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Kotliar, M.

Publications and source records attributed to Kotliar, M..

8 recordsLinked to original sources

Memory T cell rapid recall is driven by memory-specific AP-1 recruitment determined by epigenome and co-factor interactions

CD4 T cell memory is essential for long-lasting protective immunity to repeat infections. Unlike naive T cells, memory cells possess rapid recall ability to quickly produce effector molecules in response to antigen re-exposure. This ability was shown to be associated with epigenetic gene poising. Here, we examine how the activation-inducible transcription factors, AP{square}1 and NF{square}{kappa}B, regulate rapid recall gene expression. We found that AP-1 is required for their induction and that the enhanced induction of rapid recall genes in memory cells is associated with memory-specific binding of AP{square}1. Memory-specific AP{square}1 binding, in turn, is enabled by enhanced chromatin accessibility and reduced DNA methylation at regulatory elements. As the AP-1 DNA-binding motif itself does not contain methylatable CpGs, methylation likely affects the binding of AP-1 co-factors, such as ETS proteins, or accessibility of the region in general. Finally, both common and memory-specific AP{square}1/NF{square}{kappa}B binding sites show strong overlap with autoimmune and inflammatory disease risk variants, highlighting the clinical relevance of memory T cell epigenetic regulation.

immunology↗

Gene regulatory network determinants of rapid recall in human memory CD4+ T cells

Rapid recall is the hallmark of memory T cells. While naive cells require days to mount effector responses to new threats, antigen-experienced memory cells produce cytokines within hours of repeat encounter. Memory establishment and the control of rapid recall across lifespan is poorly understood. Epigenetic poising is a likely mechanism. Compared to naive, memory cells exhibit enhanced chromatin accessibility proximal to rapid recall genes, but the transcription factors (TFs) that establish, maintain and utilize these putative regulatory elements are unknown. We leverage single-nuclei (sn)multiome-seq (snRNA-seq and snATAC-seq) to characterize the dynamic activation responses of CD4+ T cell subsets and (2) reconstruct the underlying gene regulatory networks. Memory-associated TFs (MAF, PRDM1, RUNX2, SMAD3, KLF6) were predicted to orchestrate rapid recall. KLF6 binding to its predicted target genes was confirmed by ChIP-seq, while the memory-associated activities of all five factors replicated in independent scRNA-seq studies. Integrating GWAS data, we nominate CD4+ T cell populations and gene regulatory mechanisms that might underly genetic risk to immune-mediated diseases.

systems biology↗

Long-term Tolerance to Islet Transplantation via Targeted Reduction of beta cell-specific T cells.

Type 1 diabetes (T1D) results from insulin insufficiency due to the loss or dysfunction of pancreatic beta cells following T cell-mediated autoimmune attack. Currently the only long-term therapy is daily exogenous insulin replacement. The ideal curative approach is the durable restoration of functional islets via transplantation. To date the limiting factors impeding realization of this goal is the lack of a cost effective and limitless source of high-quality islets suitable for transplantation and the ability to provide long-term islet graft acceptance without prolonged need for deleterious immunosuppression. Ongoing clinical trials are testing islets derived from human induced pluripotent stem cells (iPSC); however, long-term acceptance of islet graft will require a effective therapeutic strategy to prevent engrafted islet destruction by pre-existing islet-antigen specific T cells. Here we demonstrate in the NOD mouse model for T1D that autologous islet graft acceptance can be achieved by the targeted elimination of (re)-activated islet-reactive CD4+ and CD8+ T effector (Teff) cells in the initial post-transplantation period by using a short-acting, combination therapy that results in the elimination of islet-reactive Teff cells by exacerbation of their natural DNA damage response (DDR) to drive apoptosis while at the same time maintaining endogenous Treg cells. Article HighlightsO_LIActivated beta-cell reactive CD4+ and CD8+ T effector cells undergo a profound DNA-damage response which is targetable by small molecule inhibitors of the p53 and cell cycle pathways that lead to apoptosis. C_LIO_LIThe use of a combination of MDM2 and WEE1 inhibitors, which termed "p53 potentiation with checkpoint abrogation" (PPCA), conferred significant therapeutic efficacy in treating mouse models of new onset T1D. C_LIO_LISpecific targeting of these T effector cells by PPCA results in a loss of inflammatory T cell subsets, notably proliferation CD4+ Th0 and Th1 subsets and CD8+ T effector memory cells, as determined by single cell RNA-seq studies with the preservation of T regulatory cells. C_LIO_LIWhen autologous islet grafts are given to established diabetic NOD mice, a single course of PPCA results in long-term islet graft acceptance, restoration of normoglycemia and loss of beta cell specific CD4+ and CD8+ T cells. C_LIO_LIPPCA shows promise as a potential means of estimating islet graft tolerance in T1D recipients of islet graft transplantation. C_LI

immunology↗

Accelerating Single-Cell Sequencing Data Analysis with SciDAP: A User-Friendly Approach

Single-cell (sc) RNA, ATAC and Multiome sequencing became powerful tools for uncovering biological and disease mechanisms. Unfortunately, manual analysis of sc data presents multiple challenges due to large data volumes and complexity of configuration parameters. This complexity, as well as not being able to reproduce a computational environment, affects the reproducibility of analysis results. The Scientific Data Analysis Platform (https://SciDAP.com) allows biologists without computational expertise to analyze sequencing-based data using portable and reproducible pipelines written in Common Workflow Language (CWL). Our suite of computational pipelines addresses the most common needs in scRNA-Seq, scATAC-Seq and scMultiome data analysis. When executed on SciDAP, it offers a user-friendly alternative to manual data processing, eliminating the need for coding expertise. In this protocol, we describe the use of SciDAP to analyze scMultiome data. Similar approaches can be used for analysis of scRNA-Seq, scATAC-Seq and scVDJ-Seq datasets.

bioinformatics↗

Aryl hydrocarbon receptor (AHR) and IL-13 signaling crosstalk in human keratinocytes and atopic dermatitis

Atopic dermatitis (AD) is an allergic skin disease mediated by skin barrier impairment and IL-13-driven immune response. Activation of the aryl hydrocarbon receptor (AHR) has shown promise in early clinical trials for AD, however, the mechanism by which AHR mediates this function is unknown. Herein, AHR signaling is shown to be dysregulated in AD patients using publicly available gene expression data from biopsies of patients with AD compared with controls. AHR target genes, CYP1A1, CYP1A2 and NFE2L2 were decreased in lesional skin compared with healthy control skin (p=0.001, p<1.0*10-10 and p=6.1*10-6 respectively). Single cell RNA sequencing studies demonstrated increased expression of AHR (p<1.0*10-4 and p=0.049) and decreased expression of CYP1A1 in lesional AD keratinocytes compared with healthy control keratinocytes (p=5.0*10-4 and p=0.21). AHR activation reversed IL-13-dependent gene expression of several key genes in AD pathogenesis, most notably the eosinophil chemoattractant, CCL26 (Eotaxin-3). There was substantial overlap between differentially expressed genes in keratinocytes of patients with atopic dermatitis and AHR-regulated genes. Mechanistically, there was evidence for direct transcriptional effects of AHR as its binding motifs were identified in the differentially expressed genes from lesional AD keratinocytes compared to control keratinocytes and AHR activation did not modify IL-13-dependent signal transducer and activator of transcription 6 (STAT6) translocation to the nucleus. Together these data imply that AHR modulates IL-13 downstream signaling in keratinocytes through genome-wide direct transcriptional regulatory effects.

immunology↗

KMT2D regulates thymic egress by modulating maturation and integrin expression

ObjectiveThere is a clinical need to understand how dysregulated thymocyte development, caused by pathogenic variants in the gene encoding the histone-modifying enzyme, lysine methyltransferase 2D (KMT2D), contributes to immune dysfunction, including immune deficiency, autoimmunity, and lymphoproliferative sequela, and immune-driven mortality in individuals with Kabuki syndrome type 1 (KS1). MethodsWe studied peripheral T cells and thymocytes in both individuals with KS1 and murine constitutive and conditional targeted Kmt2d in T cells and hematopoietic lineages. KMT2D target genes, identified by RNA-sequencing of murine Kmt2d-knockout single-positive thymocytes, were validated with H3K4me3 ChIP-PCR and flow cytometry. ResultsRecent thymic emigrant (RTE) and naive T cells were reduced, and memory and double-negative (DN)-T cells were expanded in human KS1 and murine models. Kmt2d loss led to Mature 1 CD8+-single positive (SP) thymocyte accumulation and a decrease in SP thymocyte egress licensing expression (normally associated with the Mature 2 phenotype). Splenomegaly is associated with hematopoietic-driven Kmt2d loss and brings to light potential overlapping phenotypes with lymphoproliferative syndromes. Finally, we identified a KMT2D-regulated cluster of integrins which likely mediates aspects of the T cell egression. ConclusionsSingle-positive thymocyte populations deficient in Kmt2d display less integrin, less maturation, and less egress licensing gene expression; thereby, altering the downstream peripheral T cell composition that contribute to the observed KS1-associated immune deficiency. T cell intrinsic Kmt2d loss increases the percentage of peripheral DNT cells potentially through dysregulated apoptotic signaling, while hematopoietic-driven Kmt2d loss predisposes to splenomegaly; therefore, loss of Kmt2d recapitulates several distinct features of lymphoproliferative syndromes.

immunology↗

maxATAC: genome-scale transcription-factor binding prediction from ATAC-seq with deep neural networks

Transcription factors read the genome, fundamentally connecting DNA sequence to gene expression across diverse cell types. Determining how, where, and when TFs bind chromatin will advance our understanding of gene regulatory networks and cellular behavior. The 2017 ENCODE-DREAM in vivo Transcription-Factor Binding Site (TFBS) Prediction Challenge highlighted the value of chromatin accessibility data to TFBS prediction, establishing state-of-the- art methods for TFBS prediction from DNase-seq. However, the more recent Assay-for- Transposase-Accessible-Chromatin (ATAC)-seq has surpassed DNase-seq as the most widely- used chromatin accessibility profiling method. Furthermore, ATAC-seq is the only such technique available at single-cell resolution from standard commercial platforms. While ATAC-seq datasets grow exponentially, suboptimal motif scanning is unfortunately the most common method for TFBS prediction from ATAC-seq. To enable community access to state-of-the-art TFBS prediction from ATAC-seq, we (1) curated an extensive benchmark dataset (127 TFs) for ATAC-seq model training and (2) built "maxATAC", a suite of user-friendly, deep neural network models for genome-wide TFBS prediction from ATAC-seq in any cell type. With models available for 127 human TFs, maxATAC is the first collection of high-performance TFBS prediction models for ATAC-seq. maxATAC performance extends to primary cells and single-cell ATAC-seq, enabling improved TFBS prediction in vivo. We demonstrate maxATACs capabilities by identifying TFBS associated with allele-dependent chromatin accessibility at atopic dermatitis genetic risk loci. Author SummaryProteins called transcription factors interpret the genome, reading both DNA sequence and chromatin state, to orchestrate gene expression across the diversity of human cell types. In any given cell type, most chromatin is "inaccessible", and only those parts of the genetic code needed or likely to be needed soon are "accessible" for transcription factor binding to affect gene expression and cellular behavior. Hundreds of transcription factors are expressed in a given cell type and context (e.g., age, disease), and knowledge of their context-specific DNA binding sites is key to uncovering how transcription factors regulate cellular behaviors in health or disease. However, experimentally profiling the >1,600 human transcription factors across all cell types and contexts is infeasible. We built a suite of computational models "maxATAC" to predict transcription factor binding from a measurement of accessible chromatin, ATAC-seq. Importantly, ATAC-seq is feasible even at single-cell resolution. Thus, this data type, in combination with maxATAC, can be used to infer transcription factor binding sites in directly-relevant cell types isolated from physiological and disease settings, enabling insights into disease mechanisms, including how genetic variants and cellular context impact transcription factor binding, gene expression patterns and disease risk.

bioinformatics↗

CWL-Airflow: a lightweight pipeline manager supporting Common Workflow Language

BackgroundMassive growth in the amount of research data and computational analysis has led to increased utilization of pipeline managers in biomedical computational research. However, each of more than 100 such managers uses its own way to describe pipelines, leading to difficulty porting workflows to different environments and therefore poor reproducibility of computational studies. For this reason, the Common Workflow Language (CWL) was recently introduced as a specification for platform-independent workflow description, and work began to transition existing pipelines and workflow managers to CWL.\n\nFindingsHere, we present CWL-Airflow, an extension for the Apache Airflow pipeline manager supporting CWL. CWL-Airflow utilizes CWL v1.0 specification and can be used to run workflows on standalone MacOS/Linux servers, on clusters, or on a variety of cloud platforms. A sample CWL pipeline for processing of ChIP-Seq data is provided.\n\nConclusionsCWL-Airflow will provide users with the features of a fully-fledged pipeline manager and an ability to execute CWL workflows anywhere Airflow can run--from a laptop to cluster or cloud environment.\n\nAvailabilityCWL-Airflow is available under Apache license v.2 and can be downloaded from https://barski-lab.github.io/cwl-airflow, http://doi.org/10.5281/zenodo.2669582, RRID: SCR_017196.

bioinformatics↗