Employing chromosome conformation capture (3C) technology, we will determine how the long-range spatial organization of the mouse Igk gene locus changes as a function of B cell development and gene activation. We will explore the modulation of interactions between known cis-acting elements in the locus before and after gene rearrangement and transcriptional activation. We will also determine which of several transcription factors that are known to interact with the enhancers are present in looped complexes. We will further determine which of several selected histone post-translational modifications are present in such looped complexes. For these studies we will use a panel of cell lines arrested at various stages of B cell differentiation, as well as primary pro-, pre- and mature-B cells from animals. We will also determine whether NF-kB or E2A (or both) are required for loop formation in pre-B cells. Finally, we will functionally elucidate a novel B-cell specific hypersensitive site at the 3'boundary of the locus that we first discovered by the 3C technology. Completion of these studies will provide new mechanistic insights on Ig gene regulation at the level of higher-order chromatin structure, transcription, recombination, and communication between cis-acting elements and trans-acting factors. The following four specific aims are proposed: 1. To elucidate separately the higher-order chromatin organization of germline and rearranged Igk alleles as a function of B cell development and transcription. 2. To determine if certain transcription factors or specific post-translational modifications of histone H3 are present in looped complexes between Igk gene enhancers and rearranged V gene or germline promoters. 3. Define the importance of NF-kB or E2A (or both) in the formation of looped complexes between cis-acting sequences in pre-B cells. 4. To functionally elucidate a novel B-cell specific hypersensitive site at the 3'boundary of the Igk gene locus.
