RESUMEN
Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas Proto-Oncogénicas , Proteínas Represoras , Transactivadores , Factores de Transcripción , Proteínas Supresoras de Tumor , Proteínas Proto-Oncogénicas/metabolismo , Animales , Transactivadores/metabolismo , Transactivadores/genética , Ratones , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ratones Endogámicos C57BL , Proteínas Cromosómicas no Histona/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Noqueados , Ensamble y Desensamble de Cromatina , Diferenciación Celular/inmunologíaRESUMEN
In this work, we describe NEAT-seq (sequencing of nuclear protein epitope abundance, chromatin accessibility and the transcriptome in single cells), enabling interrogation of regulatory mechanisms spanning the central dogma. We apply this technique to profile CD4 memory T cells using a panel of master transcription factors (TFs) that drive T cell subsets and identify examples of TFs with regulatory activity gated by transcription, translation and regulation of chromatin binding. We also link a noncoding genome-wide association study single-nucleotide polymorphism (SNP) within a GATA motif to a putative target gene, using NEAT-seq data to internally validate SNP impact on GATA3 regulation.
Asunto(s)
Cromatina , Proteínas Nucleares , Cromatina/genética , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Proteínas Nucleares/genética , TranscriptomaRESUMEN
The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TF's capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.
RESUMEN
In their recent article, Brummett and Muaygil reject Bishop et al.'s framing of the debate over standardization in clinical ethics consultation (CEC) "as one between pro-credentialing procedural and anti-credentialing phenomenological," claiming that this framing "amounts to a false dichotomy between two extreme approaches to CEC." Instead of accepting proceduralism and phenomenology as a binary, Brummett and Muaygil propose that these two views should be seen as the extreme ends of a spectrum upon which CEC should be done. However, as evidenced by several inconsistencies within their article, they have failed to fully appreciate the concern animating Bishop et al.'s proposal. Additionally, because of this failure, they do not seem to realize that credentialing ethicists for CEC will only create different problems in Saudi Arabia even as it possibly solves some of the current problems they identify. In this commentary, we highlight and clarify Brummet and Muaygil's five misunderstandings of Bishop et al. This leads us to conclude that while they claim to be advocating a middle way between proceduralism and phenomenology, in fact they would like for us to standardize another proceduralism, albeit one that incorporates some of the "qualitative" values of American bioethics.
Asunto(s)
Bioética , Consultoría Ética , Eticistas , Ética Clínica , Humanos , Estándares de Referencia , Estados UnidosRESUMEN
Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-κB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention.
Asunto(s)
COVID-19/sangre , COVID-19/inmunología , Inmunidad Innata/fisiología , Adulto , Anciano , COVID-19/genética , COVID-19/mortalidad , Estudios de Casos y Controles , Citocinas/genética , Epigénesis Genética , Femenino , Hematopoyesis , Humanos , Células Asesinas Naturales/patología , Células Asesinas Naturales/virología , Masculino , Persona de Mediana Edad , Monocitos/patología , Monocitos/virología , FN-kappa B/metabolismo , Neutrófilos/patología , Neutrófilos/virología , Proteómica , Índice de Severidad de la Enfermedad , Análisis de la Célula IndividualRESUMEN
Identifying the causes of human diseases requires deconvolution of abnormal molecular phenotypes spanning DNA accessibility, gene expression and protein abundance1-3. We present a single-cell framework that integrates highly multiplexed protein quantification, transcriptome profiling and analysis of chromatin accessibility. Using this approach, we establish a normal epigenetic baseline for healthy blood development, which we then use to deconvolve aberrant molecular features within blood from patients with mixed-phenotype acute leukemia4,5. Despite widespread epigenetic heterogeneity within the patient cohort, we observe common malignant signatures across patients as well as patient-specific regulatory features that are shared across phenotypic compartments of individual patients. Integrative analysis of transcriptomic and chromatin-accessibility maps identified 91,601 putative peak-to-gene linkages and transcription factors that regulate leukemia-specific genes, such as RUNX1-linked regulatory elements proximal to the marker gene CD69. These results demonstrate how integrative, multiomic analysis of single cells within the framework of normal development can reveal both distinct and shared molecular mechanisms of disease from patient samples.