The development of B lymphocytes requires both stage- and tissue-specific recombination of the Igh locus. It is thought that regulated changes within chromatin structure control the accessibility of the locus or locus subregion, to the common V(D)J recombinase. Elucidating the nature and regulation of changes accompanying Igh accessibility will contribute an important paradigm for the control of large, complex loci. Our previous work contributed to under- standing the organization and content of the mouse VH genes and the development of the preimmune Ig repertoire. Our most recent studies have focused on the regulation of accessibility in the context of Igh-V locus organization. To extend our studies, experiments are now proposed to test the hypotheses that 1) Igh-V locus accessibility is regulated by cis- acting elements other than the known Igh enhancers, 2) the D- proximal and D-distal regions of the Igh-V locus are regulated independently, and 3) IL-7 and pre-BCR signaling pathways determine Igh-V subregion accessibility during B cell development. Three related areas of investigation are outlined. First, we will determine if individual VH genes are associated with local elements that regulate germline transcription, a marker of locus accessibility. These experiments will utilize mice transgenic for a well-characterized germline VH gene. Second, the 5' Igh boundary will be mapped and examined for potential regulatory elements by nuclease sensitivity analysis. Third, the regulation of D-distal and D-proximal Igh-V locus accessibility will be followed during B cell development and the influence of IL-7 and pre-BCR mediated signaling will be assessed by germline transcription and by the detection of recombinase-mediated cleavage events.
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