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.

Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression

1992 ◽  
Vol 12 (10) ◽  
pp. 4251-4261 ◽  
Author(s):  
C Kingsley ◽  
A Winoto
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Multigene Family ◽  
Binding Proteins ◽  
Receptor Gene ◽  
Consensus Sequence ◽  
Gene Segment ◽  
Cell Receptor ◽  
Regulatory Elements

Analysis of a T-cell antigen receptor (TCR) alpha promoter from a variable gene segment (V) revealed a critical GT box element which is also found in upstream regions of several V alpha genes, TCR enhancer, and regulatory elements of other genes. This element is necessary for TCR gene expression and binds several proteins. These GT box-binding proteins were identified as members of a novel Sp1 multigene family. Two of them, which we term Sp2 and Sp3, were cloned. Sp2 and Sp3 contain zinc fingers and transactivation domains similar to those of Sp1. Like Sp1, Sp2 and Sp3 are expressed ubiquitously, and their in vitro-translated products bind to the GT box in TCR V alpha promoters. Sp3, in particular, also binds to the Sp1 consensus sequence GC box and has binding activity similar to that of Sp1. As the GT box has also previously been shown to play a role in gene regulation of other genes, these newly isolated Sp2 and Sp3 proteins might regulate expression not only of the TCR gene but of other genes as well.

scholarly journals Regulation of the human T-cell receptor alpha gene enhancer: multiple ubiquitous and T-cell-specific nuclear proteins interact with four hypomethylated enhancer elements.

1990 ◽  
Vol 10 (9) ◽  
pp. 4720-4727 ◽  
Author(s):  
I C Ho ◽  
J M Leiden
Keyword(s):  
T Cell ◽  
Protein Binding ◽  
Binding Sites ◽  
Nuclear Protein ◽  
Cell Receptor ◽  
Enhancer Activity ◽  
Protein Binding Sites ◽  
Human T Cell ◽  
Alpha Gene ◽  
Enhancer Function

Transcription of human T-cell receptor (TCR) alpha genes is regulated by a T-cell-specific transcriptional enhancer that is located 4.5 kilobases 3' of the C alpha gene segment. Previous studies have demonstrated that this enhancer contains at least five nuclear protein-binding sites called T alpha 1 to T alpha 5. In the studies described in this report, we have determined the molecular requirements for human TCR alpha enhancer function. In vitro mutagenesis and deletion analyses demonstrated that full enhancer activity is retained in a 116-base-pair fragment containing the T alpha 1 and T alpha 2 nuclear protein-binding sites and that both of these sites are required for full enhancer function. Functional enhancer activity requires that the T alpha 1 and T alpha 2 binding sites be separated by more than 15 and fewer than 85 base pairs. However, the sequence of this spacer region and the relative phase of the two binding sites on the DNA helix do not affect enhancer function. Deletion and mutation analyses demonstrated that the T alpha 3 and T alpha 4 nuclear protein-binding sites are not necessary or sufficient for TCR alpha enhancer activity. However, a fragment containing these two sites was able to compensate for T alpha 1 and T alpha 2 mutations that otherwise abolished enhancer activity. Electrophoretic mobility shift analyses of the TCR alpha enhancer binding proteins revealed that the T alpha 1, T alpha 3, and T alpha 4 binding proteins are expressed in a variety of T-cell and non-T-cell tumor cell lines. In contrast, one of the two T alpha 2 binding activities was detected only in T-cell nuclear extracts. The activity of the TCR alpha enhancer does not appear to be regulated solely at the level of DNA methylation on that the enhancer sequences were found to be identically hypomethylated in B and T cells as compared with fibroblasts. Taken together, these results suggest that TCR alpha enhancer activity is regulated by the interaction of multiple T-cell-specific and ubiquitous nuclear proteins with partially redundant cis-acting enhancer elements that are hypomethylated in cells of the lymphoid lineage.

scholarly journals Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression.

1992 ◽  
Vol 12 (10) ◽  
pp. 4251-4261 ◽  
Author(s):  
C Kingsley ◽  
A Winoto
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Multigene Family ◽  
Binding Proteins ◽  
Receptor Gene ◽  
Consensus Sequence ◽  
Gene Segment ◽  
Cell Receptor ◽  
Regulatory Elements

Analysis of a T-cell antigen receptor (TCR) alpha promoter from a variable gene segment (V) revealed a critical GT box element which is also found in upstream regions of several V alpha genes, TCR enhancer, and regulatory elements of other genes. This element is necessary for TCR gene expression and binds several proteins. These GT box-binding proteins were identified as members of a novel Sp1 multigene family. Two of them, which we term Sp2 and Sp3, were cloned. Sp2 and Sp3 contain zinc fingers and transactivation domains similar to those of Sp1. Like Sp1, Sp2 and Sp3 are expressed ubiquitously, and their in vitro-translated products bind to the GT box in TCR V alpha promoters. Sp3, in particular, also binds to the Sp1 consensus sequence GC box and has binding activity similar to that of Sp1. As the GT box has also previously been shown to play a role in gene regulation of other genes, these newly isolated Sp2 and Sp3 proteins might regulate expression not only of the TCR gene but of other genes as well.

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.

scholarly journals The Developmentally Regulated Expression of Twisted Gastrulation Reveals a Role for Bone Morphogenetic Proteins in the Control of T Cell Development

2002 ◽  
Vol 196 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Daniel Graf ◽  
Suran Nethisinghe ◽  
Donald B. Palmer ◽  
Amanda G. Fisher ◽  
Matthias Merkenschlager
Keyword(s):  
T Cell ◽  
Bone Morphogenetic Proteins ◽  
Cell Receptor ◽  
Mammalian Development ◽  
Double Positive ◽  
Developmentally Regulated ◽  
Twisted Gastrulation ◽  
Thymocyte Proliferation ◽  
Regulated Expression

The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4−CD8− double-negative (DN) thymocytes and their differentiation to the CD4+CD8+ double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4+ or CD8+ single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.

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 Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by compacting super-enhancers

2021 ◽  
Author(s):  
Delong Feng ◽  
Yanhong Chen ◽  
Ranran Dai ◽  
Shasha Bian ◽  
Wei Xue ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Receptor Gene ◽  
Cell Receptor ◽  
Regulation Mechanism ◽  
Thymocyte Development ◽  
Double Positive ◽  
Enhancer Activity ◽  
Cell Identity ◽  
Single Positive

Abstract CD4+ and CD8+ double-positive (DP) thymocytes are at a crucial stage during the T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRβ. Then DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, Regulatory T cells, or invariant nature kill T cells (iNKT) according to the TCR signal. Chromatin organizer SATB1 is highly expressed in DP cells and plays an essential role in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of DP thymocytes was changed, and the genes specifically highly expressed in DP cells were down-regulated. The super-enhancers regulate the expressions of the DP-specific genes, and the SATB1 deficiency reduced the super-enhancer activity. Hi-C data showed that interactions in super-enhancers and between super-enhancers and promoters decreased in SATB1 deficient thymocytes. We further explored the regulation mechanism of two SATB1-regulating genes, Ets2 and Bcl6, in DP cells and found that the knockout of the super-enhancers of these two genes impaired the development of DP cells. Our research reveals that SATB1 globally regulates super-enhancers of DP cells and promotes the establishment of DP cell identity, which helps understand the role of SATB1 in thymocyte development.

scholarly journals Adjacent, cooperative elements form a strong, constitutive enhancer in the human granulocyte-macrophage colony-stimulating factor gene [published erratum appears in Blood 1996 Oct 1;88(7):2818]

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3694-3703 ◽  
Author(s):  
SD Nimer ◽  
W Zhang ◽  
K Kwan ◽  
Y Whang ◽  
J Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Colony Stimulating Factor ◽  
Enhancer Activity ◽  
Gm Csf ◽  
Constitutive Enhancer ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Both copies of a repeated sequence CATT(A/T), located between bp -53 and -39 in the upstream region of the human GM-CSF gene, are required for mitogen-inducible promoter activity in T lymphocytes. However, the proteins that recognize this region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, and are responsible for its transcriptional regulatory activity, have not been clearly identified. Using transient transfection assays, we demonstrate that a 19-bp oligonucleotide containing the CATT(A/T) repeats has strong constitutive enhancer activity in both T cell and non-T-cell lines, even though GM-CSF is not normally constitutively expressed by these cells. A 12-bp oligonucleotide, containing only the sequence CATTAATCATTT, lacks enhancer activity indicating that the nucleotides surrounding these sequences are critical for this enhancer activity. The sequence TTTCCT, which can bind members of the ets family of transcription factors, is located just 3′ of these CATT(A/T) repeats, and mutagenesis of the CCT sequence abolishes (1) the constitutive (and mitogen inducible) enhancer activity of the 19-bp GM-CSF sequences, (2) the responsiveness to transactivation by ets-1, and (3) the ability to specifically bind ets-1 and elf-1 in electrophoretic mobility shift assays (EMSA). We demonstrate that although T cells contain nuclear proteins capable of independently recognizing the ets binding site and the CATT(A/T) repeats in EMSAs, both of these regulatory elements are required for enhancer function. The strong constitutive activity of this 19-bp region suggests that negative regulation of the GM-CSF promoter is critical for the restricted expression pattern of GM-CSF mRNA.

scholarly journals Developmentally regulated fetal thymic and extrathymic T-cell receptor gamma delta gene expression.

1990 ◽  
Vol 4 (8) ◽  
pp. 1304-1315 ◽  
Author(s):  
S R Carding ◽  
S Kyes ◽  
E J Jenkinson ◽  
R Kingston ◽  
K Bottomly ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Gamma Delta ◽  
Developmentally Regulated ◽  
Delta Gene

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins

1990 ◽  
Vol 10 (10) ◽  
pp. 5027-5035
Author(s):  
J Takeda ◽  
A Cheng ◽  
F Mauxion ◽  
C A Nelson ◽  
R D Newberry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Lymphoid Cells ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Beta Enhancer

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.

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