Peripheral subnuclear positioning suppresses Tcrb recombination and segregates Tcrb alleles from RAG2.
Allelic exclusion requires that the two alleles at antigen-receptor loci attempt to recombine variable (V), diversity (D), and joining (J) gene segments [V(D)J recombination] asynchronously in nuclei of developing lymphocytes. It previously was shown that T-cell receptor β (Tcrb) alleles frequently and stochastically associate with the nuclear lamina and pericentromeric heterochromatin in CD4(-)CD8(-) thymocytes. Moreover, rearranged alleles were underrepresented at these locations. Here we used 3D immunofluorescence in situ hybridization to identify recently rearranged Tcrb alleles based on the accumulation of the DNA-repair protein 53BP1. We found that Tcrb alleles recombine asynchronously in double-negative thymocytes and that V(D)J recombination is suppressed on peripheral as compared with central Tcrb alleles. Moreover, the recombination events that did take place at the nuclear periphery preferentially occurred on Tcrb alleles that were partially dissociated from the nuclear lamina. To understand better the mechanism by which V(D)J recombination is suppressed at the nuclear periphery, we evaluated the subnuclear distribution of recombination-activating gene 2 (RAG2) protein. We found that RAG2 abundance was reduced at the nuclear periphery. Moreover, RAG2 was distributed differently from RNA polymerase II and histone H3K4 trimethylation. Our data suggest that the nuclear periphery suppresses V(D)J recombination, at least in part, by segregating Tcrb alleles from RAG proteins.
Departments of Immunology, Radiology, and Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710.
Elizabeth A W
EA
Chan
Grace
G
Teng
Elizabeth
E
Corbett
Kingshuk Roy
KR
Choudhury
Craig H
CH
Bassing
David G
DG
Schatz
Michael S
MS
Krangel
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*
D004268:DNA-Binding Proteins; C499308:Rag2 protein, mouse; D016693:Receptors, Antigen, T-Cell, alpha-beta
T-cell development; thymus