The overall goal of this proposed research is to elucidate 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. Specifically, these experiments will address the role that chromatin structure has in 1) B cell commitment to DNA rearrangement, 2) isotype selection during heavy (H) chain class switch, 3) the simultaneous coexpression of Mu and Delta H chains and 4) the control of Ig gene expression by immunoregulatory mechanisms. 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. The general experimental strategy will be to subject the nuclei from B cell lines at different stages of development (either with or without transfected recombinant vectors) to nuclease digestion. The digested chromatin will be fractionated based on its protein composition using a) gel electrophoresis of native nucleoprotein complexes, b) the chromatin's solubility under specific ionic conditions and c) immunological techniques. 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 and DNAse I or chemical cleavage reagents will allow the localization of contact points between chromosomal proteins and specific regions of Ig genes.
