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Epstein-Barr viral (EBV) latency-associated promotersEpstein-Barr virus (EBV) infection is the cause of infectious mononucleosis and is most closely associated with tumor diseases Burkitt's lymphoma (BL) and nasopharyngeal carcinoma. EBV infection of human B lymphocytes in vitro results in B-cell proliferation and transformation into continuously growing lymphoblastoid cell lines (LCL) (for a review, see reference 42). In latently infected cells, viral genomes are maintained as multiple circular episomal copies which are replicated once per cell cycle (2, 103). Several classes of latency have been described depending on the gene expression pattern (41,77,78). In strict type I latency, represented by BL cells, viral gene expression is restricted to the two RNA polymerase III-transcribed EBER RNA genes and the EBNA1 gene (78) that is transcribed from the Q promoter (Qp) (68). The EBNA1 protein is required for the maintenance of the viral plasmid in dividing cells (45,58). In type III latency, in addition to the EBERs, EBNA-LP, -2, -3A, -3B, -3C, and -1 are expressed from the C promoter (Cp) (6), whereas LMP-1 and -2B are expressed from the bidirectional LMP1 promoter (46), and a larger splice variant of LMP-2, LMP-2A, is expressed from the TP1 promoter (36). Qp generally is supposed to be silent in type III latency (82, 105), although there is also a different view (93). Among the viral proteins expressed in latency type III, EBNA2 plays a central role in switching EBNA transcription from Wp to Cp (W to C switch) (102,104) and in the establishment and maintenance of B-cell transformation (11, 28), as EBNA2 transcriptionally activates the expression of the six nuclear antigens from the C promoter (Cp) and the membrane proteins LMP-1 and -2B from the LMP1 promoter (LMP1p), LMP-2A from the TP1 promoter, and a number of cellular proteins associated with the LCL phenotype (1,12,18,39,44,72,76,90,95,98,99,100,101,102,104,110,111). A crucial mechanism involved in the silencing of Cp and LMP1p in type I latency has been shown to be methylation of CpG dinucleotides (3,15,35,54,60,61,70,73,74,75,84,91,94). In LCL, the EBV genome is mostly free of CpG methylation, whereas in BL cells, EBV genomes are highly methylated. An essential step in understanding the differences between latency types I and III is to elucidate the patterns of methylation and in vivo protein binding of the latency promoters of EBV at nucleotide resolution. Therefore, we decided to examine Qp, Cp, and LMP1p in cells of both latency types.(The contributions of Daniel Salamon to this work were made in partial fulfillment of the requirements for a Ph.D. from Semmelweis University, Budapest, Hungary.) MATERIALS AND METHODSCell lines and tissue culture. LCL 721 is a B95-8-transformed LCL with type III phenotype (40,52,57). Rael (15,43,61) is a group I BL cell line. Mutu BLI-C1216 is a subclone of the BL line Mutu, representative of latency type I (27). Mutu BLIII-C199 is a subclone of the BL line Mutu, representative of latency type III (27). Raji cells expre...

This study was supported financially by the Swedish Research Council (A.L.H., 20324), Karolinska Institutet and the Stockholm County Council. None of the authors has any conflict of interest to declare.

Following infection with Epstein-Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4 + T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4 + T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4 + T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the downregulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4 + T cells can control the EBV-induced B-cell proliferation.infectious mononucleosis | Hodgkin lymphoma F ollowing primary EBV infection, the virus is carried by its human host over a lifetime. In healthy individuals, the latent virus resides in the memory B-cell compartment (1), in cells that do not resemble the proliferating lymphoblastoid cells (LCLs) generated by in vitro infection. The carrier cells usually do not express any virally encoded proteins other than EBNA1, which is required for the maintenance of the viral episomes. They do not express the growth transformation program and have a resting phenotype and, therefore, do not represent a direct pathogenic threat.The establishment of this type of latency by EBV is a focal point of attention, ever since the virus was discovered. According to one scenario, EBV exploits the B-cell differentiation program (2). The virus infects naïve IgD + B cells that subsequently express 9 virally encoded proteins (EBNA1 through -6 and three LMPs) and transforms the cells into immunoblasts. This expression pattern is referred to as the "growth transformation," or latency III program. The key proteins of this program are EBNA2 and LMP1. Because at least six of the nine proteins are immunogenic, the proliferating immunoblasts are readily recognized and eliminated by immune effector cells. A fraction of the infected cells migrate to the lymph node follicles and concurrently change their viral transcription program to express only three proteins: EBNA1, LMP1, and LMP2 (latency II). The LMPs facili...

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Insulin resistance and compensatory hyperinsulinemia are characteristic features of obesity and polycystic ovary syndrome, and both are associated with reduced fertility and implantation. There is little knowledge about the effect of insulin on the decidualization process and previous findings are contradictory. We investigated the effect of insulin on the regulation of forkhead box protein O1 (FOXO1), one of the most important transcription factors during decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Gene expression levels of six putative FOXO1 target genes (including insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL)) were measured with Real-Time PCR following FOXO1 inhibition or insulin treatment. PI3K inhibition was used to identify the possible mechanism behind regulation. Subcellular localization of FOXO1 was analyzed with immunofluorescence. All the genes (IGFBP1, CTGF, INSR, DCN, LEFTY2), except prolactin, were evaluated as FOXO1 target genes in decidualizing stromal cells. Insulin caused a significant dose-dependent inhibition of the verified FOXO1 target genes. It was also demonstrated that insulin regulated FOXO1 target genes by transcriptional inactivation and nuclear export of FOXO1 via PI3K pathway. However, insulin did not inhibit the morphological transformation of endometrial stromal cells via transcriptional inactivation of FOXO1. This study provides new insights on the action of insulin on the endometrium via regulation of FOXO1. It is suggested that hyperinsulinemia results in dysregulation of a high number of FOXO1 controlled genes that may contribute to endometrial dysfunction and reproductive failure. Our findings may illuminate possible reasons for unexplained infertility.

We analysed the methylation patterns of CpG dinucleotides in a bidirectional promoter region (LRS, LMP 1 regulatory sequences) of latent Epstein-Barr virus (EBV) genomes using automated fluorescent genomic sequencing after bisulfite-induced modification of DNA. Transcripts for two latent membrane proteins, LMP 1 (a transforming protein) and LMP 2B, are initiated in this region in opposite directions. We found that B cell lines and a clone expressing LMP 1 carried EBV genomes with unmethylated or hypomethylated LRS, while highly methylated CpG dinucleotides were present at each position or at discrete sites and within hypermethylated regions in LMP 1 negative cells. Comparison of high resolution methylation maps suggests that CpG methylation-mediated direct interference with binding of nuclear factors LBF 2, 3, 7, AML1/LBF1, LBF5 and LBF6 or methylation of CpGs within an E-box sequence (where activators as well as repressors can bind) is not the major mechanism in silencing of the LMP 1 promoter. Although a role for CpG methylation within binding sites of Sp1 and 3, ATF/CRE and a sis-inducible factor (SIF) cannot be excluded, hypermethylation of LRS or regions within LRS in LMP 1 negative cells suggests a role for an indirect mechanism, via methylcytosine binding proteins, in silencing of the LMP 1 promoter.