The primary focus of this revised R21 application is on defining the functions(s) of the zinc finger family member Zfp318 in the development and function of B cells. Previously we demonstrated that Zfp318 is expressed during B cell maturation and shows the highest level of expression in mature, nave follicular B cells. Zfp318 possesses 2 zinc finger domains as well as nuclear localization motifs: it is in a class of protein with RNA and/or DNA binding potential. We have created a Zfp318 conditional deletion allele and show in data included in this application and a manuscript in press that B cells lacking the protein fail to produce IgD. IgD, in nave maturing and mature B cells, is coordinately expressed with IgM via alternative splicing of the Ighm/Ighd locus. B cells lacking Zfp318 continue to express VDJ-IgM but lose the ability to generate the VDJ-IgD product. New data generated since the first submission demonstrates that Zfp318 does not function to alter the alternative splicing of the Ighm/Ighd hnRNA, but instead allows for the production of this entire hnRNA by blocking the transcription termination site at the end of the IgM- encoding exons. In the absence of Zfp318, virtually all of the Ighm transcripts terminate prior to the IgD-encoding exons. Amazingly, the Ighm/Ighd gene is the only locus in B cells that shows such altered expression profiles which means that Zfp318 has evolved to singularly control this event. The experimental plan of this application section is focused upon accomplishing three specific goals. First, we will determine if there are functional consequences to the B cell and the immune response created by the absence of Zfp318. IgD and Zfp318 have co-evolved since the development of primitive vertebrates, our Zfp318 deficient animal will provide us a unique vantage to ascertain the importance of these proteins to the immune response. Secondly we will address how Zfp318 functions. Does it bind to DNA, does it bind to RNA? The fact Zfp318 only modulates Ighm/Ighd expression indicates the protein possesses a very high level of sequence/structure specificity. And finally we will address how the regulation of the expression of the Zfp318 gene is integrated into B cell development and activation. We know from our previous studies that the expression of Zfp318 is dramatically influenced by Mef2c, a transcription factor known to modulate B cell development and activation. How Zfp318 fits into this network of expression and function is the primary goal of this last question.
B cells are required for the generation of antibodies that limit and eliminate infections. The generation of mature functional B cell requires a coordinated developmental gene expression profile. In this application we describe experiments designed to elucidate the function of a novel zinc finger binding protein, Zfp318, in the development and activation pathways of B cells.
