The Role of Epigenetics in Regulating V(D)J Recombination and Allelic Exclusion
As members of the adaptive immune response, T- and B- cells express unique antigen receptors generated from antigen receptor loci. These loci encode multiple Variable (V), Diversity (D), and Joining (J) gene segments. Through a process known as V(D)J recombination, genomic rearrangements occur to generate a unique antigen receptor proteins. During each stage of lymphocyte development, antigen receptor loci are epigenetically regulated. The epigenetic regulation promotes and inhibits V(D)J recombination through different mechanisms. To generate an antigen receptor protein, the substrates for rearrangement (recombination signal sequences, RSSs) must be made accessible to the recombination machinery. Moreover, once an antigen receptor locus has rearranged and produced a successful in-frame protein, a mechanism known as allelic exclusion prevents further recombination.
The nucleosome can positively and negatively regulate V(D)J recombination. Therefore, we defined the in vivo nucleosome organization of accessible and inaccessible RSSs on the Tcr loci. We used Tcrb and Tcra alleles which lack various cis-elements (e.g. enhancers and promoters) and terminate transcription. By comparing nucleosome organization and histone octamer occupancy, we found that accessible alleles are characterized by lower histone octamer occupancy and in some cases movement of nucleosomes. Also, we found that some these changes are mediated by transcription through the RSS. We concluded that one mechanism by which cis-elements epigenetically regulate RSS accessibility is by histone octamer loss and nucleosome repositioning and that some of these changes are mediated by transcription.
In addition, we further investigated how allelic exclusion prevents Tcrb locus recombination in CD4, CD8 double positive (DP) thymocytes. A previous study had introduced the Tcra enhancer (Eα) into the middle of the Tcrb locus to test if allelic exclusion was mediated solely by RSS accessibility. That study found that Eα could force RSS accessibility in DP thymocytes, but Vβ RSS accessibility did not overcome additional mechanisms involved in allelic exclusion. One potential mechanism that has been suggested in the literature is changes in locus conformation. Thus, we tested if RSS accessibility and locus conformation together mediate allelic exclusion. We generated two alleles that overcome changes in RSS accessibility, due to the presence of Eα and that overcome changes in locus conformation, due to a decrease in distance between Vβ and DJβ RSSs. We found that both alleles are accessible in DP thymocytes and we detected Vβ to DJβ recombination in DP thymocytes. Therefore, the epigenetic mechanisms that regulate Tcrb allelic exclusion consists of changes in RSS accessibility and changes in locus conformation.
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