RESUMO
CRISPR technology has opened a new era of genome interrogation and genome engineering. Discovered in bacteria, where it protects against bacteriophage by cleaving foreign nucleic acid sequences, the CRISPR system has been repurposed as an adaptable tool for genome editing and multiple other applications. CRISPR's ease of use, precision, and versatility have led to its widespread adoption, accelerating biomedical research and discovery in human cells and model organisms. Here we review CRISPR-based tools and discuss how they are being applied to decode the genetic circuits that control immune function in health and disease. Genetic variation in immune cells can affect autoimmune disease risk, infectious disease pathogenesis, and cancer immunotherapies. CRISPR provides unprecedented opportunities for functional mechanistic studies of coding and noncoding genome sequence function in immunity. Finally, we discuss the potential of CRISPR technology to engineer synthetic cellular immunotherapies for a wide range of human diseases.
Assuntos
Doenças Autoimunes/imunologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Infecções/imunologia , Neoplasias/imunologia , Animais , Doenças Autoimunes/genética , Sistemas CRISPR-Cas , Edição de Genes , Predisposição Genética para Doença , Variação Genética , Humanos , Imunidade , Infecções/genética , Neoplasias/genéticaRESUMO
Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transcriptoma , Biomarcadores , Sistemas CRISPR-Cas , Suscetibilidade a Doenças , Técnicas de Inativação de Genes , Marcação de Genes , Doença Enxerto-Hospedeiro/etiologia , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.
Assuntos
Diferenciação Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , Células Progenitoras Linfoides/fisiologia , Linfócitos T Reguladores/fisiologia , Timo/crescimento & desenvolvimento , Animais , Autoantígenos/imunologia , Colite/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Tolerância Imunológica/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células Progenitoras Linfoides/transplante , Camundongos , Camundongos Transgênicos , Mycobacterium tuberculosis/imunologia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Transdução de Sinais , Organismos Livres de Patógenos Específicos , Timo/citologia , Timo/imunologiaRESUMO
Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.
Assuntos
Sistemas CRISPR-Cas , Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Autoimunidade/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/biossíntese , Edição de Genes , Regulação da Expressão Gênica , Humanos , Imunoterapia , Masculino , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/prevenção & controle , Estabilidade Proteica , Reprodutibilidade dos Testes , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.
RESUMO
The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.
Assuntos
Autoimunidade/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Elementos Facilitadores Genéticos/genética , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular , Linhagem Celular , Cromatina/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/biossíntese , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Células Th17/citologia , Células Th17/imunologiaRESUMO
RasGRP1 is a Ras guanine nucleotide exchange factor, and an essential regulator of lymphocyte receptor signaling. In mice, Rasgrp1 deletion results in defective T lymphocyte development. RASGRP1-deficient patients suffer from immune deficiency, and the RASGRP1 gene has been linked to autoimmunity. However, how RasGRP1 levels are regulated, and if RasGRP1 dosage alterations contribute to autoimmunity remains unknown. We demonstrate that diminished Rasgrp1 expression caused defective T lymphocyte selection in C57BL/6 mice, and that the severity of inflammatory disease inversely correlates with Rasgrp1 expression levels. In patients with autoimmunity, active inflammation correlated with decreased RASGRP1 levels in CD4+ T cells. By analyzing H3K27 acetylation profiles in human T cells, we identified a RASGRP1 enhancer that harbors autoimmunity-associated SNPs. CRISPR-Cas9 disruption of this enhancer caused lower RasGRP1 expression, and decreased binding of RUNX1 and CBFB transcription factors. Analyzing patients with autoimmunity, we detected reduced RUNX1 expression in CD4+ T cells. Lastly, we mechanistically link RUNX1 to transcriptional regulation of RASGRP1 to reveal a key circuit regulating RasGRP1 expression, which is vital to prevent inflammatory disease.
Assuntos
Autoimunidade/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Transcrição Gênica/genética , Animais , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Histonas/genética , Histonas/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transcrição Gênica/imunologiaRESUMO
T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to â¼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to â¼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.
Assuntos
Proteínas de Bactérias/genética , Endonucleases/genética , Ribonucleoproteínas/genética , Linfócitos T/citologia , Proteínas de Bactérias/química , Linfócitos T CD4-Positivos/citologia , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eletroporação , Endonucleases/química , Técnicas de Introdução de Genes , Engenharia Genética/métodos , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucócitos Mononucleares/citologia , Receptores CXCR4/metabolismo , Ribonucleoproteínas/químicaRESUMO
Hermansky-Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism, a bleeding tendency, and sporadic pulmonary fibrosis, granulomatous colitis or infections. Nine HPS-causing genes have been identified in humans. HPS-1 is the most severe subtype with a prevalence of ~1/1800 in northwest Puerto Rico due to a founder mutation in the HPS1 gene. Mutations in HPS genes affect the biogenesis of lysosome-related organelles such as melanosomes in melanocytes and platelet dense granules. Two of these genes (HPS1 and HPS4) encode the HPS1 and HPS4 proteins, which assemble to form a complex known as Biogenesis of Lysosome-related Organelle Complex 3 (BLOC-3). We report the identification of the interacting regions in HPS1 and HPS4 required for the formation of this complex. Two regions in HPS1, spanning amino acids 1-249 and 506-700 are required for binding to HPS4; the middle portion of HPS1 (residues 250-505) is not required for this interaction. Further interaction studies showed that the N-termini of HPS1 and HPS4 interact with each other and that a discrete region of HPS4 (residues 340-528) interacts with both the N- and C-termini of the HPS1 protein. Several missense mutations found in HPS-1 patients did not affect interaction with HPS4, but some mutations involving regions interacting with HPS4 caused instability of HPS1. These observations extend our understanding of BLOC-3 assembly and represent an important first step in the identification of domains responsible for the biogenesis of lysosome-related organelles.
Assuntos
Proteínas de Transporte/metabolismo , Síndrome de Hermanski-Pudlak/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Western Blotting , Proteínas de Transporte/genética , Imunofluorescência , Fatores de Troca do Nucleotídeo Guanina , Síndrome de Hermanski-Pudlak/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Proteínas de Membrana/genética , Mutação/genética , Proteínas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mutations in ERCC6 are associated with growth failure, intellectual disability, neurological dysfunction and deterioration, premature aging, and photosensitivity. We describe siblings with biallelic ERCC6 mutations (NM_000124.2:c. [543+4delA];[2008C>T]) and brain hypomyelination, microcephaly, cognitive decline, and skill regression but without photosensitivity or progeria. DNA repair assays on cultured skin fibroblasts confirmed a defect of transcription-coupled nucleotide excision repair and increased ultraviolet light sensitivity. This report expands the disease spectrum associated with ERCC6 mutations.
Assuntos
Encéfalo/patologia , Encéfalo/fisiopatologia , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso/genética , Processamento Alternativo , Biomarcadores/metabolismo , Criança , Pré-Escolar , DNA Helicases/metabolismo , Análise Mutacional de DNA , Enzimas Reparadoras do DNA/metabolismo , Fácies , Feminino , Expressão Gênica , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Humanos , Íntrons , Imageamento por Ressonância Magnética , Masculino , Mutação , Doenças do Sistema Nervoso/diagnóstico , Linhagem , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose , IrmãosRESUMO
Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.
Assuntos
Antígenos CD28 , Antígeno CTLA-4 , Cromatina , Regulação da Expressão Gênica , Humanos , Antígeno CTLA-4/genética , Antígenos CD28/genética , Cromatina/genética , Cromatina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Sistemas CRISPR-CasRESUMO
Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are four types of OCA caused by mutations in TYR (OCA-1), OCA2 (OCA-2), TYRP1 (OCA-3), or SLC45A2 (OCA-4). Here we report 22 novel mutations in the OCA genes; 14 from a cohort of 61 patients seen as part of the NIH OCA Natural History Study and eight from a prior study at the University of Minnesota. We also include a comprehensive list of almost 600 previously reported OCA mutations along with ethnicity information, carrier frequencies, and in silico pathogenicity predictions as a supplement. In addition to discussing the clinical and molecular features of OCA, we address the cases of apparent missing heritability. In our cohort, 26% of patients did not have two mutations in a single OCA gene. We demonstrate the utility of multiple detection methods to reveal mutations missed by Sanger sequencing. Finally, we review the TYR p.R402Q temperature-sensitive variant and confirm its association with cases of albinism with only one identifiable TYR mutation.
Assuntos
Albinismo Oculocutâneo/diagnóstico , Albinismo Oculocutâneo/genética , Mutação , Albinismo Oculocutâneo/epidemiologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Estudos de Associação Genética , Variação Genética , Genótipo , Humanos , Padrões de Herança , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo , Prevalência , Relação Estrutura-AtividadeRESUMO
Genetic variants associated with human autoimmune diseases commonly map to non-coding control regions, particularly enhancers that function selectively in immune cells and fine-tune gene expression within a relatively narrow range of values. How such modest, cell-type-selective changes can meaningfully shape organismal disease risk remains unclear. To explore this issue, we experimentally manipulated species-conserved enhancers within the disease-associated IL2RA locus and studied accompanying changes in the progression of autoimmunity. Perturbing distinct enhancers with restricted activity in conventional T cells (Tconvs) or regulatory T cells (Tregs)-two functionally antagonistic T cell subsets-caused only modest, cell-type-selective decreases in IL2ra expression parameters. However, these same perturbations had striking and opposing effects in vivo , completely preventing or severely accelerating disease in a murine model of type 1 diabetes. Quantitative tissue imaging and computational modelling revealed that each enhancer manipulation impinged on distinct IL-2-dependent feedback circuits. These imbalances altered the intracellular signaling and intercellular communication dynamics of activated Tregs and Tconvs, producing opposing spatial domains that amplified or constrained ongoing autoimmune responses. These findings demonstrate how subtle changes in gene regulation stemming from non-coding variation can propagate across biological scales due to non-linearities in intra- and intercellular feedback circuitry, dramatically shaping disease risk at the organismal level.
RESUMO
Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.
RESUMO
The analysis of variants generated by exome sequencing (ES) of families with rare Mendelian diseases is a time-consuming, manual process that represents one barrier to applying the technology routinely. To address this issue, we have developed a software tool, VAR-MD (http://research.nhgri.nih.gov/software/var-md/), for analyzing the DNA sequence variants produced by human ES. VAR-MD generates a ranked list of variants using predicted pathogenicity, Mendelian inheritance models, genotype quality, and population variant frequency data. VAR-MD was tested using two previously solved data sets and one unsolved data set. In the solved cases, the correct variant was listed at the top of VAR-MD's variant ranking. In the unsolved case, the correct variant was highly ranked, allowing for subsequent identification and validation. We conclude that VAR-MD has the potential to enhance mutation identification using family based, annotated next generation sequencing data. Moreover, we predict an incremental advancement in software performance as the reference databases, such as Single Nucleotide Polymorphism Database and Human Gene Mutation Database, continue to improve.
Assuntos
Exoma , Variação Genética , Linhagem , Software , Feminino , Frequência do Gene , Humanos , Masculino , Oxigenases de Função Mista/genética , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , beta-Galactosidase/genéticaRESUMO
Transgenic Drosophila are highly useful for structure-function studies of muscle proteins. However, our ability to mechanically analyze transgenically expressed mutant proteins in Drosophila muscles has been limited to the skinned indirect flight muscle preparation. We have developed a new muscle preparation using the Drosophila tergal depressor of the trochanter (TDT or jump) muscle that increases our experimental repertoire to include maximum shortening velocity (V(slack)), force-velocity curves and steady-state power generation; experiments not possible using indirect flight muscle fibers. When transgenically expressing its wild-type myosin isoform (Tr-WT) the TDT is equivalent to a very fast vertebrate muscle. TDT has a V(slack) equal to 6.1 +/- 0.3 ML/s at 15 degrees C, a steep tension-pCa curve, isometric tension of 37 +/- 3 mN/mm(2), and maximum power production at 26% of isometric tension. Transgenically expressing an embryonic myosin isoform in the TDT muscle increased isometric tension 1.4-fold, but decreased V(slack) 50% resulting in no significant difference in maximum power production compared to Tr-WT. Drosophila expressing embryonic myosin jumped <50% as far as Tr-WT that, along with comparisons to frog jump muscle studies, suggests fast muscle shortening velocity is relatively more important than high tension generation for Drosophila jumping.
Assuntos
Biofísica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Musculares/química , Miosinas/química , Animais , Animais Geneticamente Modificados , Cálcio/química , Cálcio/metabolismo , Drosophila , Contração Muscular/fisiologia , Músculos/patologia , Mutação , Regiões Promotoras Genéticas , Isoformas de Proteínas , Estresse MecânicoRESUMO
A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic "bystander" mutations that escape detection by routine targeted genotyping assays.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Mutação , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Animais , Sequência de Bases , Células Cultivadas , Dano ao DNA , Reparo do DNA , Duplicação Gênica , Regulação da Expressão Gênica/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos Endogâmicos NOD , Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.
Assuntos
Doenças Autoimunes/genética , Doenças Cardiovasculares/genética , DNA Intergênico/genética , Elementos Facilitadores Genéticos , Mutação , Regiões Promotoras Genéticas , Alelos , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Diferenciação Celular , Cromatina , Imunoprecipitação da Cromatina/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Intergênico/metabolismo , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/imunologia , Cultura Primária de Células , Locos de Características Quantitativas , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologiaRESUMO
OBJECTIVE: Early-onset epileptic encephalopathies have been associated with de novo mutations of numerous ion channel genes. We employed techniques of modern translational medicine to identify a disease-causing mutation, analyze its altered behavior, and screen for therapeutic compounds to treat the proband. METHODS: Three modern translational medicine tools were utilized: 1) high-throughput sequencing technology to identify a novel de novo mutation; 2) in vitro expression and electrophysiology assays to confirm the variant protein's dysfunction; and 3) screening of existing drug libraries to identify potential therapeutic compounds. RESULTS: A de novo GRIN2A missense mutation (c.2434C>A; p.L812M) increased the charge transfer mediated by NMDA receptors containing the mutant GluN2A-L812M subunit. In vitro analysis with NMDA receptor blockers indicated that GLuN2A-L812M-containing NMDARs retained their sensitivity to the use-dependent channel blocker memantine; while screening of a previously reported GRIN2A mutation (N615K) with these compounds produced contrasting results. Consistent with these data, adjunct memantine therapy reduced our proband's seizure burden. INTERPRETATION: This case exemplifies the potential for personalized genomics and therapeutics to be utilized for the early diagnosis and treatment of infantile-onset neurological disease.