The goal of my proposed research continues to be the definition of the molecular details of how chromatin structure contributes to the activation of immunoglobulin (Ig) genes. The study of cultured murine cell lines with defined phenotypes of Ig gene rearrangements and expression will allow discrimination between changes in chromatin structure and composition that occur prior to Ig gene rearrangement and transcription from those which occur as a result of transcription or rearrangement. Specifically, these experiments will address the role that chromatin structure has tn (1) B cell commitment to DNA rearrangement, (2) the simultaneous coexpression of mu and delta H chains and (3) the role of enhancer and flanking sequences in establishing chromatin domains necessary for the increased Ig expression seen in plasma cells. The research can be divided into two major areas of investigation: i) Definition of B cell stage-specific chromatin alterations along endogenous Ig genes that precede and accompany rearrangement and transcription. ii) The definition of sequences (control elements) responsible for the chromatin alterations by DNA-mediated gene transfer and the use of transgenic mice. The general experimental strategy is to subject the nuclei from B cell lines at different stages of development (either with or without transfected recombinant vectors) to nuclease digestion and chemical cleavage. The digested chromatin will be fractionated based on its protein composition using a) gel electrophoresis of native nucleoprotein complexes and b) the chromatin's solubility under specific ionic conditions. Each fraction will be analyzed to determine its protein constituents, distribution of Ig gene sequences and attendant chromatin structure. The use of protein- DNA crosslinking reagents, DNAse I and chemical cleavage reagents will allow the localization of contact points between chromosomal proteins and specific regions of Ig genes. Although strong correlations between chromatin structural changes and gene activity have been found for specific Ig gene structural sequences in B cells, this study is the first to attempt to determine, at high resolution, the timing and nature of chromatin alterations within and around known control elements in differentiating B cells.
