Here we propose to continue our studies on functionally 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 previous funding cycles of this grant we discovered the following two new regions in Ig: chromatin that contain B-cell-specific and developmentally regulated DNase I hypersensitive sites (HSs): (1) A silencer (Sis) within the intervening sequence between the joining (J?) region and the most proximal V? gene, which upon V? - J? joining is either deleted or inverted and far removed from the rearranged V? gene destined to be expressed. Importantly, we showed during the last funding cycle that this element is a recombination silencer responsible for recruiting Ikaros and targeting an Igk minilocus carried on a yeast artificial chromosome (YAC) to pericentromeric heterochromatin;(2) A powerful enhancer (Ed) in the distal downstream region, which is always preserved after normal V? gene rearrangement. Importantly, we showed during the last funding cycle that this element is required for generating maximal levels of Ig: transcripts, and maximal levels of somatic hypermutation (SHM) in germinal center (GC) B cells. In this renewal we plan to focus our studies specifically on further functional delineation of the Sis element by taking advantage of the unique reagents that we have generated over the past quarter of a century under the funding of this grant.
The specific aims are: 1. To determine the impact of deleting Sis on Ig loci nuclear organization. (Parameters investigated will include pericentromeric heterochromatin localization, Igk allele contraction and looping, and heterologous and homologous Ig allele chromosome pairing.) 2. To determine distal DNA sequences that Sis interacts with that may be involved in Igk allele contraction and looping, or in heterologous or homologous Ig allele chromosome pairing. 3. To determine the impact of deleting Sis on the Ig: repertoire, with respect to V gene usage and potential RAG protein targeting mechanisms. 4. To determine the impact of separately mutating Ikaros and CTCF binding sites within Sis on Ig loci nuclear organization, distal-Sis-interacting sequences, and repertoire.
We have recently discovered a genetic region in the mouse kappa immunoglobulin gene that regulates antibody production and the diversity of antibody species that are generated during white blood cell ontogeny. This proposal will further delineate how this genetic element works at the molecular level.
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