A central process in the development of a competent immune system is the series of genomic rearrangement events that take place to produce a mature, functional immunoglobulin or T cell receptor gene. The failure of developing lymphocytes to correctly assemble their antigen receptor genes can lead to lymphoid tumors or immunodeficiency. As we previously demonstrated, the lymphoid-specific RAGI and RAG2 proteins carry out the first stage of this complex process, known as V (D) J recombination, by cleaving DNA at recombination signal sequences. V (D) J rearrangement is subject to many layers of regulation, from the simplest level of protein: DNA recognition between the RAG proteins and the signal sequences to the highly complex coordination of lineage- and stage-specific accessibility of the endogenous loci. Our goal over the next years is to understand the molecular basis for these different layers of regulation. First, we will address the requirements for assembling an active cleavage complex of RAG proteins with a pair of recombination signals on naked DNA templates. We will use a combination of genetic and biochemical approaches to ask how the RAG proteins recognize the RSS and how clevage of a signal pair is coordinated. We will also study a series of mutant derivatives of an accessory factor (mammalian HMG1 or yeast NHP6A) that is required for the assembly of a functional RAG cleavage complex, to understand this factor's role in V (D) J cleavage. Second, we will ask how chromatin structure and higher order DNA architecture can be modified to serve as a regulatable impediment to V (D) J cleavage. We will develop and analyze model substrates of increasing complexity that allow us to test what features of chromatin structure, what type of chromatin modifications and what chromatin modifying activities can serve to render a substrate more or less accessible to RAG-mediated cleavage. Third, we will use a series of genetically modified mice in which rearrangement is arrested at different stages of development to ask what chromatin modifications and modifying activities are present in vivo at endogenous antigen antigen receptor loci that, by their correlation with V (D) J cleavage, may be critical for regulating the reaction. In summary, the proposed experiments should yield a greater molecular understanding of the complex process of regulating V (D) J recombination during lymphoid development.
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