RESUMO
The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.
Assuntos
Linfócitos T CD4-Positivos , Células T de Memória , Animais , Camundongos , Memória Imunológica , Memória , Receptores de Interleucina-7 , Transativadores , Proteínas GADD45 , Antígenos de DiferenciaçãoRESUMO
The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas Cromossômicas não Histona/biossíntese , Elementos Facilitadores Genéticos , Músculo Esquelético/metabolismo , Miogenina/biossíntese , RNA não Traduzido/metabolismo , Transcrição Gênica , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Células HEK293 , Humanos , Camundongos , Músculo Esquelético/citologia , Proteína MyoD/biossíntese , Proteína MyoD/genética , Miogenina/genética , RNA não Traduzido/genética , CoesinasRESUMO
Dedicated stem cells ensure postnatal growth, repair and homeostasis of skeletal muscle. Following injury, muscle stem cells (MuSCs) exit from quiescence and divide to reconstitute the stem cell pool and give rise to muscle progenitors. The transcriptomes of pooled MuSCs have provided a rich source of information for describing the genetic programs of distinct static cell states; however, bulk microarray and RNA sequencing provide only averaged gene expression profiles, blurring the heterogeneity and developmental dynamics of asynchronous MuSC populations. Instead, the granularity required to identify distinct cell types, states, and their dynamics can be afforded by single cell analysis. We were able to compare the transcriptomes of thousands of MuSCs and primary myoblasts isolated from homeostatic or regenerating muscles by single cell RNA sequencing. Using computational approaches, we could reconstruct dynamic trajectories and place, in a pseudotemporal manner, the transcriptomes of individual MuSC within these trajectories. This approach allowed for the identification of distinct clusters of MuSCs and primary myoblasts with partially overlapping but distinct transcriptional signatures, as well as the description of metabolic pathways associated with defined MuSC states.
Assuntos
Homeostase , Músculo Esquelético/citologia , Regeneração , Análise de Célula Única/métodos , Células-Tronco/citologia , Animais , Separação Celular , Análise por Conglomerados , Biologia Computacional , Citometria de Fluxo , Genômica , Leucócitos Mononucleares/citologia , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Análise de Sequência com Séries de Oligonucleotídeos , RNA-Seq , Análise de Sequência de RNA , Software , TranscriptomaRESUMO
Mutations in the lamin A/C ( LMNA) gene cause a broad range of clinical syndromes that show tissue-restricted abnormalities of post mitotic tissues, such as muscle, nerve, heart, and adipose tissue. Mutations in other nuclear envelope proteins cause clinically overlapping disorders. The majority of mutations are dominant single amino acid changes (toxic protein produced by the single mutant gene), and patients are heterozygous with both normal and abnormal proteins. Experimental support has been provided for different models of cellular pathogenesis in nuclear envelope diseases, including changes in heterochromatin formation at the nuclear membrane (epigenomics), changes in the timing of steps during terminal differentiation of cells, and structural abnormalities of the nuclear membrane. These models are not mutually exclusive and may be important in different cells at different times of development. Recent experiments using fusion proteins of normal and mutant lamin A/C proteins fused to a bacterial adenine methyltransferase (DamID) provided compelling evidence of mutation-specific perturbation of epigenomic imprinting during terminal differentiation. These gain-of-function properties include lineage-specific ineffective genomic silencing during exit from the cell cycle (heterochromatinization), as well as promiscuous initiation of silencing at incorrect places in the genome. To date, these findings have been limited to a few muscular dystrophy and lipodystrophy LMNA mutations but seem shared with a distinct nuclear envelope disease, emerin-deficient muscular dystrophy. The dominant-negative structural model and gain-of-function epigenomic models for distinct LMNA mutations are not mutually exclusive, and it is likely that both models contribute to aspects of the many complex clinical phenotypes observed.
Assuntos
Alelos , Heterogeneidade Genética , Lamina Tipo A/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mutação/genética , FenótipoRESUMO
The genetic context of the blaNDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was investigated. Sequencing of the cosmid selected for the blaNDM-1 gene revealed the presence of two blaNDM-1 copies in the genome of P. aeruginosa MMA83 in a unique genetic environment. Additionally, mating assays, DNA-DNA hybridization, and an S1 nuclease assay strongly suggest that the blaNDM-1 gene in P. aeruginosa MMA83 is chromosome borne.
Assuntos
Cromossomos Bacterianos/genética , Dosagem de Genes , Genes Bacterianos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Sequência de Bases , Mapeamento Cromossômico , Cosmídeos , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Análise de Sequência de DNARESUMO
The transition between pluripotent and tissue-specific states is a key aspect of development. Understanding the pathways driving these transitions will facilitate the engineering of properly differentiated cells for experimental and therapeutic uses. Here, we showed that during mesoderm differentiation, the transcription factor Oct1 activated developmental lineage-appropriate genes that were silent in pluripotent cells. Using mouse embryonic stem cells (ESCs) with an inducible knockout of Oct1, we showed that Oct1 deficiency resulted in poor induction of mesoderm-specific genes, leading to impaired mesodermal and terminal muscle differentiation. Oct1-deficient cells exhibited poor temporal coordination of the induction of lineage-specific genes and showed inappropriate developmental lineage branching, resulting in poorly differentiated cell states retaining epithelial characteristics. In ESCs, Oct1 localized with the pluripotency factor Oct4 at mesoderm-associated genes and remained bound to those loci during differentiation after the dissociation of Oct4. Binding events for Oct1 overlapped with those for the histone lysine demethylase Utx, and an interaction between Oct1 and Utx suggested that these two proteins cooperate to activate gene expression. The specificity of the ubiquitous Oct1 for the induction of mesodermal genes could be partially explained by the frequent coexistence of Smad and Oct binding sites at mesoderm-specific genes and the cooperative stimulation of mesodermal gene transcription by Oct1 and Smad3. Together, these results identify Oct1 as a key mediator of mesoderm lineage-specific gene induction.
Assuntos
Células-Tronco Embrionárias , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/metabolismo , Diferenciação Celular , Sítios de Ligação , Mesoderma/metabolismo , Linhagem da CélulaRESUMO
Thymic presentation of self-antigens is critical for establishing a functional yet self-tolerant T-cell population. Hybrid peptides formed through transpeptidation within pancreatic ß-cell lysosomes have been proposed as a new class of autoantigens in type 1 diabetes (T1D). While the production of hybrid peptides in the thymus has not been explored, due to the nature of their generation, it is thought to be highly unlikely. Therefore, hybrid peptide-reactive thymocytes may preferentially escape thymic selection and contribute significantly to T1D progression. Using an antibody-peptide conjugation system, we targeted the hybrid insulin peptide (HIP) 2.5HIP toward thymic resident Langerin-positive dendritic cells to enhance thymic presentation during the early neonatal period. Our results indicated that anti-Langerin-2.5HIP delivery can enhance T-cell central tolerance toward cognate thymocytes in NOD.BDC2.5 mice. Strikingly, a single dose treatment with anti-Langerin-2.5HIP during the neonatal period delayed diabetes onset in NOD mice, indicating the potential of antibody-mediated delivery of autoimmune neoantigens during early stages of life as a therapeutic option in the prevention of autoimmune diseases.
Assuntos
Diabetes Mellitus Tipo 1 , Animais , Anticorpos , Autoantígenos , Tolerância Central , Insulina , Insulina Regular Humana , Camundongos , Camundongos Endogâmicos NOD , Peptídeos , TimoRESUMO
Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.
Assuntos
Células-Tronco Embrionárias , Transcriptoma , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
In response to various types of infection, naïve CD4+ T cells differentiate into diverse helper T cell subsets; however, the epigenetic programs that regulate differentiation in response to viral infection remain poorly understood. Demethylation of CpG dinucleotides by Tet methylcytosine dioxygenases is a key component of epigenetic programing that promotes specific gene expression, cellular differentiation, and function. We report that following viral infection, Tet2-deficient CD4+ T cells preferentially differentiate into highly functional germinal center T follicular helper (TFH) cells that provide enhanced help for B cells. Using genome-wide DNA methylation and transcription factor binding analyses, we find that Tet2 coordinates with multiple transcription factors, including Foxo1 and Runx1, to mediate the demethylation and expression of target genes, including genes encoding repressors of TFH differentiation. Our findings establish Tet2 as an important regulator of TFH cell differentiation and reveal pathways that could be targeted to enhance immune responses against infectious disease.
Assuntos
Centro Germinativo , Células T Auxiliares Foliculares , Diferenciação Celular/genética , Ativação Linfocitária , Linfócitos T Auxiliares-IndutoresRESUMO
The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Pâncreas/patologia , Linfócitos T/imunologia , Transativadores/metabolismo , Transcrição Gênica , Alelos , Sequência de Aminoácidos , Animais , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Cruzamentos Genéticos , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/prevenção & controle , Modelos Animais de Doenças , Feminino , Deleção de Genes , Células Germinativas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Linfonodos/metabolismo , Ativação Linfocitária , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Ovalbumina , Pâncreas/metabolismo , Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/patologia , Transativadores/deficiênciaRESUMO
The nuclear envelope protein lamin A is encoded by thelamin A/C(LMNA) gene, which can contain missense mutations that cause Emery-Dreifuss muscular dystrophy (EDMD) (p.R453W). We fused mutated forms of the lamin A protein to bacterial DNA adenine methyltransferase (Dam) to define euchromatic-heterochromatin (epigenomic) transitions at the nuclear envelope during myogenesis (using DamID-seq). Lamin A missense mutations disrupted appropriate formation of lamin A-associated heterochromatin domains in an allele-specific manner-findings that were confirmed by chromatin immunoprecipitation-DNA sequencing (ChIP-seq) in murine H2K cells and DNA methylation studies in fibroblasts from muscular dystrophy patient who carried a distinctLMNAmutation (p.H222P). Observed perturbations of the epigenomic transitions included exit from pluripotency and cell cycle programs [euchromatin (open, transcribed) to heterochromatin (closed, silent)], as well as induction of myogenic loci (heterochromatin to euchromatin). In muscle biopsies from patients with either a gain- or change-of-functionLMNAgene mutation or a loss-of-function mutation in theemeringene, both of which cause EDMD, we observed inappropriate loss of heterochromatin formation at theSox2pluripotency locus, which was associated with persistent mRNA expression ofSox2 Overexpression ofSox2inhibited myogenic differentiation in human immortalized myoblasts. Our findings suggest that nuclear envelopathies are disorders of developmental epigenetic programming that result from altered formation of lamina-associated domains.