ABSTRACT
Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs. How this occurs remains unclear. Here we demonstrate that in small pre-B cells, the lineage and stage-specific epigenetic reader bromodomain and WD repeat-containing protein 1 (BRWD1) reorders three-dimensional chromatin topology to affect the transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters, and coordinated this switch with Igk locus contraction. BRWD1 did so by converting chromatin-bound static to dynamic cohesin competent to mediate long-range looping. ATP-depletion revealed cohesin conversion to be the main energetic mechanism dictating dynamic chromatin looping. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.
Subject(s)
Chromatin , Cohesins , Chromatin/genetics , Precursor Cells, B-Lymphoid , Gene Expression Regulation , Cell Differentiation , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolismABSTRACT
Understanding the mechanisms promoting chromosomal translocations of the rearranging receptor loci in leukemia and lymphoma remains incomplete. Here we show that leukemias induced by aberrant activation of ß-catenin in thymocytes, which bear recurrent Tcra/Myc-Pvt1 translocations, depend on Tcf-1. The DNA double strand breaks (DSBs) in the Tcra site of the translocation are Rag-generated, whereas the Myc-Pvt1 DSBs are not. Aberrantly activated ß-catenin redirects Tcf-1 binding to novel DNA sites to alter chromatin accessibility and down-regulate genome-stability pathways. Impaired homologous recombination (HR) DNA repair and replication checkpoints lead to retention of DSBs that promote translocations and transformation of double-positive (DP) thymocytes. The resulting lymphomas, which resemble human T cell acute lymphoblastic leukemia (T-ALL), are sensitive to PARP inhibitors (PARPis). Our findings indicate that aberrant ß-catenin signaling contributes to translocations in thymocytes by guiding Tcf-1 to promote the generation and retention of replication-induced DSBs allowing their coexistence with Rag-generated DSBs. Thus, PARPis could offer therapeutic options in hematologic malignancies with active Wnt/ß-catenin signaling.
Subject(s)
Cell Transformation, Neoplastic , Genomic Instability , Hepatocyte Nuclear Factor 1-alpha , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Thymocytes , Translocation, Genetic , beta Catenin , Animals , Cell Transformation, Neoplastic/genetics , DNA Breaks, Double-Stranded , Genomic Instability/genetics , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-alpha/metabolism , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-myc/genetics , RNA, Long Noncoding/genetics , Thymocytes/pathology , Translocation, Genetic/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.
Subject(s)
Cell Proliferation , Cellular Reprogramming , Colitis, Ulcerative/metabolism , Colitis-Associated Neoplasms/metabolism , Crohn Disease/metabolism , Epigenesis, Genetic , Lymphocyte Activation , T-Lymphocytes, Regulatory/metabolism , Wnt Signaling Pathway , Animals , Case-Control Studies , Cells, Cultured , Colitis, Ulcerative/genetics , Colitis, Ulcerative/immunology , Colitis-Associated Neoplasms/genetics , Colitis-Associated Neoplasms/immunology , Crohn Disease/genetics , Crohn Disease/immunology , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-alpha/metabolism , Humans , Mice, Inbred C57BL , Mice, Transgenic , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Phenotype , T Cell Transcription Factor 1 , T-Lymphocytes, Regulatory/immunologyABSTRACT
In human lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ expansion of B cells expressing anti-vimentin antibodies (AVAs). The mechanism by which AVAs are selected is unclear. Herein, we demonstrate that AVA somatic hypermutation (SHM) and selection increase affinity for vimentin. Indeed, germline reversion of several antibodies demonstrated that higher affinity AVAs can be selected from both low affinity B cell germline clones and even those that are strongly reactive with other autoantigens. While we demonstrated affinity maturation, enzyme-linked immunosorbent assays (ELISAs) suggested that affinity maturation might be a consequence of increasing polyreactivity or even non-specific binding. Therefore, it was unclear if there was also selection for increased specificity. Subsequent multi-color confocal microscopy studies indicated that while TII AVAs often appeared polyreactive by ELISA, they bound selectively to vimentin fibrils in whole cells or inflamed renal tissue. Using a novel machine learning pipeline (CytoSkaler) to quantify the cellular distribution of antibody staining, we demonstrated that TII AVAs were selected for both enhanced binding and specificity in situ. Furthermore, reversion of single predicted amino acids in antibody variable regions indicated that we could use CytoSkaler to capture both negative and positive selection events. More broadly, our data suggest a new approach to assess and define antibody polyreactivity based on quantifying the distribution of binding to native and contextually relevant antigens.
Subject(s)
Disease Susceptibility , Immunity, Humoral , Lupus Nephritis/etiology , Machine Learning , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Autoantibodies/immunology , Autoantigens/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Lupus Nephritis/metabolism , Lupus Nephritis/pathologyABSTRACT
Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.