scholarly journals Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4+CD8+ thymocytes

2018 ◽  
Vol 216 (1) ◽  
pp. 231-243 ◽  
Author(s):  
Abani Kanta Naik ◽  
Aaron T. Byrd ◽  
Aaron C.K. Lucander ◽  
Michael S. Krangel
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Promoter Activation ◽  
Active Chromatin ◽  
Gene Assembly ◽  
Down Regulation ◽  
Hierarchical Assembly ◽  
Rag2 Gene

Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE–Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.

Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4529-4538 ◽  
Author(s):  
Steve N. Georas ◽  
John E. Cumberland ◽  
Thomas F. Burke ◽  
Rongbing Chen ◽  
Ulrike Schindler ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Regulatory Elements ◽  
Jurkat Cells ◽  
Proximal Promoter ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
Th Cells

Abstract The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4–activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

Effect of Thalidomide and Revlimid on the Gene Expression That Reveals Molecular Circuitries Involved in T Cell Co-Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2472-2472
Author(s):  
Rao Prabhala ◽  
Puneet Cheema ◽  
Masood A. Shammas ◽  
Weihua Song ◽  
Elizabeth Blanchard ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Fold Increase ◽  
Down Regulation ◽  
Activation Pathway ◽  
Molecular Events ◽  
Genome Wide ◽  
Immune Mediated ◽  
Phase Ii And Iii

Abstract Thalidomide (Thal) and its immunomodulatory analog, Revlimid (Rev) have demonstrated anti-myeloma activity in phase II and III studies and part of their activity is immune-mediated. We have previously demonstrated co-stimulatory effects of Thal and Rev on T cells mediated via B7-CD28 activation pathway. To understand the molecular events involved in T cell co-stimulation, we investigated the effects of Thal and Rev on the genome-wide gene expression profile of T cells with or without anti-CD3 activation. Normal PBMC were incubated with Thal or Rev in the presence or absence of anti-CD3 antibodies (200ng/ml) for 3 or 24 hours at 37oC and total RNA was analyzed using U133 GeneChip (Affymetrix). Although Thal and Rev do not show significant effect on T cell proliferation without CD3 stimulation, we observed up-regulation (>2-folds) of 56 genes and down-regulation of 42 genes at 3 hours, and up-regulation of 41genes and down-regulation of 35 genes at 24 hours with Rev. Significant up-regulation of IFN-α (10-folds) was observed at 3 hours. Up-regulation of chemokine CXC5 (9 fold); IFN-β (5 fold), IL-7 (5 fold) and IL-2 (3 fold); and signaling genes like JK 3 (10 folds) and MAPKK5 (5 folds) was observed at 24 hours with Rev. Following anti-CD3 activation, Rev induced significant upregulation of 59 and 48 genes and down-regulation of 126 and 61 genes at 3 and 24 hours respectively. These include down-regulation of Th2 cytokine genes IL-5(5 folds) and TGF-β (2 folds), and apoptotsis related genes, TNFSF12 (4 folds) and TNFRSF 6b (4 folds). Genes involved in B7-CD28 activation pathway and up-regulated include CXC 5 (9 folds), IL-8 (5 folds) and IL-3 (4 folds). Interestingly, p52, which is a key molecule in CD28 signaling, was elevated 18 folds. Additionally less than 2 fold increase was observed in other CD28 signaling genes like IKK, Vov, and RelA (p65). Rev also down-regulated, at 24 hours, the expression of IL-10 (6 folds), IL-17 (3 folds) and TGF-β (3 folds) which allows sustained co-stimulation. Similar changes with lesser magnitude were observed with Thal. These results provide insight into early molecular changes induced by Thal and Rev that is important for co-stimulation and suggest new molecular targets to generate effective immune responses. Moreover, these observations facilitate the pre-clinical rationale for the use of these immunomodulatory compounds to improve clinical outcome in myeloma.

Regulation of 4F2 heavy-chain gene expression during normal human T-cell activation can be mediated by multiple distinct molecular mechanisms

1988 ◽  
Vol 8 (9) ◽  
pp. 3820-3826
Author(s):  
T Lindsten ◽  
C H June ◽  
C B Thompson ◽  
J M Leiden
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Steady State ◽  
T Cell Activation ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Transcription Elongation ◽  
Fold Increase ◽  
Mrna Levels ◽  
Myristate Acetate

The 4F2 molecule belongs to the set of cell surface antigens which is induced following lectin- or antigen-mediated T-cell activation. The increase in 4F2 cell surface expression following lectin-mediated stimulation has been shown to be accompanied by a parallel increase in the steady-state levels of 4F2 heavy-chain (4F2HC) mRNA. The studies described in this report were designed to further elucidate the molecular mechanisms responsible for induction of 4F2HC gene expression following activation of normal resting human peripheral blood T cells. The low levels of mature 4F2HC mRNA in resting T cells were shown to be the result of a block to transcription elongation within the exon 1-intron 1 region of the 4F2HC gene rather than promoter inactivity. Phorbol myristate acetate stimulation of resting T cells resulted in a 20-fold increase in steady-state 4F2HC mRNA levels which was mediated by removal of this block to transcription elongation. The phorbol myristate acetate-induced increase in 4F2HC gene expression is distinct from previously described AP-1-mediated, phorbol ester-induced gene expression in that it requires new protein synthesis. Treatment of resting T cells with ionomycin plus PMA resulted in a 60-fold increase in 4F2HC mRNA levels. This induction was mediated by both an increase in promoter utilization and removal of the block to transcription elongation. Finally, by increasing the half-life of 4F2HC mRNA, cycloheximide treatment of resting T cells induced an approximately five fold increase in the levels of 4F2HC gene expression, although the physiologic significance of this mechanism remains unclear. These results demonstrate that the level of 4F2HC gene expression in normal peripheral blood T cells can be regulated by at least three distinct molecular pathways: (i) changes in promoter utilization, (ii) modulation of a block to transcription elongation, and (iii) alteration in mRNA stability.

Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4529-4538 ◽  
Author(s):  
Steve N. Georas ◽  
John E. Cumberland ◽  
Thomas F. Burke ◽  
Rongbing Chen ◽  
Ulrike Schindler ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Regulatory Elements ◽  
Jurkat Cells ◽  
Proximal Promoter ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
Th Cells

The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4–activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

scholarly journals Vascular Endothelial Growth Factor Activates Nuclear Factor of Activated T Cells in Human Endothelial Cells: a Role for Tissue Factor Gene Expression

1999 ◽  
Vol 19 (3) ◽  
pp. 2032-2043 ◽  
Author(s):  
Angel Luis Armesilla ◽  
Elisa Lorenzo ◽  
Pablo Gómez del Arco ◽  
Sara Martínez-Martínez ◽  
Arantzazu Alfranca ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vascular Endothelial Growth Factor ◽  
T Cells ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Vascular Endothelial ◽  
Nuclear Factor ◽  
Promoter Activation ◽  
Activated T Cells ◽  
Endothelial Growth Factor

ABSTRACT Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified κB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-κB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-κB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression.

scholarly journals Down-regulation of RAG1 and RAG2 gene expression in PreB cells after functional immunoglobulin heavy chain rearrangement

Immunity ◽  
1995 ◽  
Vol 3 (5) ◽  
pp. 601-608 ◽  
Author(s):  
Ulf Grawunder ◽  
Thomas M.J. Leu ◽  
David G. Schatz ◽  
Annick Werner ◽  
Antonius G. Rolink ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Down Regulation ◽  
Rag2 Gene

scholarly journals Follicular Lymphoma Cells Induce Changes in T-Cell Gene Expression and Function: Potential Impact on Survival and Risk of Transformation

2013 ◽  
Vol 31 (21) ◽  
pp. 2654-2661 ◽  
Author(s):  
Shahryar Kiaii ◽  
Andrew J. Clear ◽  
Alan G. Ramsay ◽  
Derek Davies ◽  
Ajanthah Sangaralingam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Overall Survival ◽  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
Peripheral Blood ◽  
Molecular Mechanisms ◽  
Prognostic Significance ◽  
Time Lapse ◽  
Immune Microenvironment

Purpose Previous studies have demonstrated the prognostic importance of the immune microenvironment in follicular lymphoma (FL). To investigate the molecular mechanisms during which tumor-infiltrating T cells (TILs) are altered in the FL microenvironment, we studied highly purified CD4 and CD8 TILs from lymph node biopsies at diagnosis in treatment-naive patients with FL compared with reactive tonsils and the peripheral blood of healthy donors. Patients and Methods Gene expression profiling of highly purified CD4 and CD8 TILs was performed on the Affymetrix platform. Diagnostic tissue microarrays from an independent patient set (n = 172) were used to verify protein expression and analyze any impact of TIL-expressed genes on outcome. Time-lapse imaging was used to assess T-cell motility. Results The most upregulated genes in both CD4 and CD8 TILs were PMCH, ETV1, and TNFRSF9. PMCH is not expressed in peripheral blood T cells, but expression is highly induced on culture with FL. Both CD4 and CD8 TILs from patients with FL have significantly impaired motility compared with those of healthy TILs from reactive tonsils and this can be induced on healthy T cells by FL cells. During multivariate analysis, a model incorporating the number and location of T cells expressing PMCH, NAMPT, and ETV1 showed prognostic significance for overall survival and for time to transformation. Conclusion We showed altered gene expression in TILs in FL and demonstrated that altering the immune microenvironment in FL affects overall survival and time to transformation in this disease.

scholarly journals Regulation of 4F2 heavy-chain gene expression during normal human T-cell activation can be mediated by multiple distinct molecular mechanisms.

1988 ◽  
Vol 8 (9) ◽  
pp. 3820-3826 ◽  
Author(s):  
T Lindsten ◽  
C H June ◽  
C B Thompson ◽  
J M Leiden
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Steady State ◽  
T Cell Activation ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Transcription Elongation ◽  
Fold Increase ◽  
Mrna Levels ◽  
Myristate Acetate

The 4F2 molecule belongs to the set of cell surface antigens which is induced following lectin- or antigen-mediated T-cell activation. The increase in 4F2 cell surface expression following lectin-mediated stimulation has been shown to be accompanied by a parallel increase in the steady-state levels of 4F2 heavy-chain (4F2HC) mRNA. The studies described in this report were designed to further elucidate the molecular mechanisms responsible for induction of 4F2HC gene expression following activation of normal resting human peripheral blood T cells. The low levels of mature 4F2HC mRNA in resting T cells were shown to be the result of a block to transcription elongation within the exon 1-intron 1 region of the 4F2HC gene rather than promoter inactivity. Phorbol myristate acetate stimulation of resting T cells resulted in a 20-fold increase in steady-state 4F2HC mRNA levels which was mediated by removal of this block to transcription elongation. The phorbol myristate acetate-induced increase in 4F2HC gene expression is distinct from previously described AP-1-mediated, phorbol ester-induced gene expression in that it requires new protein synthesis. Treatment of resting T cells with ionomycin plus PMA resulted in a 60-fold increase in 4F2HC mRNA levels. This induction was mediated by both an increase in promoter utilization and removal of the block to transcription elongation. Finally, by increasing the half-life of 4F2HC mRNA, cycloheximide treatment of resting T cells induced an approximately five fold increase in the levels of 4F2HC gene expression, although the physiologic significance of this mechanism remains unclear. These results demonstrate that the level of 4F2HC gene expression in normal peripheral blood T cells can be regulated by at least three distinct molecular pathways: (i) changes in promoter utilization, (ii) modulation of a block to transcription elongation, and (iii) alteration in mRNA stability.

scholarly journals Interleukin (IL)-4 inhibits IL-10 to promote IL-12 production by dendritic cells

2005 ◽  
Vol 201 (12) ◽  
pp. 1899-1903 ◽  
Author(s):  
Yongxue Yao ◽  
Wei Li ◽  
Mark H. Kaplan ◽  
Cheong-Hee Chang
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Dendritic Cells ◽  
Cell Differentiation ◽  
B Cells ◽  
Histone Acetylation ◽  
Cell Lineage ◽  
Down Regulation ◽  
Th1 Cell ◽  
Th2 Cell

Interleukin (IL)-4 is known to be the most potent cytokine that can initiate Th2 cell differentiation. Paradoxically, IL-4 instructs dendritic cells (DCs) to promote Th1 cell differentiation. We investigated the mechanisms by which IL-4 directs CD4 T cells toward the Th1 cell lineage. Our study demonstrates that the IL-4–mediated induction of Th1 cell differentiation requires IL-10 production by DCs. IL-4 treatment of DCs in the presence of lipopolysaccharide or CpG resulted in decreased production of IL-10, which was accompanied by enhanced IL-12 production. In IL-10–deficient DCs, the level of IL-12 was greatly elevated and, more importantly, the ability of IL-4 to up-regulate IL-12 was abrogated. Interestingly, IL-4 inhibited IL-10 production by DCs but not by B cells. The down-regulation of IL-10 gene expression by IL-4 depended on Stat6 and was at least partly caused by decreased histone acetylation of the IL-10 promoter. These data indicate that IL-4 plays a key role in inducing Th1 cell differentiation by instructing DCs to produce less IL-10.

NK Cells From CLL Patients Exhibit Down-Regulation Of Interferon Response Genes That Can Be Reversed With Lenalidomide

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4131-4131
Author(s):  
John C. Riches ◽  
Ajanthah Sangaralingam ◽  
Tracy Chaplin ◽  
Fabienne McClanahan ◽  
Sameena Iqbal ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Interferon Response ◽  
Down Regulation ◽  
Inducible Genes ◽  
Increased Susceptibility

Abstract Chronic lymphocytic leukemia (CLL) is associated with profound defects in immune function, resulting in failure of anti-tumor immunity and increased susceptibility to infection. We have previously demonstrated alterations in gene expression profiles of T cells from CLL patients, which translate into functional defects in T-cell immune synapse formation, motility and cytotoxicity (Gorgun et al. JCI 2005; Ramsay et al. JCI 2008, Blood 2013). However a comparison of the transcriptome of natural killer (NK) cells from CLL patients and controls has not been investigated. NK cells were isolated from the peripheral blood of patients with CLL and healthy donors, followed by gene expression profiling using the Affymetrix U133Plus2.0 platform. 117 probes showed a >2-fold decrease in expression while only 18 probes showed a >2-fold increase in expression (adjusted p-value < 0.05) in CLL NK cells compared to healthy donor NK cells. Strikingly, 52 out of the 117 significantly down-regulated probes (44.4%) were for interferon-inducible genes including STAT1 (Signal Transducers and Activator 1), SOCS1 (Suppressor of cytokine signaling 1), interferon regulatory factor genes IRF7 and IRF9, and oligoadenylate synthetase genes OAS1, OAS2, and OAS3. The majority of these genes were inducible by both type 1 and type 2 interferons. Many of these genes have been implicated in host immunity to viral infections, and so it is possible that decreased NK-cell responsiveness to interferon contributes to the increased susceptibility of CLL patients to viruses. Notably, there was also altered expression of signaling pathways in common with T cells from CLL patients, with dysregulation of the cytoskeleton genes RAB3GAP1, RAB38, and EPHA1 and down-regulation of JUN mirroring the dysregulated JNK-signaling and the altered actin cytoskeleton pathways we have found in T cells from CLL patients. These changes were not due to differences in the relative frequencies of CD56DIM and CD56BRIGHTNK cells. Lenalidomide has significant clinical activity in CLL. It is not directly toxic to tumor cells in vitro, but instead is thought to activate anti-tumor immunity and block pro-tumor micro-environmental factors. NK-cell proliferation has been shown to correlate with clinical response to lenalidomide in CLL (Chanan Khan et al. BJ Haem 2011). Therefore, we investigated the effect of lenalidomide treatment on the gene expression profiles of NK cells from CLL patients in comparison to healthy controls. PBMCs from CLL patients or healthy controls were cultured in the presence of 1μM lenalidomide or vehicle control for 48 hours, followed by NK-cell isolation, RNA extraction and gene expression profiling. There were striking differences in the effect of lenalidomide on NK cells from CLL patients compared with healthy NK cells. In CLL NK cells, lenalidomide repaired the down-regulation of interferon-inducible genes, by increasing the expression of genes such as OAS3, IFIT1, IFI44L, IFIT3, OAS1, PDK4, and ACTN1. Pathway analysis highlighted the effect of lenalidomide on inducing interferon signaling, showing significant activation of interferon α, γ, and λ as upstream regulators. While many of the interferon-inducible genes were up-regulated >3-fold in CLL NK cells, only OAS3 was significantly up-regulated in healthy NK cells with lenalidomide. Furthermore, the gene for IFNγ, IFNG, was actually significantly down-regulated in healthy NK cells by this agent. Lenalidomide also significantly down-regulated the expression of 5 genes encoding killer-cell immunoglobulin-like receptors (KIRs): KIR2DL1, KIR2DL2, KIR2DS3, KIR2DS4, and KIR3DL2, in healthy NK cells, but did not significantly down-regulate KIR genes in the CLL NK-cell dataset. Lenalidomide treatment did have some overlapping effects on CLL and healthy NK cells, including up-regulation of genes ARL11, CYFIP, and CORO1B that regulate the actin cytoskeleton pathway. In conclusion, NK cells from CLL patients have down-regulation of interferon response genes and pathways known to regulate normal immune function in response to bacteria and viruses. Lenalidomide has a differential effect on CLL and healthy NK cells: in CLL NK cells it repairs defective interferon responses, whereas in healthy NK cells it down-regulates inhibitory pathways. Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

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