scholarly journals Poor Quality Vβ Recombination Signal Sequences Enforce TCRβ Allelic Exclusion by Limiting the Frequency of Vβ Recombination

2020 ◽  
Author(s):  
Glendon S. Wu ◽  
Katherine S. Yang-Iott ◽  
Morgann A. Reed ◽  
Katharina E. Hayer ◽  
Kyutae D. Lee ◽  
...  
Keyword(s):  
B Lymphocyte ◽  
Feedback Inhibition ◽  
Signal Sequence ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
Poor Quality ◽  
Functional Gene ◽  
Monoallelic Expression ◽  
Allelic Exclusion ◽  
Recombination Signal

SUMMARYMonoallelic expression (allelic exclusion) of T and B lymphocyte antigen receptor genes is achieved by the assembly of a functional gene through V(D)J recombination on one allele and subsequent feedback inhibition of recombination on the other allele. There has been no validated mechanism for how only one allele of any antigen receptor locus assembles a functional gene prior to feedback inhibition. Here, we demonstrate that replacement of a single Vβ recombination signal sequence (RSS) with a better RSS increases Vβ rearrangement, reveals Tcrb alleles compete for utilization in the αβ T cell receptor (TCR) repertoire, and elevates the fraction of αβ T cells expressing TCRβ protein from both alleles. The data indicate that poor qualities of Vβ RSSs for recombination with Dβ and Jβ RSSs enforces allelic exclusion by stochastically limiting the incidence of functional Vβ rearrangements on both alleles before feedback inhibition terminates Vβ recombination.

scholarly journals Poor quality Vβ recombination signal sequences stochastically enforce TCRβ allelic exclusion

2020 ◽  
Vol 217 (9) ◽  
Author(s):  
Glendon S. Wu ◽  
Katherine S. Yang-Iott ◽  
Morgann A. Klink ◽  
Katharina E. Hayer ◽  
Kyutae D. Lee ◽  
...  
Keyword(s):  
Immune Responses ◽  
Feedback Inhibition ◽  
Poor Quality ◽  
The Other ◽  
Monoallelic Expression ◽  
Allelic Exclusion ◽  
Epigenetic Mechanisms ◽  
Recombination Signal Sequences ◽  
Cardinal Feature

The monoallelic expression of antigen receptor (AgR) genes, called allelic exclusion, is fundamental for highly specific immune responses to pathogens. This cardinal feature of adaptive immunity is achieved by the assembly of a functional AgR gene on one allele, with subsequent feedback inhibition of V(D)J recombination on the other allele. A range of epigenetic mechanisms have been implicated in sequential recombination of AgR alleles; however, we now demonstrate that a genetic mechanism controls this process for Tcrb. Replacement of V(D)J recombinase targets at two different mouse Vβ gene segments with a higher quality target elevates Vβ rearrangement frequency before feedback inhibition, dramatically increasing the frequency of T cells with TCRβ chains derived from both Tcrb alleles. Thus, TCRβ allelic exclusion is enforced genetically by the low quality of Vβ recombinase targets that stochastically restrict the production of two functional rearrangements before feedback inhibition silences one allele.

Promoter Element for Transcription of Unrearranged T-Cell Receptor β-Chain Gene in Pro-T Cells

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3017-3025 ◽  
Author(s):  
Raymond T. Doty ◽  
Dong Xia ◽  
Suzanne P. Nguyen ◽  
Tanya R. Hathaway ◽  
Dennis M. Willerford
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
B Lymphocyte ◽  
Developmental Regulation ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
Chain Gene ◽  
Germline Transcription ◽  
Lineage Specificity ◽  
T Cell Lines

Abstract The hallmark of T- and B-lymphocyte development is the rearrangement of variable (V), diversity (D), and joining (J) segments of T-cell receptor (TCR) and immunoglobulin (Ig) genes to generate a diverse repertoire of antigen receptor specificities in the immune system. The process of V(D)J recombination is shared in the rearrangement of all seven antigen receptor genes and is controlled by changes in chromatin structure, which regulate accessibility to the recombinase apparatus in a lineage- and stage-specific manner. These chromatin changes are linked to transcription of the locus in its unrearranged (germline) configuration. To understand how germline transcription of the TCRβ-chain gene is regulated, we determined the structure of germline transcripts initiating near the Dβ1 segment and identified a promoter within this region. The Dβ1 promoter is active in the presence of the TCRβ enhancer (Eβ), and in this context, exhibits preferential activity in pro-T versus mature T-cell lines, as well as T- versus B-lineage specificity. These studies provide insight into the developmental regulation of TCRβ germline transcription, one of the earliest steps in T-cell differentiation.

scholarly journals CompletecisExclusion upon Duplication of the Eμ Enhancer at the Immunoglobulin Heavy Chain Locus

2015 ◽  
Vol 35 (13) ◽  
pp. 2231-2241 ◽  
Author(s):  
Nadine Puget ◽  
Claire Leduc ◽  
Zéliha Oruc ◽  
Mohammed Moutahir ◽  
Marc Le Bert ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Critical Role ◽  
Antigen Receptor ◽  
Immunoglobulin Heavy Chain ◽  
Antisense Transcription ◽  
Regulatory Elements ◽  
Monoallelic Expression ◽  
Allelic Exclusion ◽  
Chain Locus ◽  
Heavy Chain Locus

Developing lymphocytes somatically diversify their antigen-receptor loci through V(D)J recombination. The process is associated with allelic exclusion, which results in monoallelic expression of an antigen receptor locus. Variouscis-regulatory elements control V(D)J recombination in a developmentally regulated manner, but their role in allelic exclusion is still unclear. At the immunoglobulin heavy chain locus (IgH), the Eμ enhancer plays a critical role in V(D)J recombination. We generated a mouse line with a replacement mutation in the constant region of the locus that duplicates the Eμ enhancer and allows premature expression of the γ3 heavy chain. Strikingly, IgM expression was completely and specifically excluded incisfrom the mutant allele. Thiscisexclusion recapitulated the main features of allelic exclusion, including differential exclusion of variable genes. Notably, sense and antisense transcription within the distal variable domain and distal VH-DJHrecombination were inhibited.cisexclusion was established and stably maintained despite an active endogenous Eμ enhancer. The data reveal the importance of the dynamic, developmental stage-dependent interplay betweenIgHlocus enhancers and signaling in the induction and maintenance of allelic exclusion.

scholarly journals Level of human TCRBV3S1 (V beta 3) expression correlates with allelic polymorphism in the spacer region of the recombination signal sequence.

1994 ◽  
Vol 179 (5) ◽  
pp. 1707-1711 ◽  
Author(s):  
D N Posnett ◽  
C S Vissinga ◽  
C Pambuccian ◽  
S Wei ◽  
M A Robinson ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Signal Sequence ◽  
Cell Receptor ◽  
Allelic Polymorphism ◽  
Nucleotide Position ◽  
Recombination Signal Sequence ◽  
Recombination Signal ◽  
Cis Acting ◽  
Human T Cell

One of the causes of variations in the expressed human T cell receptor (TCR) BV (V beta) repertoire is genetic variation in the germline DNA. Herein evidence is provided that allelic polymorphism may affect recombination frequency for a specific V gene. Two alleles of the TCR BV3 differ only at a single nucleotide position (C/T) within the 23-bp spacer region of the recombination signal sequence. These alleles are associated with variable percentages of BV3 cells in the peripheral blood, as shown in families and in unrelated normal donors. Individuals homozygous for allele 2 have a mean of 8.1% BV3 cells, heterozygous individuals have a mean of 4.7% BV3 cells, and homozygotes for allele 1 have a mean of 1.2% BV3 cells in CD3+ CD4+ peripheral blood T cells. Since the correlation is tight in unrelated individuals and other genetic differences were not found in the vicinity of BV3, we suggest that the spacer region sequence itself modifies recombination efficiency. This allelic system provides an example of a novel mechanism by which cis-acting genetic elements may affect recombination in a natural in vivo system.

Promoter Element for Transcription of Unrearranged T-Cell Receptor β-Chain Gene in Pro-T Cells

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3017-3025 ◽  
Author(s):  
Raymond T. Doty ◽  
Dong Xia ◽  
Suzanne P. Nguyen ◽  
Tanya R. Hathaway ◽  
Dennis M. Willerford
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
B Lymphocyte ◽  
Developmental Regulation ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
Chain Gene ◽  
Germline Transcription ◽  
Lineage Specificity ◽  
T Cell Lines

The hallmark of T- and B-lymphocyte development is the rearrangement of variable (V), diversity (D), and joining (J) segments of T-cell receptor (TCR) and immunoglobulin (Ig) genes to generate a diverse repertoire of antigen receptor specificities in the immune system. The process of V(D)J recombination is shared in the rearrangement of all seven antigen receptor genes and is controlled by changes in chromatin structure, which regulate accessibility to the recombinase apparatus in a lineage- and stage-specific manner. These chromatin changes are linked to transcription of the locus in its unrearranged (germline) configuration. To understand how germline transcription of the TCRβ-chain gene is regulated, we determined the structure of germline transcripts initiating near the Dβ1 segment and identified a promoter within this region. The Dβ1 promoter is active in the presence of the TCRβ enhancer (Eβ), and in this context, exhibits preferential activity in pro-T versus mature T-cell lines, as well as T- versus B-lineage specificity. These studies provide insight into the developmental regulation of TCRβ germline transcription, one of the earliest steps in T-cell differentiation.

scholarly journals T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells.

1996 ◽  
Vol 16 (2) ◽  
pp. 609-618 ◽  
Author(s):  
F Livak ◽  
D G Schatz
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Signal Sequence ◽  
Cell Receptor ◽  
Recombination Reaction ◽  
Dna Molecules ◽  
Coding Region ◽  
Recombination Signal ◽  
By Products

In addition to the assembled coding regions of immunoglobulin and T-cell receptor (TCR) genes, the V(D)J recombination reaction can in principle generate three types of by-products in normal developing lymphocytes: broken DNA molecules that terminate in a recombination signal sequence or a coding region (termed signal or coding end molecules, respectively) and DNA molecules containing fused recombination signal sequences (termed reciprocal products). Using a quantitative Southern blot analysis of the murine TCR alpha locus, we demonstrate that substantial amounts of signal end molecules and reciprocal products, but not coding end molecules, exist in thymocytes, while peripheral T cells contain substantial amounts of reciprocal products. At the 5' end of the J alpha locus, 20% of thymus DNA exists as signal end molecules. An additional 30 to 40% of the TCR alpha/delta locus exists as remarkably stable reciprocal products throughout T-cell development, with the consequence that the TCR C delta region is substantially retained in alpha beta committed T cells. The disappearance of the broken DNA molecules occurs in the same developmental transition as termination of expression of the recombination activating genes, RAG-1 and RAG-2. These findings raise important questions concerning the mechanism of V(D)J recombination and the maintenance of genome integrity during lymphoid development.

scholarly journals Rag1D600A, a novel catalytically inactive RAG mouse model

2019 ◽  
Author(s):  
Jason B Wong ◽  
Jane A Skok
Keyword(s):  
Mouse Model ◽  
Signal Sequence ◽  
Antigen Receptor ◽  
Model System ◽  
Transgenic Expression ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Acidic Residues ◽  
Dna Nicking ◽  
Mouse Model System

AbstractThe RAG complex (RAG1 and RAG2) can bind to recombination signal sequences of antigen receptor loci gene segments and coordinate V(D)J recombination which is the primary method of generating antigen receptor diversity. Previous biochemistry studies discovered RAG1 D600, D708 and E962 residues as essential for catalytic DNA nicking and hairpin forming activity of the RAG complex. Neutralization of each of the acidic residues does not impair DNA binding to recombination signal sequence containing DNA substrates, but cleavage of the substrates is severely compromised. These three acidic residues are thought to comprise a DDE motif that is responsible for binding to a divalent cation that is necessary for cleavage activity. Although a Rag1-/-; RAG1-D708A transgenic mouse model system has been used to study dynamics of RAG activity, transgenic expression may not precisely mimic expression from the endogenous locus. In order to improve upon this model, we created Rag1D600A mice that lack B and T cells and demonstrate a developmental block at the pro-B and DN stages, respectively. Thus, Rag1D600A mice provide a novel mouse model system for studying the poorly understood noncanonical functions of RAG1.

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