Transcriptional regulation of the Epstein-Barr virus latent membrane protein 1 gene in B cells
Abstract: Epstein-Barr Virus (EBV) is a ubiquitous human B-lymphotrobic herpesvirus that latently infects over 90 percent of the world´s population. EBV infection is usually benign in healthy people. However, EBV is the causative agent of infectious mononucleosis, and is strongly associated with an increasing number of human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and immunoblastic lymphomas in immunocomprised people. EBV has the ability to immortalize B cells in vitro and stimulate B-cell proliferation. Mutagenesis of the viral genome has defined a subset of five genes required for transformation (EBNA1, -2, -3, -6 and LMP1). The LMP1 gene is of particular interest since it is an oncogene that is involved in the transformation as well as proliferation of B cells latently infected by EBV. The aim of this thesis is to investigate the molecular mechanisms of transcriptional regulation of the LMP1 gene in B cells. The LMP1 promoter is controlled by both positive and negative transcriptional cis-elements and the gene is inactive without inducers. EBNA2 can overcome the repression of the LMP1 promoter. By deletion analyses, two distinct LMP1 regulatory sequence (LRS) regions, -106 to +40 and -176 to -136, relative to the transcriptional initiation site, were shown to contribute to the EBNA2-responsiveness in LRS. In the distal EBNA2 responsive region, both the octamer motif (bound by a POU domain protein) and the PU box-binding site (bound by a PU.1 factor) were critical for the EBNA2 induction process. Cooperation between the factors binding to these two sites is required for full EBNA2 transactivation, and EBNA2 may be targeted to the LMP1 promoter via interacting with the POU domain protein. In the promoter-proximal region (LRS-106/+40), mutational analyses showed that both an Sp site and an ATF/CRE site are important in the EBNA2-dependent activation of the LMP1 promoter. Overexpression of Sp1 and ATF1/CREB1 mediates activation of the LMP1 promoter independently of EBNA2 via their respective binding sites, whereas EBNA2-induced activation occurs through a direct contact between EBNA2 and ATF-2/c-Jun heterodimer via the ATF/CRE site. We have also shown that a silencing element overlaps with a transcriptional enhancer element in an LRS sequence that contains an E-box-homologous motif. Transient co-transfection analyses showed that USF proteins confer EBNA2-independent activity on the LMP1 promoter via the E-box site and that this activation was downregulated by the Max-Mad1-mSin3A factors. In addition to the E-box site, we have also identified an adjacent Ikaros site that binds Ikaros factors, which also functions as a repressive element. The repression exerted by the factors binding to the E-box site and the Ikaros site was released by an inhibitor of histone deacetylation, Trichostatin A (TSA), indicating that histone deacetylation plays an important role in repression mediated by these factors. EBNA2 relieved the repression through an indispensable response element in the -107/-95 LRS region, which contains a functional AP-2 site. AP-2 factors cooperated with EBNA2 to overcome the repression exerted by the E-box and the Ikaros site binding factors, which led to the activation of the LMP1 promoter in reporter plasmids.
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