DNA containing an unmethylated CpG dinucleotide flanked by two purines on the 5' side and two pyrimidines on the 3' side (CpG motif"""""""") causes potent B cell activation. This is manifested by expression of early activation genes such as egr-1 and c-fos, secretion of immunoglobulin and IL-6, and entry into the cell cycle both in vitro and in vivo. This activation synergizes with signals through the B cell antigen receptor (BCR). Such unmethylated CpG motifs occur more than twenty times as often in microbial DNA as in vertebrates. Bacterial DNA activates B cells, but vertebrate DNA does not. Thus, lymphocyte activation by CpG motifs may be an important immune defense mechanism since it distinguishes between microbial and self DNA and appears to effectively promote antigen-specific immunity. This CpG motif is nearly identical to the binding site for the CREB/ATF family of transcription factors, the CRE. CREB/ATF proteins can transcriptionally regulate many genes, including egr-1, c-fos, and IL-6. CpG ODN can bind one or more CREB/ATF proteins, and specifically compete the binding of CREB/ATF to the CRE. These data raise the possibility that the effects of CpG ODN may result from their interactions with one or more CREB/ATF proteins. The broad goals of the present proposal are first, to determine how synergy occurs between the CpG DNA and the BCR signaling pathways; and second, to determine the molecular mechanism through which CpG DNA induces the transcription of egr-1, c-fos, and IL-6. The first specific aim will determine whether the CpG DNA and BCR signaling pathways interact through proximal or more distal activation steps. The second specific aim will elucidate the molecular mechanism(s) through which CpG ODN induces egr-1, c-fos, and IL-6 transcription using in vitro transcription assays, and transfection of promoter reporter gene constructs into B cells. The third specific aim will first, identify, characterize, and, if necessary, clone CREB/ATF or other B cell proteins that bind CpG ODN; and second, determine how this binding affects the properties of these proteins using gel shift and supershift assays, western and southwestern blots, immunoprecipitation, and assessment of changes in phosphorylation, associated proteins, or DNA binding activity. Completion of these studies will improve the understanding of the mechanisms regulating lymphocyte activation. These studies also have implications for possible unintended immune activation resulting from the use of """"""""antisense' ODN and for human gene therapy and DNA vaccines containing CpG motifs. These studies will determine the mechanism of immune regulation by a promising new class of immunomodulators.
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