED subunits affects promoters linked to multiple enhancers d Cohesin is required to tether regulatory DNA; mMED and Pol II are not

Key Points• Normally, engrafting HSCs reside and oscillate within confined bone marrow niches.• HSCs harvested from mice bearing acute infection are migratory and interact with larger niches.Hematopoietic stem cells (HSCs) maintain the turnover of mature blood cells during steady state and in response to systemic perturbations such as infections. Their function critically depends on complex signal exchanges with the bone marrow (BM) microenvironment in which they reside, but the cellular mechanisms involved in HSC-niche interactions and regulating HSC function in vivo remain elusive. We used a natural mouse parasite, Trichinella spiralis, and multipoint intravital time-lapse confocal microscopy of mouse calvarium BM to test whether HSC-niche interactions may change when hematopoiesis is perturbed. We find that steady-state HSCs stably engage confined niches in the BM whereas HSCs harvested during acute infection are motile and therefore interact with larger niches. These changes are accompanied by increased long-term repopulation ability and expression of CD44 and CXCR4. Administration of a CXCR4 antagonist affects the duration of HSC-niche interactions. These findings suggest that HSC-niche interactions may be modulated during infection. IntroductionThe function of hematopoietic stem cells (HSCs) depends on their interaction with complex niches within the bone marrow (BM) 1,2 and must readily adjust to meet the changing needs for differentiated cells during situations of stress, such as the immune responses generated by infectious agents.3-5 The current working hypothesis is that changes in HSC-niche interactions may cause changes in HSC function and therefore a major challenge in the field is to understand the specific cellular behaviors (eg, duration of cell-cell/cell-matrix interactions) resulting in different HSC fates (quiescence vs proliferation, self-renewal vs differentiation). Direct visualization of HSCs in vivo and over time is the ideal approach to monitor the nature of the cellular interactions between individual stem cells and their niches and can be achieved by intravital microscopy of mouse calvarium BM. 6,7 We studied a naturally occurring, nonlethal mouse model of infection initiated by ingestion of the parasitic nematode Trichinella spiralis, the infectious agent causing trichinosis. Ingested T spiralis reside for several days in the intestinal epithelium, where they mature, proliferate, and release larvae, which migrate into the circulatory system and invade the skeletal muscle, where they form cysts and survive for the life of the host.8 Migration of parasites causes extensive tissue damage and intense inflammation over the early phase of infection (weeks 1-3), which is ameliorated by the induction of regulatory cytokines during the chronic phase of the disease. 9,10Here we show that acute T spiralis infection affects hematopoietic dynamics and boosts HSC function. Using time-lapse intravital microscopy of transplanted HSCs, we show that acute infection is accompanied by changes in HSC-nic...

It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression–particularly in relation to histone modifications and cell factors involved–the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells.

Allday and collaborators demonstrate that the EBV transcription factor and oncoprotein EBNA3C directly induces the expression of AID and somatic mutations in B cells, providing a mechanism linking infection and lymphoma induction.

We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters – widely reported to have cell transformation-associated activity – are regulated by EBNA3A and EBNA3C. Utilising a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours – including lymphoma/leukemia. ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28kb upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in increased expression of the cyclin-dependent kinase inhibitor p57KIP2, a well-established target of miR-221/miR-222. MiR blocking experiments confirmed that miR-221/miR-222 target p57KIP2 expression in LCLs. In contrast, EBNA3A and EBNA3C are necessary to silence the tumour suppressor cluster miR-143/miR-145, but here ChIP-seq suggests that repression is probably indirect. This miR cluster is frequently down-regulated or deleted in human cancer, however, the targets in B cells are unknown. Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.

The oncogenic Epstein Barr virus (EBV) infects the majority of the human population and usually persists within its host for life without symptoms. The EBV oncoproteins nuclear antigen 3A (EBNA3A) and 3C (EBNA3C) are required for B cell transformation in vitro and are expressed in EBV associated immunoblastic lymphomas in vivo. In order to address the necessity of EBNA3A and EBNA3C for persistent EBV infection in vivo, we infected NOD-scid γcnull mice with reconstituted human immune system components (huNSG mice) with recombinant EBV mutants devoid of EBNA3A or EBNA3C expression. These EBV mutants established latent infection in secondary lymphoid organs of infected huNSG mice for at least 3 months, but did not cause tumor formation. Low level viral persistence in the absence of EBNA3A or EBNA3C seemed to be supported primarily by proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression without elevated lytic replication. In vitro, EBNA3A and EBNA3C deficient EBV infected B cells could be rescued from apoptosis through CD40 stimulation, mimicking T cell help in secondary lymphoid tissues. Thus, even in the absence of the oncogenes EBNA3A and 3C, EBV can access a latent gene expression pattern that is reminiscent of EBV persistence in healthy virus carriers without prior expression of its whole growth transforming program.

Infectious mononucleosis, caused by infection with the human gamma-herpesvirus Epstein-Barr virus (EBV), manifests with one of the strongest CD8+ T-cell responses described in humans. The resulting T-cell memory response controls EBV infection asymptomatically in the vast majority of persistently infected individuals. Whether and how dendritic cells (DCs) contribute to the priming of this near-perfect immune control remains unclear. Here we show that of all the human DC subsets, plasmacytoid DCs (pDCs) play a central role in the detection of EBV infection in vitro and in mice with reconstituted human immune system components. pDCs respond to EBV by producing the interferon (IFN) subtypes α1, α2, α5, α7, α14, and α17. However, the virus curtails this type I IFN production with its latent EBV gene products EBNA3A and EBNA3C. The induced type I IFNs inhibit EBV entry and the proliferation of latently EBV-transformed B cells but do not influence lytic reactivation of the virus in vitro. In vivo, exogenous IFN-α14 and IFN-α17, as well as pDC expansion, delay EBV infection and the resulting CD8+ T-cell expansion, but pDC depletion does not significantly influence EBV infection. Thus, consistent with the observation that primary immunodeficiencies compromising type I IFN responses affect only alpha- and beta-herpesvirus infections, we found that EBV elicits pDC responses that transiently suppress viral replication and attenuate CD8+ T-cell expansion but are not required to control primary infection.

The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator has provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II but failed to reveal the structure of the Tail module that contains most subunits targeted by activators and repressors. Here we present a molecular model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals that the previously unresolved Tail module, which includes a number of metazoan specific subunits, interacts extensively with core Mediator and has the potential to influence its conformation and interactions.