Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA137455 | ENA |
REPOSITORIES: ENA
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Project description:The present study reports an unbiased analysis of the cytotoxic T cell serine-threonine phosphoproteome using high resolution mass spectrometry. Approximately 2,000 phosphorylations were identified in CTLs of which approximately 450 were controlled by TCR signaling. A significantly overrepresented group of molecules identified in the phosphoproteomic screen were transcription activators, co-repressors and chromatin regulators. A focus on the chromatin regulators revealed that CTLs have high expression of the histone deacetylase HDAC7 but continually phosphorylate and export this transcriptional repressor from the nucleus. HDAC7 dephosphorylation results in its nuclear accumulation and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. The screening of the CTL phosphoproteome thus reveals intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators in CTLs and determine the CTL functional program. We used Affymetrix microarray analysis to explore the molecular basis for the role of HDAC7 in CTLs and the impact of GFP-HDAC7 phosphorylation deficient mutant expression on the CTL transcriptional profile. In vitro generated P14 TCR cytotoxic T cells were retrovirally infected with a construct encoding GFP-HDAC7 phosphorylation deficient mutant, sorted in base of GFP expression (GFP positive and GFP negative) and processed for microarray analysis in three biological replicas.
2011-03-13 | E-GEOD-27092 | biostudies-arrayexpress
Project description:The present study reports an unbiased analysis of the cytotoxic T cell serine-threonine phosphoproteome using high resolution mass spectrometry. Approximately 2,000 phosphorylations were identified in CTLs of which approximately 450 were controlled by TCR signaling. A significantly overrepresented group of molecules identified in the phosphoproteomic screen were transcription activators, co-repressors and chromatin regulators. A focus on the chromatin regulators revealed that CTLs have high expression of the histone deacetylase HDAC7 but continually phosphorylate and export this transcriptional repressor from the nucleus. HDAC7 dephosphorylation results in its nuclear accumulation and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. The screening of the CTL phosphoproteome thus reveals intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators in CTLs and determine the CTL functional program. We used Affymetrix microarray analysis to explore the molecular basis for the role of HDAC7 in CTLs and the impact of GFP-HDAC7 phosphorylation deficient mutant expression on the CTL transcriptional profile.
2011-03-13 | GSE27092 | GEO
Project description:Title of Publication: Histone Deacetylase 7 Regulates Cell Survival and TCR Signaling in CD4/CD8 Double-Positive Thymocytes Abstract of publicaton: CD4/CD8 double-positive (DP) thymocytes express the transcriptional repressor Histone Deacetylase 7 (HDAC7), a class IIa HDAC that is exported from the cell nucleus after T cell receptor (TCR) engagement. Through signal-dependent nuclear export, class IIa HDACs such as HDAC7 mediate signal-dependent changes in gene expression that are important to developmental fate decisions in multiple tissues. We report that HDAC7 is exported from the cell nucleus during positive selection in thymocytes, and regulates genes mediating the coupling between TCR engagement and downstream events that determine cell survival. Thymocytes lacking HDAC7 are inefficiently positively selected due to a severely shortened lifespan and exhibit a truncated repertoire of TCR J segments. The expression of multiple important mediators and modulators of the response to TCR engagement is altered in HDAC7-deficient thymocytes, resulting in increased tonic MAP kinase activity that contributes to the observed loss of viability. Remarkably, the activity of Protein Kinase D, the kinase that mediates nuclear export of HDAC7 in response to TCR signaling, is also increased in HDAC7-deficient thymocytes, suggesting that HDAC7 nuclear export governs a self-sustaining auto-excitatory loop. These experiments add to the understanding of the life/death decision in thymic T cell development, define a novel function for class IIa HDACs, and point to a novel feed-forward mechanism whereby these molecules regulate their own state and mediate stable developmental transitions. Goal of Microarray experiment: We did these experiments to determine how alteration of the function of HDAC7, a site-specific and signal-dependent repressor of transcription, changes gene expression in CD4/CD8 DP thymocytes. Three biological replicate samples were prepared for each non-wild type mouse strain (Samples 7-18). Six biological replicates were prepared for the wild type strain (Samples 1-6). Total RNA was prepared from isolated CD4/CD8 Double-positive thymocytes and labeled with the Affymetrix Whole-Transcript labeling protocol. Labeled probes were hybridized to one Affymetrix Mouse Gene 1.0ST array each. Data from .cel files were normalized using RMA, in normalization groups representing each of the binary comparisons made. These binary comparisons between sample groups represent gene expression changes due to loss of HDAC7 (samples 1-3 vs. 7-9), transgenic expression of an HDAC7-VP16 fusion protein (samples 4-6 vs. 10-12), positive thymic selection (samples 1-6 vs. samples 11-15), and negative thymic selection (Samples 11-15 vs. samples 16-18).
2011-04-06 | E-GEOD-26488 | biostudies-arrayexpress
Project description:Abstract of publicaton: CD4/CD8 double-positive (DP) thymocytes express the transcriptional repressor Histone Deacetylase 7 (HDAC7), a class IIa HDAC that is exported from the cell nucleus after T cell receptor (TCR) engagement. Through signal-dependent nuclear export, class IIa HDACs such as HDAC7 mediate signal-dependent changes in gene expression that are important to developmental fate decisions in multiple tissues. We report that HDAC7 is exported from the cell nucleus during positive selection in thymocytes, and regulates genes mediating the coupling between TCR engagement and downstream events that determine cell survival. Thymocytes lacking HDAC7 are inefficiently positively selected due to a severely shortened lifespan and exhibit a truncated repertoire of TCR Jalpha segments. The expression of multiple important mediators and modulators of the response to TCR engagement is altered in HDAC7-deficient thymocytes, resulting in increased tonic MAP kinase activity that contributes to the observed loss of viability. Remarkably, the activity of Protein Kinase D, the kinase that mediates nuclear export of HDAC7 in response to TCR signaling, is also increased in HDAC7-deficient thymocytes, suggesting that HDAC7 nuclear export governs a self-sustaining auto-excitatory loop. These experiments add to the understanding of the life/death decision in thymic T cell development, define a novel function for class IIa HDACs, and point to a novel feed-forward mechanism whereby these molecules regulate their own state and mediate stable developmental transitions. Title of manuscript: Nuclear Export of Histone Deacetylase 7 During Thymic Selection Mediates Immune Self-tolerance. abstract of manuscript: Histone Deacetylase 7 (HDAC7) is a TCR signal-dependent regulator of differentiation that is highly expressed in CD4/CD8 double-positive (DP) thymocytes. Here we examine the effect of blocking TCR-dependent nuclear export of HDAC7 during thymic selection, through expression of a signal-resistant mutant of HDAC7 (HDAC7-?P) in thymocytes. We find that HDAC7-?P Transgenic thymocytes exhibit a profound block in negative thymic selection, but can still undergo positive selection, resulting in the escape of autoreactive T cells into the periphery. Gene expression profiling reveals a comprehensive suppression of the negative selection-associated gene expression program in DP thymocytes, associated with a defect in the activation of MAP kinase pathways by TCR signals. The consequence of this block in vivo is a lethal autoimmune syndrome involving the exocrine pancreas and other abdominal organs. These experiments establish a novel molecular model of autoimmunity and cast new light on the relationship between thymic selection and immune self-tolerance. Goal of Microarray experiment: We did these experiments to determine how alteration of the function of HDAC7, a site-specific and signal-dependent repressor of transcription, changes gene expression in CD4/CD8 DP thymocytes.
2011-04-06 | GSE26488 | GEO
Project description:Comparison of transcriptional profile of TCR stimulated P14-TCR wild-type and P14-PKD2 null murine lymph node cells
2012-02-29 | GSE33942 | GEO
Project description:The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identified 642 RBPs in the thymus and focused on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 was downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation required Stim1/Stim2 and CaMK4 expression and involved Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly bound RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome revealed strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes showed reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype was recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.
2023-03-31 | MSV000091606 | MassIVE
Project description:The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labelling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3,505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription and translation. In other cell types PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.
2014-10-03 | PXD001076 | Pride
Project description:Comparison of transcriptional profile of TCR stimulated P14-TCR wild-type and P14-PKD2 null murine lymph node cells Lymph node cells from 3 biological replicates (3 wild-type and 3 PKD2 null mice) were isolated and left unstimulated or stimulated for 4 hours with antigenic peptide (gp33-41) prior to RNA extraction and hybridization to Affymetrix microarray.
2012-02-29 | E-GEOD-33942 | biostudies-arrayexpress
Project description:Lysosome-related organelles have versatile functions including protein and lipid degradation, signal transduction, and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal/lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system.. Here, we describe a novel human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated-protein-kinase (MAPK) signaling to late endosomes, is critical for the function of neutrophils, B-cells, cytotoxic T-cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3â UTR of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a novel role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity. Experiment Overall Design: EBV-transformed B cell lines from 2 parents and 2 affected children
2008-06-14 | E-GEOD-6322 | biostudies-arrayexpress
Project description:Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how hypoxia configures the proteome of CD8+ cytotoxic T lymphocytes (CTLs). We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8+ T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts. For this project, P14 cytotoxic T cells (CTLs) were generated from T cells from the spleens of mice. CD8+ T cells were activated for 2 days with the P14-T cell receptor (TCR) cognate ligand (peptide gp33-41 from Lymphocytic Choriomeningitis Virus, LCMV) in the presence of 20 ng/ml IL 2 (Proleukin) and 2 ng/ml IL-12 (Peprotech). Cells were grown in a humidified incubator with a gas atmosphere of 18% oxygen and 5% CO2 and a temperature of 37�C. After 48 hours, the activated CD8+ T cells were removed from TCR stimulation and were cultured in RPMI 1640, with 10% foetal bovine serum (FBS), supplemented with 100 units/ml penicillin-G, 100 �g/ml streptomycin, 50 �M-mercaptoethanol and 20 ng/ml IL-2 (Proleukin). Each day, the T cells were counted and split to 500,000 cells per ml with fresh IL-2 being added to a final concentration of 20 ng/ml. CTLs were differentiated in IL-2 for 4 days, before being counted and re-suspended at a concentration of 300,000 cells per ml in RPMI with 10% foetal bovine serum, 50 units/ml penicillin-G, 50 �g/ml streptomycin, 50 �M-mercaptoethanol and 20 ng/ml IL-2. A volume of 8 mls of the cell suspension was plated per well of a 6 well plate, and the CTLs were either maintained in a gas atmosphere of 18% oxygen and 5% CO2 or transferred to an atmosphere of 1% O2 and 5% CO2 in a Galaxy 48 R incubator (Eppendorf) for 24 hours. Three biological replicates (CTLs generated from three separate spleens) were analysed in this experiment."
2021-10-20 | PXD026223 | Pride