Our goal is to understand at the molecular level the mechanism of activation of growth regulatory genes that function in human B lymphocytes. The lymphocyte receptor for IgE (FCERII or CD23) plays pivotal roles in B cell activation, Epstein-Barr virus (EBV) induced B cell immortalization, and regulation of the IgE immune response. The EBNA2 gene of EBV, interleukin 4 (IL-4), and some other mitogenic effectors induce the expression of FCERII in lymphocytes, while IL-4 and interferon-gamma (IFN-gamma) induce its expression in monocytes. This proposed research seeks to determine the molecular mechanism of viral and cytokine-induced transcriptional regulation of this gene in those cellular contexts in which its regulated expression serves the functions listed above. We will use several EBV-negative Burkitt lymphoma (BL) cell lines that show enhanced expression of FCERII following in vitro conversion with the B95-8 strain of EBV, or following treatment with IL4, to study FCERII gene expression in B cells. The monocytic leukemia cell line U937 will be used for analogous studies of FCERII regulation in monocytes. In order to identify potential regulatory sequences of the FCERII gene that are influenced by EBV, we have defined those distinct chromatin structural features of transcriptionally active FCERII alleles by analysis of the pattern of distinct DNaseI hypersensitive sites present in EBV-converted BL cell lines compared with their EBV-negative parent lines. We will construct mutants of the EBNA2 gene in order both to identify that portion of the molecule responsible for transcriptional transactivation of FCERII and to study the molecular mechanism of transactivation by the EBNA2 gene product. A reporter """"""""minigene"""""""" that includes the FCERII promoter will be modified by deletion of specific 5' flanking and intragenic regions. These constructs will be transfected into experimental cell contexts and expression of distinct FCERII transcripts from these constructs will be determined by quantitative RNase protection assays. By performing these assays in EBV-converted BL cells, IL-4 treated BL cells, IL-4 treated U937 cells, and IFN-gamma treated U937 cells we will define the sequence elements responsive to EBV and cytokines in these cells. Gel shift analysis and methylation interference assays will be used to identify protein factors binding to functional regulatory sequences defined by these assays and those identified with the presence of specific DNasel hypersensitive sites. Core sequences that are responsible for regulatory protein binding will be used as probes to clone regulatory genes from cDNA phage expression libraries.
| Schubach, W H; Horvath, G; LeVea, C et al. (1997) Chromatin structure of the lymphocyte Fc epsilon receptor gene (CD23): identification of an upstream transcriptional enhancer. J Immunol 158:2228-35 |
| Sauder, C; Gotzinger, N; Schubach, W H et al. (1996) Mutational analysis of the Epstein-Barr virus nuclear antigen 2 by far-Western blotting and DNA-binding studies. J Gen Virol 77 ( Pt 5):991-6 |
| Tsui, S; Schubach, W H (1994) Epstein-Barr virus nuclear protein 2A forms oligomers in vitro and in vivo through a region required for B-cell transformation. J Virol 68:4287-94 |
| Schubach, W H; Horvath, G; Spoth, B et al. (1991) Expression of Epstein-Barr virus nuclear antigen 2 in insect cells from a baculovirus vector. Virology 185:428-31 |
