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.
Project description:Determine the biochemical cause of impaired development of Themis-deficient thymocytes. We isolated three thymocyte populations (three populations: CD4+CD8+CD5loCD69lo, CD4+CD8+CD5+CD69+, and CD4+CD8-CD24hiH2Klo) from Themis-deficient and WT mice. 8 or 9 individual biological replicates were analyzed for each population. An aliquot of cDNA from each sample was pooled and used as a reference.
Project description:Notch1 signaling is an important regulator of cell fate in early stages of T cell development. Because Notch receptors can function redundantly, we sought an approach for inhibiting all endogenous Notch signaling in thymocytes. Upon ligand engagement, Notch receptors undergo two successive proteolytic cleavages, the second involving a Presenilin (PS) containing complex with -secretase activity that releases a small intracellular fragment of Notch. This activated form of Notch (NotchIC) translocates from the membrane to the nucleus, where it acts as a transcription factor, inducing the expression of multiple target genes . Since Presenilins are required for the activation of all four mammalian forms of Notch, we generated mice with deletions of both Presenilin1 and Presenilin2 genes, the only genes encoding Presenilin in the mouse genome. To target Notch inactivation specifically to developing T cells, we introduced Cd4-Cre to mediate Presenilin gene deletion in a tissue- and stage-specific manner. Direct target genes of Notch signaling are largely unknown, and are likely to be cell lineage and stage specific. Therefore in order to identify potential Notch target genes , we compared RNA of thymocytes at the CD4+CD8+ stage of development from H-2b transgenic 5CC7 TCR/ RAG2-deficient / Presenilin1flox/flox /PS2-/- mice, with and without Cd4-Cre. Keywords: presenilin, thymocytes, mouse
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).
Project description:T cell-specific deletion of PTEN induces premalignancy in CD4+ CD8+ (DP) immature T cells in the thymus, which progresses to the development of mature CD4+ T cell lymphomas in the lymph nodes and spleen. As part of a screen to identify factors that inhibit progression to malignancy, we compared miRNA expression in premalignant PTEN-deficient DP thymocytes versus wild-type controls. DP thymocytes were collected by cell sorting from three 9-week-old, premalignant T cell-specific PTEN-deficient mice (tPTEN-/-) and three littermate controls. miRNA expression was assessed relative to a reference pool generated from an equal mixture of all samples.
Project description:We wanted to test the role of mammalian E proteins E2A and HEB in the development of T cells. Using a conditional deletion system in which these proteins are deleted at the DP stage of T cell development, we compared DP thymocytes deficient for E2A, HEB or both to wild-type thymocytes
Project description:Purpose : The goal of this study is to compare the gene expression profiles between wild-type (WT) and CIC-deficient DP thymocytes to understand the molecular mechanisms of CIC regulation of T cell development, especially in DP thymocytes. Methods : DP thymocytes (lin-CD4+CD8+TCRlo (Lineage : CD11b, CD11c, CD19, NK1.1, Gr-1, γδTCR, and TER119)) from thymus were sorted by a MoFlo-XDP (Beckman Coulter). Total RNA was extracted using RiboEX (GeneAll) according to the manufacturer's instructions. The library for mRNA sequencing was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina) and sequencing was performed with NovaSeq 6000 (Illumina). Results : Trimmed reads were mapped to the mouse reference genome (mm10 RefSeq) with HISAT2, and the transcripts were assembled using StringTie. A total of 482 differentially expressed genes (DEGs; fold change > 2 and P-value < 0.05) were generated using edgeR. The 263 upregulated genes included known CIC target genes such as Etv1, Etv4, Etv5, Spry4, Spred1, Dusp4, and Dusp6. Conclusions : Our study represents the first comparative analysis of DP thymocytes from WT and hematopoietic lineage cell-specific Cic deficient mice. We concluded that CIC deficiency leads to impaired positive and negative selection, and TCR signaling in DP cells. The attenuation of TCR signaling is caused by derepression of CIC target genes involved in dephosphorylation of ERK.