Reorganization of Higher-Order Chromatin Structures During Igk Gene Activation

Ut Southwestern Medical Center, Dallas TX

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Abstract

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