RESUMEN
Although invariant Vα14+ natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ double-positive (DP) thymocytes, the developmental origin of CD4-CD8- double-negative (DN) NKT cells still remains unresolved. Here we provide definitive genetic evidence obtained, through studies of mice with DP-stage-specific ablation of expression of the gene encoding the recombinase component RAG-2 (Rag2) and by a fate-mapping approach, that supports the proposal of the existence of an alternative developmental pathway through which a fraction of DN NKT cells with strong T-helper-type-1 (TH1)-biased and cytotoxic characteristics develop from late DN-stage thymocytes, bypassing the DP stage. These findings provide new insight into understanding of the development of NKT cells and propose a role for timing of expression of the invariant T cell antigen receptor in determining the functional properties of NKT cells.
Asunto(s)
Células T Asesinas Naturales/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Timocitos/fisiología , Animales , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Citocinas/metabolismo , Citotoxicidad Inmunológica/genética , Proteínas de Unión al ADN/genética , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células TH1/inmunologíaRESUMEN
Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell-specific deficiency in Uhrf1 (Uhrf1(fl/fl)Cd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1(fl/fl)Cd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.
Asunto(s)
Colitis/inmunología , Colon/inmunología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Epigénesis Genética , Proteínas Nucleares/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Proteínas Potenciadoras de Unión a CCAAT , Puntos de Control del Ciclo Celular , Proliferación Celular , Células Cultivadas , Clostridium/inmunología , Colitis/genética , Colon/microbiología , Metilación de ADN , Perfilación de la Expresión Génica , Interleucina-2 , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microbiota/inmunología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Simbiosis/inmunología , Ubiquitina-Proteína Ligasas , Regulación hacia ArribaRESUMEN
Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diabetes Mellitus Tipo 1/congénito , Diarrea/genética , Factores de Transcripción Forkhead/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades del Sistema Inmune/congénito , Inflamación/genética , Linfocitos T Reguladores/fisiología , Alelos , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Análisis Mutacional de ADN , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diarrea/inmunología , Factores de Transcripción Forkhead/genética , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Humanos , Enfermedades del Sistema Inmune/genética , Enfermedades del Sistema Inmune/inmunología , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación Missense/genética , Especificidad de Órganos/genéticaRESUMEN
Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that â¼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.
Asunto(s)
Linfocitos B/metabolismo , Células Madre Multipotentes/metabolismo , Transcripción Genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Linaje de la Célula/genética , Células Cultivadas , Epigénesis Genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Código de Histonas , Factor 4 Similar a Kruppel , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX5/fisiología , Análisis de la Célula Individual , Transactivadores/fisiología , TranscriptomaRESUMEN
In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5 These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.
Asunto(s)
Linfocitos B/citología , Transformación Celular Neoplásica/genética , Epigénesis Genética/genética , Silenciador del Gen , Proteínas del Grupo Polycomb/metabolismo , Linfocitos T/citología , Animales , Linaje de la Célula , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Cadenas Pesadas de Inmunoglobulina/genética , Ratones Endogámicos C57BL , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Complejo Represivo Polycomb 1/genética , Regiones Promotoras Genéticas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Allergic asthma is an inflammatory disease characterized by lung eosinophilia controlled by type 2 cytokines. Cysteine proteases are potent triggers of allergic inflammation by causing barrier disruption in lung epithelial cells inducing the elevation of interleukin-5 (IL-5) and IL-13 from natural helper (NH) cells, a member of ILC2s, which leads to lung eosinophilia. In this study, we found that basophils play a crucial role in NH cell-mediated eosinophilic inflammation induced by protease allergens. Conditional deletion of basophils caused a resolution of the papain-induced eosinophilia and mucus production. Resolution of eosinophilia was also observed in mice lacking IL-4 specifically in basophils, indicating that basophil-derived IL-4 enhanced expression of the chemokine CCL11, as well as IL-5, IL-9, and IL-13 in NH cells, thus attracting eosinophils. These results demonstrate that IL-4 from basophils has an important role in the NH-derived cytokine and chemokine expression, subsequently leading to protease allergen-induced airway inflammation.
Asunto(s)
Basófilos/inmunología , Eosinófilos/inmunología , Interleucina-13/inmunología , Interleucina-4/inmunología , Interleucina-5/inmunología , Animales , Asma/inmunología , Quimiocina CCL11/biosíntesis , Interleucina-13/biosíntesis , Interleucina-4/deficiencia , Interleucina-4/genética , Interleucina-5/biosíntesis , Interleucina-9/biosíntesis , Interleucina-9/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/inmunología , Eosinofilia Pulmonar/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
BACKGROUND: Jasmonates (JAs) mediate trade-off between responses to both biotic and abiotic stress and growth in plants. The Arabidopsis thaliana HISTONE DEACETYLASE 6 is part of the CORONATINE INSENSITIVE1 receptor complex, co-repressing the HDA6/COI1-dependent acetic acid-JA pathway that confers plant drought tolerance. The decrease in HDA6 binding to target DNA mirrors histone H4 acetylation (H4Ac) changes during JA-mediated drought response, and mutations in HDA6 also cause depletion in the constitutive repressive marker H3 lysine 27 trimethylation (H3K27me3). However, the genome-wide effect of HDA6 on H4Ac and much of the impact of JAs on histone modifications and chromatin remodelling remain elusive. RESULTS: We performed high-throughput ChIP-Seq on the HDA6 mutant, axe1-5, and wild-type plants with or without methyl jasmonate (MeJA) treatment to assess changes in active H4ac and repressive H3K27me3 histone markers. Transcriptional regulation was investigated in parallel by microarray analysis in the same conditions. MeJA- and HDA6-dependent histone modifications on genes for specialized metabolism; linolenic acid and phenylpropanoid pathways; and abiotic and biotic stress responses were identified. H4ac and H3K27me3 enrichment also differentially affects JAs and HDA6-mediated genome integrity and gene regulatory networks, substantiating the role of HDA6 interacting with specific families of transposable elements in planta and highlighting further specificity of action as well as novel targets of HDA6 in the context of JA signalling for abiotic and biotic stress responses. CONCLUSIONS: The findings demonstrate functional overlap for MeJA and HDA6 in tuning plant developmental plasticity and response to stress at the histone modification level. MeJA and HDA6, nonetheless, maintain distinct activities on histone modifications to modulate genetic variability and to allow adaptation to environmental challenges.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Histona Desacetilasa 6 , Acetilación , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Histona Desacetilasa 6/genética , Histona Desacetilasa 6/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/genética , MetilaciónRESUMEN
Suppression of Meis genes in the distal limb bud is required for proximal-distal (PD) specification of the forelimb. Polycomb group (PcG) factors play a role in downregulation of retinoic acid (RA)-related signals in the distal forelimb bud, causing Meis repression. It is, however, not known whether downregulation of RA-related signals and PcG-mediated proximal gene repression are functionally linked. Here, we reveal that PcG factors and RA-related signals antagonize each other to polarize Meis2 expression along the PD axis in mouse. Supported by mathematical modeling and simulation, we propose that PcG factors are required to adjust the threshold for RA-related signaling to regulate Meis2 expression. Finally, we show that a variant Polycomb repressive complex 1 (PRC1), incorporating PCGF3 and PCGF5, represses Meis2 expression in the distal limb bud. Taken together, we reveal a previously unknown link between PcG proteins and downregulation of RA-related signals to mediate the phase transition of Meis2 transcriptional status during forelimb patterning.
Asunto(s)
Miembro Anterior/embriología , Proteínas de Homeodominio/metabolismo , Esbozos de los Miembros/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Tretinoina/metabolismo , Animales , Miembro Anterior/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sitios Genéticos , Ratones , Transducción de SeñalRESUMEN
Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.
Asunto(s)
Antígenos CD/genética , Cadherinas/genética , Moléculas de Adhesión Celular/genética , Diferenciación Celular/genética , Proteínas HMGB/genética , Hematopoyesis/genética , Factores de Transcripción SOXF/genética , Animales , Aorta/crecimiento & desarrollo , Aorta/metabolismo , Cadherinas/antagonistas & inhibidores , Moléculas de Adhesión Celular/antagonistas & inhibidores , Embrión de Mamíferos , Células Endoteliales/citología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas HMGB/antagonistas & inhibidores , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Embarazo , ARN Interferente Pequeño/farmacología , Factores de Transcripción SOXF/antagonistas & inhibidoresRESUMEN
Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the ß subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg.
Asunto(s)
Blastocisto/citología , Desarrollo Embrionario , Receptores de Glicina/fisiología , Cigoto/citología , Secuencia de Aminoácidos , Animales , Blastocisto/metabolismo , Femenino , Glicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Transcriptoma , Cigoto/metabolismoRESUMEN
The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas F-Box/química , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/química , Receptores de Superficie Celular/química , Arabidopsis/química , Arabidopsis/genética , Cruzamientos Genéticos , Cinética , Mutación , Reguladores del Crecimiento de las Plantas , Raíces de Plantas , Unión Proteica , Ingeniería de Proteínas , Plantones , Transducción de Señal , TransgenesRESUMEN
Polycomb group (PcG) proteins play a pivotal role in silencing developmental genes and help to maintain various stem and precursor cells and regulate their differentiation. PcG factors also regulate dynamic and complex regional specification, particularly in mammals, but this activity is mechanistically not well understood. In this study, we focused on proximal-distal (PD) patterning of the mouse forelimb bud to elucidate how PcG factors contribute to a regional specification process that depends on developmental signals. Depletion of the RING1 proteins RING1A (RING1) and RING1B (RNF2), which are essential components of Polycomb repressive complex 1 (PRC1), led to severe defects in forelimb formation along the PD axis. We show that preferential defects in early distal specification in Ring1A/B-deficient forelimb buds accompany failures in the repression of proximal signal circuitry bound by RING1B, including Meis1/2, and the activation of distal signal circuitry in the prospective distal region. Additional deletion of Meis2 induced partial restoration of the distal gene expression and limb formation seen in the Ring1A/B-deficient mice, suggesting a crucial role for RING1-dependent repression of Meis2 and likely also Meis1 for distal specification. We suggest that the RING1-MEIS1/2 axis is regulated by early PD signals and contributes to the initiation or maintenance of the distal signal circuitry.
Asunto(s)
Miembro Anterior/embriología , Proteínas de Homeodominio/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Hibridación in Situ , Masculino , Ratones , Ratones Mutantes , Complejo Represivo Polycomb 1/genética , Embarazo , Ácido Retinoico 4-Hidroxilasa , Tretinoina/farmacología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.
Asunto(s)
Butiratos/metabolismo , Diferenciación Celular , Colon/inmunología , Colon/microbiología , Fermentación , Simbiosis , Linfocitos T Reguladores/citología , Acetilación/efectos de los fármacos , Traslado Adoptivo , Animales , Butiratos/análisis , Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Colitis/tratamiento farmacológico , Colitis/patología , Colon/citología , Colon/metabolismo , Secuencia Conservada , Femenino , Factores de Transcripción Forkhead/genética , Vida Libre de Gérmenes , Histonas/metabolismo , Homeostasis/efectos de los fármacos , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Recuento de Linfocitos , Espectroscopía de Resonancia Magnética , Masculino , Metaboloma , Ratones , Regiones Promotoras Genéticas/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
ESET/SETDB1, one of the major histone methyltransferases, catalyzes histone 3 lysine 9 (H3K9) trimethylation. ESET is critical for suppressing expression of retroviral elements in embryonic stem cells; however, its role in the immune system is not known. We found that thymocyte-specific deletion of ESET caused impaired T cell development, with CD8 lineage cells being most severely affected. Increased apoptosis of CD8 single-positive cells was observed, and TCR-induced ERK activation was severely inhibited in ESET(-/-) thymocytes. Genome-wide comprehensive analysis of mRNA expression and H3K9 trimethylation revealed that ESET regulates expression of numerous genes in thymocytes. Among them, FcγRIIB, whose signaling can inhibit ERK activation, was strongly and ectopically expressed in ESET(-/-) thymocytes. Indeed, genetic depletion of FcγRIIB in ESET(-/-) thymocytes rescued impaired ERK activation and partially restored defective positive selection in ESET(-/-) mice. Therefore, impaired T cell development in ESET(-/-) mice is partly due to the aberrant expression of FcγRIIB. Collectively, to our knowledge, we identify ESET as the first trimethylated H3K9 histone methyltransferase playing a crucial role in T cell development.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Apoptosis , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genoma , N-Metiltransferasa de Histona-Lisina/deficiencia , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Regiones Promotoras Genéticas , Receptores de IgG/genética , Receptores de IgG/metabolismo , Timocitos/inmunología , Timocitos/fisiologíaRESUMEN
Reprogramming of antigen-specific T lymphocytes into induced pluripotent stem cells (iPSCs) and their subsequent re-differentiation has enabled expansion of functional T lymphocytes in vitro, thus opening up new approaches for immunotherapy of cancer and other diseases. In this study, we have established a robust protocol to reprogram human invariant NKT (Vα24+ iNKT) cells, which have been shown to act as cellular adjuvants and thus exert anti-tumor activity in mice and humans, and to re-differentiate the iNKT cell-derived iPSCs into functional iNKT cells. These iPSC-derived iNKT cells (iPS-Vα24+ iNKT cells) can be activated by ligand-pulsed dendritic cells (DCs) and produce a large amount of interferon-γ upon activation, as much as parental Vα24+ iNKT cells, but exhibit even better cytotoxic activity against various tumor cell lines. The iPS-Vα24+ iNKT cells possess significant anti-tumor activity in tumor-bearing mice and can activate autologous NK cells upon activation by ligand-pulsed DCs in the NOG mouse model in vivo, further extending their therapeutic potential. This study thus provides a first proof of concept for the clinical application of human iPS-Vα24+ iNKT cells for cancer immunotherapy. Stem Cells 2016;34:2852-2860.
Asunto(s)
Antineoplásicos/metabolismo , Células T Asesinas Naturales/citología , Receptores de Antígenos de Linfocitos T/metabolismo , Regeneración , Animales , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/citología , Activación de Linfocitos , Ratones , Células T Asesinas Naturales/metabolismoRESUMEN
This study tested the hypothesis that oocyte-derived paracrine factors (ODPFs) regulate miRNA expression in mouse granulosa cells. Expression of mmu-miR-322-5p (miR-322) was higher in mural granulosa cells (MGCs) than in cumulus cells of the Graafian follicles. The expression levels of miR-322 decreased when cumulus cells or MGCs were co-cultured with oocytes denuded of their cumulus cells. Inhibition of SMAD2/3 signaling by SB431542 increased miR-322 expression by cumulus-oocyte complexes (COCs). Moreover, the cumulus cells but not the MGCs in Bmp15(-/-)/Gdf9(+/-) (double-mutant) mice exhibited higher miR-322 expression than those of wild-type mice. Taken together, these results show that ODPFs suppress the expression of miR-322 in cumulus cells. Gene ontology analysis of putative miR-322 targets whose expression was detected in MGCs with RNA-sequencing suggested that multiple biological processes are affected by miR-322 in MGCs. These results demonstrate that ODPFs regulate miRNA expression in granulosa cells and that this regulation may participate in the differential control of cumulus cell versus MGC functions. Therefore, the ODPF-mediated regulation of cumulus cells takes place at both transcriptional and post-transcriptional levels.
Asunto(s)
Células del Cúmulo/metabolismo , Células de la Granulosa/metabolismo , MicroARNs/metabolismo , Oocitos/metabolismo , Animales , Proteína Morfogenética Ósea 15/genética , Proteína Morfogenética Ósea 15/metabolismo , Técnicas de Cocultivo , Células del Cúmulo/citología , Femenino , Regulación de la Expresión Génica , Células de la Granulosa/citología , Factor 9 de Diferenciación de Crecimiento/genética , Factor 9 de Diferenciación de Crecimiento/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , Oocitos/citologíaRESUMEN
Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII), thereby manifesting intermediate steps for the establishment of transcriptional activation. Here, using genetics and DRB, we show that mammalian Absent, small, or homeotic discs 1-like (Ash1l), a member of the trithorax group proteins, methylates Lys36 of histone H3 to promote the establishment of Hox gene expression by counteracting Polycomb silencing. Importantly, we found that Ash1l-dependent Lys36 di-, tri-methylation of histone H3 in a coding region and exclusion of Polycomb group proteins occur independently of transcriptional elongation in embryonic stem (ES) cells, although both were previously thought to be consequences of transcription. Genome-wide analyses of histone H3 Lys36 methylation under DRB treatment have suggested that binding of the retinoic acid receptor (RAR) to a certain genomic region promotes trimethylation in the RAR-associated gene independent of its ongoing transcription. Moreover, DRB treatment unveils a parallel response between Lys36 methylation of histone H3 and occupancy of either Tip60 or Mof in a region-dependent manner. We also found that Brg1 is another key player involved in the response. Our results uncover a novel regulatory cascade orchestrated by Ash1l with RAR and provide insights into mechanisms underlying the establishment of the transcriptional activation that counteracts Polycomb silencing.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteínas del Grupo Polycomb/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/genética , Animales , Cromatina/genética , Proteínas de Unión al ADN/genética , Diclororribofuranosil Benzoimidazol/farmacología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Silenciador del Gen , Histona Acetiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/genética , Humanos , Lisina/genética , Metilación , Proteínas Nucleares/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/genética , Factores de Transcripción/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
RNA sequencing (RNA-seq) provides information not only about the level of expression of individual genes but also about genomic sequences of host cells. When we use transcriptome data with whole-genome single nucleotide polymorphism (SNP) variant information, the allele frequency can show the genetic composition of the cell population and/or chromosomal aberrations. Here, I show how SNPs in mRNAs can be used to evaluate RNA-seq experiments by focusing on RNA-seq data based on a recently retracted paper on stimulus-triggered acquisition of pluripotency (STAP) cells. The analysis indicated that different types of cells and chromosomal abnormalities might have been erroneously included in the dataset. This re-evaluation showed that observing allele frequencies could help in assessing the quality of samples during a study and with retrospective evaluation of experimental quality.
Asunto(s)
Frecuencia de los Genes , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Animales , Aberraciones Cromosómicas , Simulación por Computador , Bases de Datos Genéticas , Factores de Crecimiento de Fibroblastos/metabolismo , Genotipo , Humanos , Control de Calidad , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodos , Células Madre/metabolismoRESUMEN
To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested, only Sox17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs, thereby regulating hematopoietic development from hESCs/iPSCs.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/fisiología , Animales , Diferenciación Celular/fisiología , División Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/citología , Células Endoteliales/fisiología , Sangre Fetal/citología , Fibroblastos/citología , Hematopoyesis/genética , Humanos , Lentivirus/genética , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Transducción Genética/métodosRESUMEN
Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.