Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of #483 T-ALL clone FACS sorted from BM and spleen

ABSTRACT: #483 T-ALL cells were transplanted into C57BL6/J mice. Engraftment was monitored by FACS analysis of peripheral blood obtained by retroorbital blood collection. 20 days after transplantation EGFP+ T-ALL cells were sorted from the bone marrow and spleen of three individual mice. Total RNA was prepared using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) and quality of RNA was assessed using the Agilent 2100 Bioanalyzer. RNA was reverse transcribed and amplified using the Affy GeneChip 3' IVT Express Kit. Fragmented and labeled cDNA was hybridized to Affymetrix Mouse Genome 430 2.0 GeneChip arrays. We compared the global gene expression profile of the #483 T-ALL to normal, CD4+CD8+ thymocytes from published dataset that shared the same mouse strain (C57BL6/J), RNA isolation and amplification technology and microarray platform (Li et al., 2008, GSE12948). The .CEL files from the Microarray experiments obtained from GEO were preprocessed together with the .CEL files from the #483 T-ALL clone using RMA. The complete dataset representing the #483 T-ALL and the CD4+CD8+ double positive thymocytes from Series GSE12948 (re-processed using RMA), is linked below as a supplementary file. #483 T-ALL cells were transplanted into 3 sublethally (7.5 Gy) irradiated C57BL6/J mice. 20 days after Tx EGFP+ T-ALL cells were isolated and subjected to Microarray Analysis. Each mouse serves as a biological replicate.

ORGANISM(S): Mus musculus

SUBMITTER: Adrian Schwarzer

PROVIDER: E-GEOD-41385 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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).

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Gene expression profiling of #483 T-ALL clone FACS sorted from BM and spleen

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