Here we propose to continue our studies on elucidating cis-acting regulatory elements in the mouse ? immunoglobulin (Ig) gene locus. Namely, the elements that either inhibit or target locus accessibility to the recombinational and transcriptional machinery in B- and T-lymphocytes. During the last funding cycle of this grant we discovered the following three new regions in chromatin that contain B cell specific and developmentally regulated nuclease hypersensitive sites (HSs): (1) A silencer (Sis) within the intervening sequence (IS) between the joining (J) region and the most proximal V region, which upon V-J joining is either deleted or inverted and far removed from the rearranged V gene destined to be expressed; (2) A powerful enhancer (Ed) in the distal downstream region, which is always preserved after normal V gene rearrangement; and (3) Pre-B cell specific germline V gene HSs at or near recombination signal sequences (RSSs), termed HSRss. ? We plan on elucidating the functions of these HSs in this renewal application by addressing the following three major aims: ? 1. To functionally elucidate the Sis, with emphasis on its potential negative regulatory roles in B- and T-lymphocytes. Parameters to be investigated will include: gene rearrangement levels; allelic exclusion; germline and rearranged gene transcription levels; B- vs T-cell specificity for rearrangement; and developmental timing of V-J joining. ? 2. To functionally elucidate the Ed, with emphasis on its potential positive role in up-regulating transcription upon B cell differentiation and stimulation. Parameters to be investigated will include: transcription levels of rearranged genes; and V-recombining sequence (RS) recombination. ? 3. To investigate potential mechanisms for generation of HSs at or near germline VK gene RSSs in pre-B cells. Parameters to be investigated will include: the relationships and requirements of adjacent E-boxes and bound E2A proteins; and clonality and mono- or bi-allelic nature. ? ? ? ?
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