Project description:Comparison of epigenome and Tcf1 occupancy between control and Tcf1/Lef1-deficient CD8 T cells Control mice or those are deficient for Tcf1 and Lef1 transcription factors (deleted by CD4-Cre) were used to isolate thymocytes. The thymocytes were surface-stained to identify TCRbeta high, CD69â, CD24â CD8+ subsets. These cells were sorted for ChIPseq analysis of various histone marks. Control mice or those are deficient for Tcf1 (deleted by CD4-Cre) were used to isolate thymocytes. The splenocytes were surface-stained to identify TCRbeta high, CD8+ subsets. These cells were sorted for ChIPseq analysis of Tcf1 binding locations.
Project description:Regulatory T (Treg) cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a lineage-defining protein. In mature Treg cells, the Foxp3 core promoter is unmethylated indicating that this area could harbor a transcription factor complex to initiate or repress gene expression, respectively. We used an unbiased method to identify Foxp3-promoter-binding transcription factors (TFs) by inverted chromatin immunoprecipitation (IP) followed by quantitative mass spectrometry. We identified several candidate factors which showed Foxp3-promoter suppressive capacity, one of which was T-cell factor 1 (Tcf1). Using viral overexpression and CRISPR/Cas knockout studies, we identified Tcf1 as a repressor of Foxp3 expression in primary conventional CD4 T cells (Tconv). In Tcf1-deficient animals, increased levels of Foxp3intermediateCD25negative T cells were identified in secondary lymphoid tissues, implicating that Tcf1 protects Foxp3-negative T cells from inadvertent Foxp3 expression.
Project description:This SuperSeries is composed of the following subset Series: GSE14232: Transcriptome analysis of freshly sorted and expanded regulatory and conventional T cells GSE14233: Detection of differentially methylated regions in CD4+CD25+CD45RA+ regulatory T-cells and conventional CD4+CD25- T-cells GSE14234: Histone H3 Lysine 4 mono-, di- and trimethyl and CTCF in CD4+CD25+CD45RA+ regulatory and conventional CD4+CD25- T-cells Refer to individual Series
Project description:The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1-/- mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1-/- mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1-/- mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell‚Äìspecific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus. Using the Tcf1-/- DeltaVII/DeltaVII knockout mouse (Verbeek et al. Nature 1995), thymocytes of 17 mice (5 control Tcf+/-, 4 Tcf-/- and 8 Tcf-/- with thymic lymphoma) were homogenized for RNA isolation using Qiagen RNeasy minicolumns. The quantity and quality of total RNA was determined using spectrophotometry (Nanodrop) and an Agilent Bioanalyzer. One ¬µg of RNA was used to generate cRNA using Affymetrix One cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA, USA), after which the samples were biotinylated using an Affymetrix IVT labeling kit (Affymetrix). The samples were hybridized overnight at 42¬?C to GeneChip mouse genome 430 2.0 Arrays (Affymetrix). Washing and staining steps were performed on a Fluidics station 450, and the Genechips were scanned using a GeneChip scanner 3000 (Affymetrix) at the Department of Immunology, Erasmus Medical Center. Raw data were normalized and summarized using Robust Multichip Average (RMA) method. The experiment consists of 5 control Tcf+/- thymi, 4 Tcf-/- thymi and 8 Tcf-/- thymus samples with thymic lymphoma.
Project description:In this study, we compared the proteomes of mouse CD4+Foxp3+ regulatory T cells (Treg) and CD4+Foxp3- conventional T cells (Tconv) in order to build a data set of proteins differentially regulated in these two cell populations. The data set contains mass spectrometry results from the analysis of 7 biological replicates of Treg/Tconv cell samples purified by flow cytometry, each experiment performed from a pool of 4-5 mice. Global proteomic analysis of each sample was performed by single-run nanoLC-MS/MS, using chromatographic separation of peptides on 50cm C18 reverse-phase columns, with either a 480min gradient on LTQ-Velos orbitrap mass spectrometer (replicates 1 and 2) or a 300min gradient on Q-Exactive orbitrap mass spectrometer (replicates 3-7). Several MS injection replicates were performed for some experiments, leading to 27 raw files composing the data set. The detailed description of each analysis (file name, sample type, biological replicate number, MS technical replicate number, MS instrument used, sample name in MaxQuant ouput) is given in the table “Files list.txt”.