scholarly journals Regulation of V(D)J Recombination by Transcriptional Promoters

1999 ◽  
Vol 19 (4) ◽  
pp. 2773-2781 ◽  
Author(s):  
Michael L. Sikes ◽  
Cristina C. Suarez ◽  
Eugene M. Oltz
Keyword(s):  
Gene Segment ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
Inducible Promoter ◽  
Distal Enhancer ◽  
Critical Function ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Primary Determinant ◽  
Antigen Receptor Loci

ABSTRACT Enhancer elements potentiate the rearrangement of antigen receptor loci via changes in the accessibility of gene segment clusters to V(D)J recombinase. Here, we show that enhancer activity per se is insufficient to target T-cell receptor β miniloci for DβJβ recombination. Instead, a promoter situated 5′ to Dβ1 (PDβ) was required for efficient rearrangement of chromosomal substrates. A critical function for promoters in regulating gene segment accessibility was further supported by the ability of heterologous promoters to direct rearrangement of enhancer-containing substrates. Importantly, activation of a synthetic tetracycline-inducible promoter (Ptet) positioned upstream from the Dβ gene segment was sufficient to target recombination of miniloci lacking a distal enhancer element. The latter result suggests that DNA loops, generated by interactions between flanking promoter and enhancer elements, are not required for efficient recognition of chromosomal gene segments by V(D)J recombinase. Unexpectedly, the Ptet substrate exhibited normal levels of rearrangement despite its retention of a hypermethylated DNA status within the DβJβ cluster. Together, our findings support a model in which promoter activation, rather than intrinsic properties of enhancers, is the primary determinant for regulating recombinational accessibility within antigen receptor loci.

scholarly journals Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core-binding factors.

1994 ◽  
Vol 14 (1) ◽  
pp. 473-483 ◽  
Author(s):  
C Hernandez-Munain ◽  
M S Krangel
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Transcriptional Activation ◽  
Cell Receptor ◽  
Enhancer Activity ◽  
Control Of Gene Expression ◽  
Enhancer Element ◽  
The Core ◽  
Core Site ◽  
Factor C

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor (TCR) delta gene. The 30-bp minimal enhancer element denoted delta E3 carries a core sequence (TGTGGTTT) that binds a T-cell-specific factor, and that is necessary but not sufficient for transcriptional activation. Here we demonstrate that the transcription factor c-Myb regulates TCR delta enhancer activity through a binding site in delta E3 that is adjacent to the core site. Both v-Myb and c-Myb bind specifically to delta E3. The Myb site is necessary for enhancer activity, because a mutation that eliminates Myb binding abolishes transcriptional activation by the delta E3 element and by the 370-bp TCR delta enhancer. Transfection of cells with a c-Myb expression construct upregulates delta E3 enhancer activity, whereas treatment of cells with an antisense c-myb oligonucleotide inhibits delta E3 enhancer activity. Since intact Myb and core sites are both required for delta E3 function, our data argue that c-Myb and core binding factors must cooperate to mediate transcriptional activation through delta E3. Efficient cooperation depends on the relative positioning of the Myb and core sites, since only one of two overlapping Myb sites within delta E3 is functional and alterations of the distance between this site and the core site disrupt enhancer activity. Cooperative regulation by c-Myb and core-binding factors is likely to play an important role in the control of gene expression during T-cell development.

Annotation Of Pseudogenous Gene Segments By Massively Parallel Sequencing Of Rearranged Lymphocyte Receptor Loci

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3480-3480
Author(s):  
Ryan O Emerson ◽  
Anna Sherwood ◽  
Harlan Robins ◽  
Christopher S Carlson ◽  
Mark Rieder
Keyword(s):  
Immune System ◽  
T Cell ◽  
B Cells ◽  
Gene Segment ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
T And B Cells ◽  
Employment Equity ◽  
Immune Receptor ◽  
Equity Ownership

Abstract The adaptive immune system generates a remarkable breadth of antigen-specific T cell receptors (TCRs) and B cell receptor (BCRs) by combinatoric shuffling of gene segments, enabling the immune system to recognize a diverse and unpredictable set of antigens. To generate this enormous diversity, lymphocytes undergo somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) region gene segments, which collectively encode the CDR3 region along with non-templated deletion or insertion of nucleotides at the V-D, and D-J junctions. Many of the V, D, and J gene segments at immune receptor loci are annotated as non-functional due to defects in primary sequence, motifs necessary for rearrangement, or chromosome position. However, full annotation of functional, pseudogene, or ORF, has proven elusive due to the random and unpredictable nature of non-templated deletions and insertions, the huge space of potential receptors, and the large V and J gene families. We amplified genomic DNA using a highly multiplexed PCR assay that targeted the rearranged immunoglobulin heavy chain (IGH) and T cell receptor beta (TCRB) receptor locus. Deep DNA sequencing allowed us to characterize the TCRB and IGH immune repertoire from mature T and B cells in a large cohort of healthy adults. Random chance predicts that less than one-third of somatic rearrangements at the TCRB and IGH loci will lead to transcripts with key motifs in-frame and no premature stops (i.e. a productive antigen receptor). Selection during lymphocyte maturation ensures that all mature T and B cells carry at least one rearrangement coding for a productive receptor, with a second allele rearranging in some cells that can be out of frame or include a V, D or J pseudogene segment (i.e. non-productive receptor). We classified each V, D, and J gene segment as functional or pseudogene based on the proportion of in-frame rearrangements and on 3-nt periodicity in the length of the CDR3 hypervariable coding region. Based on these data we were able to conclusively annotate the functional status of each gene segment in the complex TCRB and IGH immune receptor loci. In TCRB, we found 2 presumed-functional gene segments that are clearly pseudogenes; in IgH we found 3 presumed-functional genes that are actually pseudogenes and 1 annotated pseudogene that is clearly functional. In both loci we identified numerous cases of gene segment functional/pseudogene status segregating among healthy subjects, indicating that genotype at the TCR and IGH loci may be a source of substantial differences in the naive antigen receptor repertoire between individuals. These results have helped us to more accurately characterize the genetic landscape underpinning somatic rearrangement of antigen receptors, a seminal event in the generation of robust antigen-specific immune responses. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Rieder:Adaptive Biotechnologies: Employment, Equity Ownership.

Regulation of the T-cell receptor delta enhancer by functional cooperation between c-Myb and core-binding factors

1994 ◽  
Vol 14 (1) ◽  
pp. 473-483
Author(s):  
C Hernandez-Munain ◽  
M S Krangel
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Transcriptional Activation ◽  
Cell Receptor ◽  
Enhancer Activity ◽  
Control Of Gene Expression ◽  
Enhancer Element ◽  
The Core ◽  
Core Site ◽  
Factor C

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor (TCR) delta gene. The 30-bp minimal enhancer element denoted delta E3 carries a core sequence (TGTGGTTT) that binds a T-cell-specific factor, and that is necessary but not sufficient for transcriptional activation. Here we demonstrate that the transcription factor c-Myb regulates TCR delta enhancer activity through a binding site in delta E3 that is adjacent to the core site. Both v-Myb and c-Myb bind specifically to delta E3. The Myb site is necessary for enhancer activity, because a mutation that eliminates Myb binding abolishes transcriptional activation by the delta E3 element and by the 370-bp TCR delta enhancer. Transfection of cells with a c-Myb expression construct upregulates delta E3 enhancer activity, whereas treatment of cells with an antisense c-myb oligonucleotide inhibits delta E3 enhancer activity. Since intact Myb and core sites are both required for delta E3 function, our data argue that c-Myb and core binding factors must cooperate to mediate transcriptional activation through delta E3. Efficient cooperation depends on the relative positioning of the Myb and core sites, since only one of two overlapping Myb sites within delta E3 is functional and alterations of the distance between this site and the core site disrupt enhancer activity. Cooperative regulation by c-Myb and core-binding factors is likely to play an important role in the control of gene expression during T-cell development.

Regulation of human microsomal prostaglandin E synthase-1 by IL-1β requires a distal enhancer element with a unique role for C/EBPβ

2012 ◽  
Vol 443 (2) ◽  
pp. 561-571 ◽  
Author(s):  
Jewell N. Walters ◽  
Justin S. Bickford ◽  
Kimberly J. Newsom ◽  
Dawn E. Beachy ◽  
Sarah J. Barilovits ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Specific Binding ◽  
Proximal Promoter ◽  
Prostaglandin E ◽  
Distal Enhancer ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Enhancer Binding Protein ◽  
Hypersensitive Sites ◽  
Distal Site

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1β (interleukin 1β) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near −8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPβ (CCAAT/enhancer-binding protein β) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPβ binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPβ in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPβ highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

scholarly journals Influence of a CTCF-Dependent Insulator on Multiple Aspects of Enhancer-Mediated Chromatin Organization

2015 ◽  
Vol 35 (20) ◽  
pp. 3504-3516 ◽  
Author(s):  
Garima Varma ◽  
Pratishtha Rawat ◽  
Manisha Jalan ◽  
Manjula Vinayak ◽  
Madhulika Srivastava
Keyword(s):  
T Cell ◽  
Cell Receptor ◽  
Regulatory Elements ◽  
Genomic Context ◽  
Developmentally Regulated ◽  
Enhancer Activity ◽  
Vdj Recombination ◽  
Chromatin Configuration ◽  
Genetically Manipulated ◽  
Antigen Receptor Loci

Developmental stage-specific enhancer-promoter-insulator interactions regulate the chromatin configuration necessary for transcription at various loci and additionally for VDJ recombination at antigen receptor loci that encode immunoglobulins and T-cell receptors. To investigate these regulatory interactions, we analyzed the epigenetic landscape of the murine T-cell receptor β (TCRβ) locus in the presence and absence of an ectopic CTCF-dependent enhancer-blocking insulator, H19-ICR, in genetically manipulated mice. Our analysis demonstrated the ability of the H19-ICR insulator to restrict several aspects of enhancer-based chromatin alterations that are observed during activation of the TCRβ locus for transcription and recombination. The H19-ICR insulator abrogated enhancer-promoter contact-dependent chromatin alterations and additionally prevented Eβ-mediated histone modifications that have been suggested to be independent of enhancer-promoter interaction. Observed enhancer-promoter-insulator interactions, in conjunction with the chromatin structure of the Eβ-regulated domain at the nucleosomal level, provide useful insights regarding the activity of the regulatory elements in addition to supporting the accessibility hypothesis of VDJ recombination. Analysis of H19-ICR in the heterologous context of the developmentally regulated TCRβ locus suggests that different mechanisms proposed for CTCF-dependent insulator action might be manifested simultaneously or selectively depending on the genomic context and the nature of enhancer activity being curtailed.

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