One of the primary goals of our study is to understand the roles played by the transcription factor, IRF8 in hematopoietic cells. In the mouse, studies have focused on dendritic cells, B cells and macrophages which are complemented by analyses of human B cell lines and normal B cells. We have utilized flow cytometry, gene expression profiling using microarrays, high-throughput real time quantitative RT-PCR and mass spectrometry for this owrk. Studies of IRF8 in B cells demonstrated that the gene is expressed at low levels in naive cells, increases to highest levels in germinal center (GC) B cells and then is strikingly downregulated in plasma cells. IRF8 contributes to the transcriptinal regulation of two genes critically involved in B cell differentiation and function, BCL6 and AID. Similar results were obtained using either mouse or human cells. IRF8 also appears to collaborate with BCL6 in suppressing the activities of p53 and, secondarily, p21, making it possible for B cells to tolerate double stranded DNA breaks that occur physiologically during the processes of somatic hypermutation and class switch recombination. By the use of ChIP-chip technology, we have identified over 280 other targets of IRF8 that are common to human and mouse GC B cells. As part of longterm collaborative studies with the Gabriele laboratory, we have shown that IRF8 is critically involved in the ability of splenic and bone marrow macrophages to take up and present antigens to CD4 T cells with both functions markedly affected only in peripheral mature dendritic cells. This provides another in a string of findings that identify IRF8 as a central player in the regulation of innate immunity. Mass spectroscopic studies of macrophages for IRF8 partner proteins, done in collaboration with Dr. Ozato's laboratory (NICHD), identified interactions with Ro52, otherwise known as TRIM21. Ro52 is a ubiquitin E3 ligase that ubiquitinates IRF8 in vitro and in vivo. Further, studies of IRF8-Ro52 coexression showed that the transcriptonal activity of IRF8 was significantly increased. We have generated a gene reporter mouse for TRIM21 that will be invaluable for clarifying its role in IRF8 function as well as characterizing its normal patterns of expression. In addition, it will be of interest to see if lack of this autoantigen has any effect on the immune system of mice. Regulation of gene function by ubiquitination and deubiquitination has become a subject of increasing prominence and importance. We have recently shown that a deubiquitinating enzyme, DUB1, is differentially and tightly regulated during early B cell development. The mechanisms govering DUB1 expression and its targets are an area of intensive study. A quite separate but productive area of investigation relates to the ability of antibodies to function as enzymes, cleaving proteins with high acitivity and specificity. In collaborative efforts with the Gabibov laboratory, we have found that mice and humans with autoimmune disease are predisposed to generate so called """"""""abzymes"""""""" and that these molecules may not only contribute to disease pathogenesis in multiple sclerosis, but may serve as diagnostic and prognostic markers. In addition, it may be possible to direct antibodies with these enzymatic features to microbial targets that woud cripple pathogens such as HIV. Finally, we have been working with mice mutagenized with ENU to identify novel genes that induce leukocytosis or leukopenia. These mice were generated as part of a NHLBI program to identify genes involved in cardiac, respiratory, or hematopietic differentiation and function. We have identified the abnormalities associated with two mutations as affecting expression of IL7 and Tnf. These mutations effectvely yield null alleles in C57BL/6 mice. While conventional knockouts of these alleles were made previously, they do not exist on a pure inbred background or without neo selection makers that may confound phenotypes. These mice will permit clean assessments of the biology resulting from deficiency of either gene.
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