RESUMEN
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.
Asunto(s)
Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación Viral de la Expresión Génica/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Células Cultivadas , Cromatina/genética , Inmunoprecipitación de Cromatina , Interacciones Huésped-Parásitos/genética , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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 28 kb 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.
Asunto(s)
Transformación Celular Neoplásica/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , MicroARNs/genética , Linfocitos B/virología , Western Blotting , Inmunoprecipitación de Cromatina , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/biosíntesis , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunoprecipitación , MicroARNs/biosíntesis , Oncogenes , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Nicho de Células Madre/fisiología , Triquinelosis/metabolismo , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/metabolismo , Ratones , Microscopía Confocal , Receptores CXCR4/inmunología , Receptores CXCR4/metabolismo , Imagen de Lapso de Tiempo , Trichinella spiralis , Triquinelosis/inmunologíaRESUMEN
Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.