scholarly journals The RAG-2 Inhibitory Domain Gates Accessibility of the V(D)J Recombinase to Chromatin

2018 ◽  
Vol 38 (15) ◽  
Author(s):  
Alyssa Ward ◽  
Gita Kumari ◽  
Ranjan Sen ◽  
Stephen Desiderio
Keyword(s):  
B Cell ◽  
Point Mutation ◽  
Antigen Receptor ◽  
Plant Homeodomain ◽  
Inhibitory Domain ◽  
Rag 1 ◽  
Antigen Receptor Loci

ABSTRACTAccessibility of antigen receptor loci to RAG is correlated with the presence of H3K4me3, which binds to a plant homeodomain (PHD) in the RAG-2 subunit and promotes V(D)J recombination. A point mutation in the PHD, W453A, eliminates binding of H3K4me3 and impairs recombination. The debilitating effect of the W453A mutation is ameliorated by second-site mutations that locate an inhibitory domain in the interval from residues 352 through 405 of RAG-2. Disruption of the inhibitory domain stimulates V(D)J recombination within extrachromosomal substrates and at endogenous antigen receptor loci. Association of RAG-1 and RAG-2 with chromatin at theIgHlocus in B cell progenitors is dependent on recognition of H3K4me3 by the PHD. Strikingly, disruption of the inhibitory domain permits association of RAG with theIgHlocus in the absence of H3K4me3 binding. Thus, the inhibitory domain acts as a gate that prohibits RAG from accessing theIgHlocus unless RAG-2 is engaged by H3K4me3.

scholarly journals A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

1993 ◽  
Vol 13 (10) ◽  
pp. 6223-6230 ◽  
Author(s):  
E M Oltz ◽  
F W Alt ◽  
W C Lin ◽  
J Chen ◽  
G Taccioli ◽  
...  
Keyword(s):  
Heat Shock ◽  
B Cell ◽  
Cell Line ◽  
Primary Role ◽  
Transcriptional Enhancer ◽  
Recombination Activating Genes ◽  
Rag Proteins ◽  
Rag 1 ◽  
Antigen Receptor Loci

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

scholarly journals Control of V(D)J Recombination through Transcriptional Elongation and Changes in Locus Chromatin Structure and Nuclear Organization

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Beatriz del Blanco ◽  
Vanina García ◽  
Alberto García-Mariscal ◽  
Cristina Hernández-Munain
Keyword(s):  
Chromatin Structure ◽  
Antigen Receptor ◽  
Open Chromatin ◽  
Recombination Reaction ◽  
Transcriptional Elongation ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Rag 1 ◽  
Antigen Receptor Loci

V(D)J recombination is the assembly of gene segments at the antigen receptor loci to generate antigen receptor diversity in T and B lymphocytes. This process is regulated, according to defined developmental programs, by the action of a single specific recombinase complex formed by the recombination antigen gene (RAG-1/2) proteins that are expressed in immature lymphocytes. V(D)J recombination is strictly controlled by RAG-1/2 accessibility to specific recombination signal sequences in chromatin at several levels: cellular lineage, temporal regulation, gene segment order, and allelic exclusion. DNA cleavage by RAG-1/2 is regulated by the chromatin structure, transcriptional elongation, and three-dimensional architecture and position of the antigen receptor loci in the nucleus. Cis-elements specifically direct transcription and V(D)J recombination at these loci through interactions with transacting factors that form molecular machines that mediate a sequence of structural events. These events open chromatin to activate transcriptional elongation and to permit the access of RAG-1/2 to their recombination signal sequences to drive the juxtaposition of the V, D, and J segments and the recombination reaction itself. This chapter summarizes the advances in this area and the important role of the structure and position of antigen receptor loci within the nucleus to control this process.

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination

1993 ◽  
Vol 13 (10) ◽  
pp. 6223-6230
Author(s):  
E M Oltz ◽  
F W Alt ◽  
W C Lin ◽  
J Chen ◽  
G Taccioli ◽  
...  
Keyword(s):  
Heat Shock ◽  
B Cell ◽  
Cell Line ◽  
Primary Role ◽  
Transcriptional Enhancer ◽  
Recombination Activating Genes ◽  
Rag Proteins ◽  
Rag 1 ◽  
Antigen Receptor Loci

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

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