Expression profiling of double negative thymocytes
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE7784 | GEO | 2007/05/17
SECONDARY ACCESSION(S): PRJNA99807
REPOSITORIES: GEO
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Project description:In order to understand the molecular mechanisms of DN thymocyte development, it may be also of use to clarify how these developmental processes are regulated in terms of their entire gene expression, to which cell differentiation is ultimately ascribed. In the current study, we approached this issue by investigating gene expression profiles in discrete subsets of DN thymocytes under development, in which DN2, DN3, and DN4 thymocytes were sorted and subjected to expression profiling analysis with high-density oligonucleotide microarrays. Experiment Overall Design: The DN2, DN3, and DN4 populations were FACS-sorted from DN thymocytes harvested from four C57BL/6 mice and analyzed by Affymetrix® Mouse Genome 430 2.0 Array® for gene expression. Four independent experiments were performed using 16 mice.
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Project description:Mitochondria and endoplasmic reticulum contacts (MERCs) regulate multiple cellular processes including cell survival and differentiation. Based on the observations that MERCs were specifically enriched in the CD4-CD8- double negative (DN) stage, we studied their role in early thymocyte development. We found that T-cell-specific knockout of Hspa9, which encodes GRP75, a chaperone mediates MERC formation by assembling the IP3R-GRP75-VDAC complex, impaired DN3 thymocyte viability and resulted in thymocyte developmental arrest at the DN3-DN4 transition. Mechanistically, GRP75 deficiency induced mitochondrial stress, releasing mitochondrial DNA (mtDNA) into the cytosol and triggering the type I interferon (IFN-I) response. IFN-I pathway contributed to both the impairment of cell survival and DN3-DN4 transition blockage, while increased lipid peroxidation (LPO) played a major role downstream of IFN-I. Thus, our study reveals the essential role of GRP75-dependent MERCs in early thymocyte development and uncovers the governing facts of cellular survival and differentiation in the DN stage.
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Project description:β -selection imposes a considerable demand for new protein synthesis of the newly rearranged Tcrβ gene and the multiple factors that execute the transcriptional and metabolic programs demanded by DN thymocyte proliferation. However, how proteome homeostasis or “proteostasis” is regulated during thymocyte development is largely unknown. Here, we show that the endoplasmic reticulum (ER)- associated degradation (ERAD), but not the unfolded protein response (UPR), is the master regulator of physiological ER proteostasis in immature DN thymocytes. The ERAD machinery was critically required for successful β-selection of DN3 thymocytes and consequently, ERAD deficiency impeded αβ T cell development. The Sel1L-Hrd1 complex is the most conserved branch of mammalian ERAD machinery. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired αβ T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. This study revealed the stringent protein quality control through the SEL1L-ERAD pathway is required for successful b-selection and the development of the ab T cells that mediate adaptive immunity.
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Project description:T cell development proceeds in a series of developmental stages, which is precisely orchestrated by multiple signaling and molecular networks. Here we found a zinc finger protein Zfp335 intrinsically controls DN to DP transition, as T cell-specific deficiency in Zfp335 leads to a substantial accumulation of DN3 along with reduction of DP, CD4+ and CD8+ thymocytes. This developmental blockade at DN stage results from the impaired intracellular TCRβ expression as well as increased susceptibility to apoptosis in thymocytes. Transcriptomic and ChIP-seq analyses revealed a direct regulation of transcription factors Bcl6 and Rorc by Zfp335. Importantly, enhanced expression of TCRβ and Bcl6/RorγT restores the developmental defect during DN3 to DN4 transition and improves thymocytes survival, respectively. These findings identify a critical role of Zfp335 in controlling T cell development by maintaining intracellular TCR expression-mediated β-selection and independently activating cell survival signaling.
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Project description:T cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related, and the activation of lymphocyte-specific programs. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor interacts with the Polycomb Repressive Complex 2 (PRC2) in CD4-CD8- thymocytes, and allows its binding to >200 developmentally-regulated genes, many of which are expressed in hematopoietic stem cells. Loss of Ikaros in CD4-CD8- cells leads to diminished histone H3 Lys27 (H3K27) trimethylation and ectopic expression of these genes. Ikaros binding triggers PRC2 recruitment and H3K27 trimethylation. Furthermore, Ikaros interacts with PRC2 independently of the Nucleosome Remodeling and Deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells. Genome-wide comparison of different histone modifications, Ikaros, Suz12 and NuRD binding in different stages of T cell development in WT and Ikaros mutant mice. Profiling of H3K27me3 in DN1, DN2, DN3, DN4 and DP thymocytes and hematopoietic stem and progenitor cells (LSK cells) of WT and Ikaros mutant mice. Profiling of H3K4me3 and H3ac in WT and Ikaros mutant DP thymocytes. Global analysis of Ikaros binding in WT DN3, DN4 and DP cells, Suz12 binding in WT and Ikaros mutant DN3 cells, and Mta2 and Mi2beta binding in WT DN3 cells. Genome-wide profiling of Ikaros binding and H3K27me3 upon Ikaros activation in Ikaros-deficient leukemic T cells.
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Project description:The noncluster homeodomain containing gene, HOX11/TLX1 (TLX1) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell Acute Lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of TLX1 in T cells. The oncogenic potential of TLX1 was demonstrated in IgHµ-TLX1Tg mice, which developed mature B cell lymphoma after a long latency period suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed IgHµ-TLX1Tg mice with PrkdcScid/Scid mice; To identify the molecular pathways dysregulated in the earliest stages of TLX1-induced transformation, we used Affimetrix microarrays to compare gene expression profiling of premaliganant thymocytes (6~8 weeks old): DN1, DN2 and DN3 stages from HOX11 transgenic PrkdcScid/Scid (HOXSCID) mice with the same stage thymocytes from the sex and age matched control PrkdcScid/Scid (SCID) mice. Expression analysis of IgH-TLX1TgPrkdcScid/Scid thymocytes revealed dysregulated expression of cell cycle, apoptotic, mitotic spindle and anaphase-promoting complex genes in double negative (DN) 2 and DN3 stage thymocytes. Moreover, DN1, DN2 and DN3 TLX1-expressing thymocytes showed downregulated expression of ribosomal and mitochondria ribosomal protein genes. Four groups of double mutant IgH-HOX11TgPrkdcScid/Scid mice and four groups of control PrkdcScid/Scid mice were sacrificed at the age of 6~8 weeks and the fresh thymocytes were FACS sorted by markers CD25 and CD44 to DN1, DN2 and DN3 stages. To minimize the sample variability caused by individual differences among animals, about 2 ~5 the same phenotype, stage and sex thymocytes were pooled (total 104 ~ 105 cells) and used as one sample each for RNA extraction and hybridization on Affymetrix microarrays.
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Project description:The (pro)renin receptor (PRR) was originally thought to be important in regulating blood pressure via the renin-angiotensin system (RAS), however it is emerging that PRR is instead generally essential for cellular development via its involvement in Wnt signalling. Here, we have specifically deleted PRR from T cells, which require Wnt for their development. T cell-specific PRR-knockout (cKO) mice (ATP6AP2flox/y;Lck-CRE) had a significant decrease in thymic cellularity, corresponding with a 100-fold decrease in the number of CD4+ and CD8+ thymocytes, and a large increase in double negative (DN) precursors. Further characterisation of DN T cell progenitors revealed that deletion of PRR impaired T cell development at multiple stages; including transition from DN3(CD25+CD44-) - DN4(CD25-CD44-), DN4-CD4+ CD8+ double positive (DP), DP-intermediate single positive (CD3-CD8+ ISP) cells. We performed gene expression analysis on sorted DN3 T cells, which indicated that PRR cKO T cells have perturbations in key cellular pathways essential at the DN3 stage, such as RNA processing and translation. Our study identifies a new role for PRR in multiple facets of T cell development, and gives further support to the notion that PRR is generally essential for cellular development.
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Project description:The noncluster homeodomain containing gene, HOX11/TLX1 (TLX1) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell Acute Lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of TLX1 in T cells. The oncogenic potential of TLX1 was demonstrated in IgHµ-TLX1Tg mice, which developed mature B cell lymphoma after a long latency period suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed IgHµ-TLX1Tg mice with PrkdcScid/Scid mice; To identify the molecular pathways dysregulated in the earliest stages of TLX1-induced transformation, we used Affimetrix microarrays to compare gene expression profiling of premaliganant thymocytes (6~8 weeks old): DN1, DN2 and DN3 stages from HOX11 transgenic PrkdcScid/Scid (HOXSCID) mice with the same stage thymocytes from the sex and age matched control PrkdcScid/Scid (SCID) mice. Expression analysis of IgH-TLX1TgPrkdcScid/Scid thymocytes revealed dysregulated expression of cell cycle, apoptotic, mitotic spindle and anaphase-promoting complex genes in double negative (DN) 2 and DN3 stage thymocytes. Moreover, DN1, DN2 and DN3 TLX1-expressing thymocytes showed downregulated expression of ribosomal and mitochondria ribosomal protein genes.
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