Allelic exclusion functions at certain T-cell receptor (TCR) and immunoglobulin loci to restrict developing lymphocytes to produce a single productive rearrangement. At the TCR beta locus, this involves a feedback signal that inhibits V-beta to DJ-beta rearrangement in the face of continued expression of recombinase. This suggests regulation at the level of substrate accessibility, but to date, this has not been confirmed and mechanistic insights have been limited. By comparing the acetylation of histone 3 in TCR beta locus chromatin of double negative (DN) thymocytes that have yet to receive an allelic exclusion signal and double positive (DP) thymocytes that have already received this signal, we demonstrate a change in the structure of V-beta chromatin associated with allelic exclusion. We propose to evaluate the regulation of V-beta chromatin structure and the role of chromatin structure in V-beta rearrangement and allelic exclusion.
In Specific Aim I, we will characterize chromatin structure across the TCR beta locus in DN and DP thymocyte to evaluate the role of global vs. local chromatin regulation in allelic exclusion. We will map acetylation, accessibility, subnuclear organization, and promoter structure. The chromatin environment created by V-beta promoter function may be critical recombination and may be a critical parameter for allelic exclusion.
Specific Aim II, we will test a role for regulated V-beta promoter activity in V-beta rearrangement and allelic exclusion by assaying promoter function in a transgenic reporter devoid of enhancer elements, and by deleting promoters from the endogenous locus by homologous recombination. Although there is a change V-beta chromatin structure that is associated with allelic exclusion, the process could in theory be enforced by a non-chromatin based mechanism.
In Specific Aim III we will address a causal role for chromatin structure by overriding the structural transition and ask whether we simultaneously override allelic exclusion. We will do so by supplying a germline V-beta promote copy of a local copy of E-beta using a knock-in approach. Allelic exclusion must inhibit rearrangement even on VDJ-beta rearranged allele in which the promoters of upstream V-beta segments are in proximity of E-beta.
In Specific Aim I V we will study chromatin structure on a VDJ-beta rearranged allele, and will evaluate any role for the promoter of the rearranged V-beta segment in establishing that structure, by knock-in with or the rearranged V-beta segment with or without its associated promoter. These studies should provide insights into roles for developmentally regulated chromatin opening, enhancer activity and promoter function in V-beta rearrangement and allelic exclusion.
Showing the most recent 10 out of 26 publications
