The somatic assembly of antigen receptor genes by V(D)J recombination is essential for the generation of diverse antigen receptor repertoires on T and B lymphocytes. V(D)J recombination at the T cell receptor β (Tcrb) locus is strictly regulated and is activated and inactivated during T lymphocyte development in a manner that promotes allelic exclusion. However the mechanisms that regulate Tcrb locus recombination and allelic exclusion are only partly understood. This project will address unexplored aspects of Tcrb locus regulation that are of fundamental importance for the integrity of the Tcrb locus V(D)J recombination program.
Specific Aim I will test the hypothesis that appropriate developmental activation of Tcrb locus chromatin for V(D)J recombination requires mechanisms that open chromatin as well as mechanisms that suppress the influence of nearby repressive chromatin. A retroviral LTR has been localized to a discrete boundary between repressive and active chromatin domains, and this LTR or an adjacent LINE appears to drive transcription across the active chromatin domain. To evaluate the importance of these elements for Tcrb locus regulation, the LTR/LINE region will be functionally dissected to discriminate components contributing to chromatin barrier and transcriptional activity. Functional significance for Tcrb gene recombination in vivo will be assessed by using gene targeting to eliminate components required for barrier function, promoter activity, or both.
Specific Aim II will test the hypothesis that Tcrb association with the nuclear lamina suppresses Vβ-to-DJβrecombination in a manner that promotes asynchronous allelic recombination, thereby setting the stage for allelic exclusion. Interactions between Tcrb alleles and the nuclear lamina will be probed using four-color, three dimensional immuno-fluorescence in situ hybridization (3D Immuno-FISH) and DamID to evaluate whether the Tcrb locus adopts distinct conformations at the nuclear lamina that are suppressive or permissive for Vβ-to-DJβ recombination. A suppressive influence of the nuclear lamina on V(D)J recombination will then be directly assessed by recruiting to the nuclear lamina a TCR locus that is normally free of this compartment. This will be accomplished by using gene targeting to introduce a lac operator (lacO) array into the Tcra/Tcrd locus and by expressing lac repressor (lacI) as a fusion protein with a component of the nuclear lamina. Successful completion of this work should provide insights into the mechanisms that regulate both Dβ-to-Jβand Vβ-to-DJβ recombination and insights into allelic regulation that will be valuable for understanding the recombination programs at other TCR and Ig loci. A successful outcome should also provide fundamental insights into retroelement and barrier-type insulator function that will impact our understanding of similar elements that are widely distributed across the genome, and important and broadly applicable insights into the role of the nuclear lamina as a regulator of gene activity.
Health relatedness V(D)J recombination is essential for the development of a functional immune system. Failure of V(D)J recombination can cause immunodeficiency and disregulated V(D)J recombination can result in autoimmunity or chromosomal translocations that contribute to tumorigenesis. These studies will elucidate basic mechanisms that contribute to normal immune system development and pathologies such as immunodeficiency, autoimmunity and cancer.
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