Bacterial DNA containing unmethylated CpG dinucleotides (CpG DNA) has recently been recognized as an important immune-activating agent and holds strong promise for therapeutic treatment of cancer, allergy, and infection. Although it is known now that CpG DNA functions through a signaling cascade involving Toll-like receptor 9 and associated proteins, culminating in activation of transcription factors NF-kappaB and AP-1, as well as subsequent induction of various immune-regulatory genes, studies on how host cells differentiate CpG DNA from non-CpG DNA or methylated CpG DNA have been relatively limited. My long-term goal is to contribute fundamental knowledge in molecular mechanisms of host immune responses and their role in treatment of disease. The objective of this project is to define mechanism(s) by which CpG DNA induces expression of inflammatory mediators in mouse macrophages. The central hypothesis of this research project is that the capacity of being methylated enables CpG DNA to compete with host cell genomic DNA as a substrate for DNA methyltransferase I and causes reduced methylation of genomic DNA, which leads to elevated gene expression (possibly through enhancing accessibility of upstream regulatory regions of these genes to activated transcription factors). Since our preliminary data indicate CpG DNA induces rapid expression of transcription factor C/EBPdelta, which has binding sites in the promoters of a number of proinflammatory genes, we also hypothesize that C/EBPdelta mediates inflammatory gene induction in response to CpG DNA. To test these hypotheses, the following specific aims are proposed: 1. To define the role of CpG DNA in regulation of mouse macrophage inflammatory gene expression within the framework of intracellular methylation events. 2. To determine the extent to which treatment of macrophages with CpG DNA results in reduced methylation of 5' regulatory regions of specific inflammatory genes and, as a result, causes increased expression of these genes. 3. To determine the role of transcription factor C/EBPdelta in induction of inflammatory genes in response to CpG DNA. The successful completion of this research will contribute to the development of CpG DNA as a novel therapeutic agent against cancer, allergy, and infection.
