Study of RFX1 and RFX3 binding sites in the mouse genome
Ontology highlight
ABSTRACT: The aim of the study was to identify RFX1 and RFX2 binding sites in mouse pancreatic beta cells. Chromatin immunoprecipitation experiments were performed with the mouse MIN6 beta cell line and antibodies raised against RFX1 and RFX3. Immunoprecipitated DNA was sequenced using the Genome Analyzer II (Illumina). Antibodies were described in Reith W, Ucla C, Barras E, Gaud A, Durand B, Herrero-Sanchez C, Kobr M, Mach B : RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins. Mol Cell Biol 1994;14:1230–1244
Project description:A disulfide-stabilized MHC class I molecule (HLA-A*02:01(Y84C/A139C), short dsA2) was characterized by native mass spectrometry. dsA2 was shown to be structurally intact in absence of any peptide whereas its wild type form was not. Furthermore, it was observed that the mutation did not disrupt the binding of cognate peptides.
Project description:The medullary thymic epithelial cells (mTECs) express virtually all autoantigens of the body. This phenomenon was then termed promiscuous gene expression (PGE). A large set of autoantigen genes (but not all) is controlled by the transcriptional modulator Autoimmune regulator (Aire) in mTECs. These autoantigens represent all tissues and organs in the thymus and it is implicated in the negative selection of autoreactive thymocytes, and consequantly preventing autoreactive autoimmune reactions and autoimmune diseases (e.g. type 1 diabetes mellitus, systemic lupus erythematosus). Thus, we are looking at gene expression in thse cells because it is very important to better understand the molecular basis of central immune tolerance to normal tissues and organs. The aim of this study is to evaluate the possible link between the expression of the transcriptional regulator Aire, the genetic background of mouse strains and the promiscuous gene expression in mTECs.
Project description:To determine the modulation of gene expression of C57BL/6 and DBA/2 BMDLs in the presence of living intracellular Leishmania amazonensis amastigotes A genome-wide transcriptional analysis was performed by comparing the gene expression profiles of control DLs and live amastigote-hosting DLs from both mouse strains. Dendritic Leucocytes were generated in vitro from bone marrow progenitors (C57Bl/6 and DBA/2 mice). Leishmania amazonensis amastigotes were purified from mouse cutaneous lesions and were added to DL cultures. After 24h, and following a sorting procedure, only BMDls housing living amastigotes were selected for total RNA extraction. Three Biological replicates per condition were run.
Project description:Using highthroughput mass spectrometry, we probed the sixreadingframe translation of human B cells’ transcriptome. We report that about 10% of MHC Iassociated peptides (MAPs) derive from noncanonical reading frames. These socalled cryptic MAPs originate from allegedly non- coding genomic sequences and from outofframe translation. Their biogenesis and properties differ in many ways from those of conventional MAPs. Thus, cryptic MAPs come from very short proteins with atypical Ctermini, and they are coded by transcripts bearing long UTRs selectively enriched in destabilizing elements. Cryptic MAPs increase the complexity of the immunopeptidome and represent an heretofore unexploited source of potential tumorspecific epitopes. In a more general context, the features of cryptic MAPs suggest that mRNA instability is instrumental in the biogenesis of MAPs. Associated RNA-seq accession: GSE67174 .
Project description:Using proteogenomics, we identified and analyzed 25,172 major histocompatibility complex class I-associated peptides (MAPs) isolated from B lymphocytes of 18 individuals who collectively expressed 27 HLA-A,B allotypes. While 58% of genes were the source of 1-64 MAPs per gene, 42% of genes were not represented in the immunopeptidome. Overall, we estimate the immunopeptidome presented by 27 HLA-A,B allotypes covered only 17% of exomic sequences expressed in subjects’ cells. We identified several features of transcripts and proteins that enhance MAP production. From these data we built a logistic regression model that predicts with high accuracy whether a gene from our dataset or from independent datasets would generate MAPs. Our results show preferential selection of MAPs from a limited repertoire of gene products with distinct features. The notion that the immune system can monitor MAPs covering only a fraction of the protein coding genome has profound implications in autoimmunity and cancer immunology.
Project description:Regulatory T cells (Treg cells) expressing the forkhead family transcription factor Foxp3 are critical mediators of dominant immune tolerance to self. Most Treg cells constitutively express the high-affinity interleukin 2 (IL-2) receptor alpha-chain (CD25); however, the precise function of IL-2 in Treg cell biology has remained controversial. To directly assess the effect of IL-2 signaling on Treg cell development and function, we analyzed mice containing the Foxp3gfp knock-in allele that were genetically deficient in either IL-2 (Il2-/-) or CD25 (Il2ra-/-). We found that IL-2 signaling was dispensable for the induction of Foxp3 expression in thymocytes from these mice, which indicated that IL-2 signaling does not have a nonredundant function in the development of Treg cells. Unexpectedly, Il2-/- and Il2ra-/- Treg cells were fully able to suppress T cell proliferation in vitro. In contrast, Foxp3 was not expressed in thymocytes or peripheral T cells from Il2rg-/- mice. Gene expression analysis showed that IL-2 signaling was required for maintenance of the expression of genes involved in the regulation of cell growth and metabolism. Thus, IL-2 signaling seems to be critically required for maintaining the homeostasis and competitive fitness of Treg cells in vivo. Experiment Overall Design: We isolated Foxp3+ CD4+ T cells from Il2-/- mice treated with PBS or recombinant IL-2 for gene expression analysis using whole-genome oligonucleotide microarrays. To identify gene expression changes resulting from IL-2 deficiency and subsequent IL-2 stimulation in Treg cells, we directly compared expression profiles with each other and with expression profiles of Foxp3+ CD4+ T cells isolated from wild-type mice.
Project description:F6 murine leukaemic cells were transfected either with MHC II or with GFP and transcriptionally profiled by RNA-seq. Triplicate samples for each genotype were sequenced. This is part of ongoing investigation of a possible cell-intrinsic role of MHC II B cells.
Project description:We microdissected each embryo region from 6-micron paraffin sections using the Leica AS LMD system to identify all genes active in different embryo region of an SRB seed containing globular-stage embryos. Experiment Overall Design: Globular-stage embryo regions were isolated using the Leica AS LMD system. Total RNA was amplified and hybridized with Affymetrix Soybean Genome GeneChip Arrays.
Project description:Somatic mutations in cancer are a potential source of cancer specific neoantigens. Acute myeloid leukemia (AML) has common recurrent mutations shared between patients in addition to private mutations specific to individuals. We hypothesized that neoantigens derived from recurrent shared mutations would be attractive targets for future immunotherapy and sought to study the Class I and II HLA ligandomes of thirteen primary AML tumor samples and two AML cell lines (OCI-AML3 and MV4-11) using mass spectrometry. We identified two endogenous, mutation-bearing HLA Class I ligands from NPM1, which are predicted to bind the common HLA haplotypes, HLA-A*03:01 and HLA-A*02:01 respectively. We further derived CD8+ T cells from healthy donor peripheral blood samples which bound mutant-peptide loaded A*03:01 and A*02:01 tetramers, suggesting a new source of NPM1 mutation-specific T cell receptors (TCRs) for future evaluation. Since NPM1 is mutated in approximately one-third of patients with AML, the finding of endogenous NPM1 neoantigens supports future studies evaluating immunotherapeutic approaches against this target, for this subset of patients with AML.
Project description:Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. Such neoantigens have been implicated in response to immunotherapies including immune checkpoint blockade, yet their identification and validation remains challenging. Here we discover neoantigens in human mantle cell lymphomas using an integrated strategy for genomic and proteomic tumor antigen discovery that interrogates peptides presented within the tumor major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically identify neoantigen peptides in diagnostic tumor specimens from 17 patients. Remarkably, the 52 discovered neoantigenic peptides were invariably derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we could identify MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. The immunoglobulin variable region somatic mutations were almost exclusively presented by MHC-II. We found T-cells specific for an immunoglobulin-derived neoantigen in the blood of a patient using MHC-II tetramers, and these T-cell clones expanded in frequency following tumor vaccination. These results demonstrate that an integrative approach combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.