Cis-acting elements within T cell receptor (TCR) and immunoglobulin loci impart developmental regulation to V(D)J recombination by functioning as region- and developmental stage-specific modifiers of chromatin structure. The TCR alpha/delta locus provides a valuable model to unravel the complex mechanisms underlying this regulation, since it contains distinct sets of gene segments activated according to distinct developmental programs, and there is much known about the roles of two prominent enhancers, E-delta and E-alpha. However, full accounting for the complex locus dynamics requires better understanding of the cis-elements with which the enhancers collaborate and the mechanisms by which cis-elements translate activity into changes in chromatin structure and function. We will produce and analyze genetically manipulated versions of the endogenous murine TCR alpha/delta locus and transgenic reporter substrates to address mechanisms of regulation.
In Aim I, we will assess the role of germline promoters as regulators of J-alpha accessibility and usage. Previous data indicate E-alpha to collaborate with the TEA promoter to regulate accessibility of 5' Ja segments and imply its interaction with additional elements to regulated more 3' Jas. We have identified a promoter that is a strong candidate for such a regulator, and will test this by gene targeting at the endogenous TCR alpha/delta locust. The role of germline transcription in accessibility and V(D)J recombination is unclear, We propose that enhancer- and promoter distal accessibility may depend critically on transcriptional elongation, and will test this in Aim II by inserting a transcriptional terminator into the Ja region. The factors determining TCR alpha/delta V gene usage are not understood.
In Aim III, we will ask whether developmental changes in V segment chromatin structure determine whether members of the TCR alpha/delta V segment pool function as V-alpha's, V-deltas, or both. E-delta interacts with germline TCR delta promoters to provide accessibility for V(D)J recombination but does not activate germline TCR-a lpha promoters.
In Aim I V, we will use transgenic reporters to functionally characterize germline J-delta promoters and to investigate the mechanisms restricting E-delta from activating the germline TCR alpha promoter TEA. The function of enhancer-associated nuclear matrix attachment regions (MARs) is unclear.
In Aim V we will analyze human E-delta MAR function within a transgenic reporter, and murine E-delta MAR function within the endogenous locus, to test a dual function model in which these elements activate in DN thymocytes and repress in DP thymocytes, thereby contributing critically to developmental regulation by E-delta.
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