The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells

2020 ◽  
Vol 5 (51) ◽  
pp. eabb1455 ◽  
Author(s):  
Kazuko Miyazaki ◽  
Hitomi Watanabe ◽  
Genki Yoshikawa ◽  
Kenian Chen ◽  
Reiko Hidaka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
T Cell ◽  
B Cells ◽  
Cell Lineage ◽  
Cell Receptor ◽  
Regulatory Elements ◽  
Mouse Strains ◽  
Specific Gene ◽  
T And B Cells

Cell type–specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage–specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage–specific enhancer (R-TEn) and the two B cell–specific elements, R1B and R2B. By generating mice lacking either R-TEn or R1B and R2B, we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn, we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules.

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 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.

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.

scholarly journals Identifying Transcription Regulatory Elements in the Human and Mouse Genomes Using Tissue-specific Gene Expression Profiles

2007 ◽  
Vol 4 (2) ◽  
pp. 1-23
Author(s):  
Amitava Karmaker ◽  
Kihoon Yoon ◽  
Mark Doderer ◽  
Russell Kruzelock ◽  
Stephen Kwek
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Tissue Specific Gene ◽  
Human And Mouse

Summary Revealing the complex interaction between trans- and cis-regulatory elements and identifying these potential binding sites are fundamental problems in understanding gene expression. The progresses in ChIP-chip technology facilitate identifying DNA sequences that are recognized by a specific transcription factor. However, protein-DNA binding is a necessary, but not sufficient, condition for transcription regulation. We need to demonstrate that their gene expression levels are correlated to further confirm regulatory relationship. Here, instead of using a linear correlation coefficient, we used a non-linear function that seems to better capture possible regulatory relationships. By analyzing tissue-specific gene expression profiles of human and mouse, we delineate a list of pairs of transcription factor and gene with highly correlated expression levels, which may have regulatory relationships. Using two closely-related species (human and mouse), we perform comparative genome analysis to cross-validate the quality of our prediction. Our findings are confirmed by matching publicly available TFBS databases (like TRANFAC and ConSite) and by reviewing biological literature. For example, according to our analysis, 80% and 85.71% of the targets genes associated with E2F5 and RELB transcription factors have the corresponding known binding sites. We also substantiated our results on some oncogenes with the biomedical literature. Moreover, we performed further analysis on them and found that BCR and DEK may be regulated by some common transcription factors. Similar results for BTG1, FCGR2B and LCK genes were also reported.

scholarly journals Exploring and analysing single cell multi-omics data with VDJView

2019 ◽  
Author(s):  
Jerome Samir ◽  
Simone Rizzetto ◽  
Money Gupta ◽  
Fabio Luciani
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
B Cells ◽  
Hypothesis Testing ◽  
Single Cell ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Specific Gene ◽  
T And B Cells ◽  
Gene Signatures

Abstract Background Single cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information.Results We developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8+ T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians.Conclusions VDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview .

scholarly journals Exploring and analysing single cell multi-omics data with VDJView

2020 ◽  
Author(s):  
Jerome Samir ◽  
Simone Rizzetto ◽  
Money Gupta ◽  
Fabio Luciani
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
B Cells ◽  
Hypothesis Testing ◽  
Single Cell ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Specific Gene ◽  
T And B Cells ◽  
Gene Signatures

Abstract Background Single cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information.Results We developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8+ T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians.Conclusions VDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview .

scholarly journals Expression and Function of Tec, Itk, and Btk in Lymphocytes: Evidence for a Unique Role for Tec

2004 ◽  
Vol 24 (6) ◽  
pp. 2455-2466 ◽  
Author(s):  
Michael G. Tomlinson ◽  
Lawrence P. Kane ◽  
Jennifer Su ◽  
Theresa A. Kadlecek ◽  
Marianne N. Mollenauer ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Th2 Cells ◽  
Receptor Signaling ◽  
T And B Cells ◽  
Nfat Activation ◽  
Cell Receptor Signaling

ABSTRACT The Tec protein tyrosine kinase is the founding member of a family that includes Btk, Itk, Bmx, and Txk. Btk is essential for B-cell receptor signaling, because mutations in Btk are responsible for X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice, whereas Itk is involved in T-cell receptor signaling. Tec is expressed in both T and B cells, but its role in antigen receptor signaling is not clear. In this study, we show that Tec protein is expressed at substantially lower levels in primary T and B cells relative to Itk and Btk, respectively. However, Tec is up-regulated upon T-cell activation and in Th1 and Th2 cells. In functional experiments that mimic Tec up-regulation, we find that Tec overexpression in lymphocyte cell lines is sufficient to induce phospholipase Cγ (PLC-γ) phosphorylation and NFAT (nuclear factor of activated T cells) activation. In contrast, overexpression of Btk, Itk, or Bmx does not induce NFAT activation. Tec-induced NFAT activation requires PLC-γ, but not the adapters LAT, SLP-76, and BLNK, which are required for Btk and Itk to couple to PLC-γ. Finally, we show that the unique effector function for Tec correlates with a unique subcellular localization. We hypothesize that Tec functions in activated and effector T lymphocytes to induce the expression of genes regulated by NFAT transcription factors.

scholarly journals WASp-deficient B cells play a critical, cell-intrinsic role in triggering autoimmunity

2011 ◽  
Vol 208 (10) ◽  
pp. 2033-2042 ◽  
Author(s):  
Shirly Becker-Herman ◽  
Almut Meyer-Bahlburg ◽  
Marc A. Schwartz ◽  
Shaun W. Jackson ◽  
Kelly L. Hudkins ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Cell Lineage ◽  
Cell Receptor ◽  
Mixed Chimerism ◽  
Chimeric Mice ◽  
Autoantibody Production

Patients with the immunodeficiency Wiskott-Aldrich syndrome (WAS) frequently develop systemic autoimmunity. Here, we demonstrate that mutation of the WAS gene results in B cells that are hyperresponsive to B cell receptor and Toll-like receptor (TLR) signals in vitro, thereby promoting a B cell–intrinsic break in tolerance. Whereas this defect leads to autoantibody production in WAS protein–deficient (WASp−/−) mice without overt disease, chimeric mice in which only the B cell lineage lacks WASp exhibit severe autoimmunity characterized by spontaneous germinal center formation, class-switched autoantibodies, renal histopathology, and early mortality. Both T cell help and B cell–intrinsic TLR engagement play important roles in promoting disease in this model, as depletion with anti-CD4 antibodies or generation of chimeric mice with B cells deficient in both WASp and MyD88 prevented development of autoimmune disease. These data highlight the potentially harmful role for cell-intrinsic loss of B cell tolerance in the setting of normal T cell function, and may explain why WAS patients with mixed chimerism after stem cell transplantation often develop severe humoral autoimmunity.

An Endothelial Cell-Specific Regulatory Mutation in Galgt2 Is the Major Cause of Prolonged aPTT in Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 253-253 ◽  
Author(s):  
Jill Johnsen ◽  
Gallia Levy ◽  
David Ginsburg
Keyword(s):  
Gene Expression ◽  
Endothelial Cell ◽  
Haplotype Block ◽  
Transcriptional Start Site ◽  
Sequence Divergence ◽  
Regulatory Elements ◽  
Mouse Strains ◽  
Specific Gene ◽  
Regulatory Mutation ◽  
Expression Program

Abstract The regulation of endothelial cell gene expression plays a central role in maintaining hemostatic balance. However, little is known about the key sequence elements responsible for the control of the endothelial gene expression program. We previously identified the cause of low levels of von Willebrand factor (VWF) in the inbred mouse strain RIIIS/J as a regulatory mutation in the gene encoding an N-acetylgalactosaminyltransferase, GALGT2. This low VWF allele, which we termed Mvwf1, results from a tissue-specific switch in expression program from intestinal epithelium to vascular endothelium. The ectopic expression of Galgt2 in vascular endothelial cells results in aberrant post-translational modification of VWF, leading to accelerated clearance. The specific regulatory DNA sequences responsible for this remarkable lineage-specific transcriptional switch were mapped genetically to an ~300kb region surrounding the Galgt2 gene. To further define the critical regulatory sequences, we have generated transgenic mice using three overlapping C57BL/6J bacterial artificial chromosomes (BACs) encompassing the Galgt2 gene. All three transgenes directed wild-type (C57BL/6J) GI epithelial expression of Galgt2, mapping the GI specific regulatory elements to an 84kb interval shared by these three BACs. Direct sequencing of RIIIS/J over >80kb flanking the transcriptional start site identified a region of 2–3% sequence divergence from C57BL/6J beginning ~25kb upstream of exon 1 and extending ~10kb into intron 1. This region contains several insertions and deletions ranging from 120bp to 6.4kb. We are currently testing chimeric RIIIS/J:C57BL/6J BACs to fine map the critical regulatory elements responsible for the switch. PCR genotyping of 49 mouse strains identified a total of 9 strains that have RIIIS/J polymorphisms within the region of high sequence divergence. Extensive sequencing confirmed a common haplotype block of at least 55kb in length that is shared by all 9 Mvwf1 strains that also exhibit the GI epithelial to endothelial switch in Galgt2 expression. These data identify an ancient founder allele present in a number of mouse strains and retained at a low level within the wild mouse population. In a survey of the Jackson Phenome Database, Mvwf1 strains accounted for 5 of the 6 highest aPTTs, establishing this single allele as the most common cause of prolonged aPTTs in laboratory mice. The prevalence of this allele also suggests that it has been maintained through positive selective pressure. In summary, we have identified a common allele leading to low VWF levels in the mouse. We have characterized a haplotype block shared by all Mvwf1 mice that begins at least 35kb upstream of the Galgt2 transcriptional start site and overlaps the 84kb interval that confers wild-type Galgt2 expression. Continued analysis of these sequences should provide important new insights into endothelial cell-specific gene expression and the more general problem of tissue specific gene regulation.

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