RESUMO
XIST (X-inactive specific transcript) long noncoding RNA (lncRNA) is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female preimplantation embryos without triggering X chromosome silencing. The XACT (X-active coating transcript) lncRNA coaccumulates with XIST on active X chromosomes and may antagonize XIST function. Here, we used human embryonic stem cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during preimplantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and dampens the transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional before XCI, confirms the existence of a transient process of X chromosome dosage compensation and reveals that XCI and dampening rely on the same set of factors.
RESUMO
The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.
Assuntos
Trofoblastos , Humanos , Trofoblastos/metabolismo , Trofoblastos/citologia , Metilação de DNA , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Modelos Biológicos , Implantação do Embrião , Diferenciação Celular , Epigênese Genética , Transcriptoma/genética , Proteômica/métodosRESUMO
With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.
Assuntos
Sistemas Microfisiológicos , Políticas , Humanos , Desenvolvimento de MedicamentosRESUMO
The past decade has seen expeditious developments in our ability to grow and maintain a variety of human cells and tissues, with properties closely mimicking those in the human body. Prominent researchers and entrepreneurs from all over the world assembled in Hyderabad, India to discuss developments in this field that have not only aided fundamental understanding of organ development and disease processes but have served as good physiological models for toxicity testing and drug development. The speakers presented ingenious, cutting-edge technology and forward-thinking ideas. This report presents the salient aspects of their discussions, highlights the importance of identifying unmet needs, and discusses setting of standards that will help regulatory approvals as we move into a new era, with nominal animal use in research and effective drug discovery.
Assuntos
Dispositivos Lab-On-A-Chip , Sistemas Microfisiológicos , Animais , Humanos , Descoberta de Drogas , ÍndiaRESUMO
X chromosome activity is a defining attribute of naive pluripotency, with naive pluripotency being a rare context in which both X chromosomes of females are active. RNA-fluorescence in situ hybridization (RNA-FISH) is a powerful tool to determine the transcriptional status of specific genes with allelic and single-cell resolution and has been widely used in the context of X chromosome inactivation, the process ensuring dosage compensation for X-linked genes between sexes in mammals. RNA-FISH using genomic or intronic probes allows the detection of newly synthesized transcripts at the site of transcription. This technique is invaluable for appreciating the putative heterogeneity in the expression profiles within cell populations. RNA-FISH has the added advantage of allowing the visualization of gene transcription in a spatial perspective. Here, we provide a detailed protocol describing the application of RNA-FISH to detect nascent X-linked transcripts in female naive human embryonic stem cells to assess their X chromosome status, along with another complementary technique, DNA-FISH.
Assuntos
Células-Tronco Pluripotentes , Animais , Feminino , Humanos , Hibridização in Situ Fluorescente , RNA Longo não Codificante/genética , Cromossomo X , Inativação do Cromossomo X/genéticaRESUMO
Candida albicans, a human pathogen, carries an expanded family of Zn(II)2Cys6 transcription factors. A CTG clade-specific protein Zcf32 and its closely related protein Upc2, a well-conserved transcription factor across the various fungal species, belong to this family of proteins. Unlike Upc2, Zcf32 is poorly studied in C. albicans. Here, we examined roles played by these two related transcription factors in biofilm development and virulence of C. albicans. Our data show that the null mutants of each of Zcf32 or Upc2 form better biofilms than the wild-type suggesting that both of them negatively regulate the biofilm development. While acting as negative regulators of biofilm formation, these two transcription factors target a different set of biofilm genes. A mouse model of candidiasis reveals that zcf32/zcf32 was hypervirulent, while upc2/upc2 shows compromised virulence compared to the wild-type. Notably, the absence of Zcf32 enhances detrimental inflammation brought about by TNFα, IFNß and IFNγ. upc2/upc2 failed to generate a similar feedback, instead demonstrated an elevated anti-inflammatory (IL4 and IL10) host response. Taking together, we show how a recently evolved transcription factor Zcf32 retained functional resemblance with a more ubiquitous member Upc2 but also functionally diverged from the latter in the regulation of biofilm development and virulence of the pathogen.
Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Candida albicans/genética , Candidíase/patologia , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/metabolismo , Animais , Candida albicans/patogenicidade , Candidíase/microbiologia , Modelos Animais de Doenças , Deleção de Genes , Inflamação/patologia , Camundongos Endogâmicos BALB C , Fatores de Transcrição/genética , VirulênciaRESUMO
Tissue repair is a complex process that necessitates an interplay of cellular processes, now known to be dictated by epigenetics. Intriguingly, macrophages are testimony to a large repertoire of evolving functions in this process. We identified a role for BMP signaling in regulating macrophage responses to Candida albicans infection during wound repair in a murine model. In this study, the RNA binding protein, AU-rich element-binding factor 1, was posttranslationally destabilized to bring about ubiquitin ligase, NEDD4-directed activation of BMP signaling. Concomitantly, PI3K/PKCδ mobilized the rapid phosphorylation of BMP-responsive Smad1/5/8. Activated BMP pathway orchestrated the elevated recruitment of EZH2 at promoters of genes assisting timely wound closure. In vivo, the repressive H3K27 trimethylation was observed to persist, accompanied by a robust upregulation of BMP pathway upon infection with C. albicans, culminating in delayed wound healing. Altogether, we uncovered the signaling networks coordinated by fungal colonies that are now increasingly associated with the infected wound microbiome, resulting in altered wound fate.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Candida albicans/fisiologia , Candidíase/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Macrófagos/fisiologia , Cicatrização , Animais , Candidíase/metabolismo , Modelos Animais de Doenças , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Transdução de SinaisRESUMO
Mycobacteria propelled modulation of host responses is of considerable interest in the face of emerging drug resistance. Although it is known that Abl tyrosine kinases affect entry and persistence of mycobacteria, mechanisms that couple c-Abl to proximal signaling pathways during immunity are poorly understood. Loss-of-function of c-Abl through Imatinib, in a mouse model of tuberculosis or RNA interference, identified bone morphogenesis protein (BMP) signaling as its cellular target. We demonstrate that c-Abl promotes mycobacterial survival through epigenetic modification brought about by KAT5-TWIST1 at Bmp loci. c-Abl-BMP signaling deregulated iNOS, aggravating the inflammatory balance. Interestingly, BMP signaling was observed to have far-reaching effects on host immunity, as it attenuated TLR3 pathway by engaging miR27a. Significantly, these events were largely mediated via WhiB3 and DosR/S/T but not SecA signaling pathway of mycobacteria. Our findings suggest molecular mechanisms of host pathways hijacked by mycobacteria and expand our understanding of c-Abl inhibitors in potentiating innate immune responses.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-abl/genética , Tuberculose/genética , Tuberculose/metabolismo , Proteína 1 Relacionada a Twist/genética , Regiões 3' não Traduzidas , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Montagem e Desmontagem da Cromatina , Epigênese Genética , Feminino , Interações Hospedeiro-Patógeno , Masculino , Camundongos , Modelos Biológicos , Interferência de RNA , Espécies Reativas de Oxigênio , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
NQO1 and TRXR1 are important host reductases implicated in the regulation of inflammation and apoptosis. Although the transcriptional machinery governing these processes have been extensively investigated, the associated epigenetic regulatory events remain unclear. Here, we report that SET8, a histone H4 lysine 20 monomethylase (H4K20me1), is highly induced during Mycobacterium tuberculosis infection that orchestrates immune evasion strategies through the induction of NQO1 and TRXR1 in vivo. SET8, along with FoxO3a, mediates an active NQO1-PGC1-α complex, which promotes the anti-inflammatory M2 macrophage phenotype, and assists TRXR1-regulated arrest of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Strikingly, the loss-of-function analysis in an in vivo mouse tuberculosis model further corroborated the pivotal role of SET8-responsive NQO1 and TRXR1 in mycobacterial survival. Thus, augmenting host immune responses against Mycobacterium tuberculosis by harnessing the SET8-NQO1/TRXR1 axis with its specific and potent inhibitors could lead to promising host-directed therapeutic adjuvants for tuberculosis treatment.
Assuntos
Epigênese Genética , Histona-Lisina N-Metiltransferase/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/imunologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Humanos , Evasão da Resposta Imune , Leucócitos Mononucleares/microbiologia , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Células RAW 264.7 , Reprodutibilidade dos Testes , Transdução de Sinais , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Tuberculose/microbiologiaRESUMO
Foamy macrophages (FM)s harbor lipid bodies that not only assist mycobacterial persistence within the granulomas but also are sites for intracellular signaling and inflammatory mediators which are essential for mycobacterial pathogenesis. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs during mycobacterial infection are not clear. Here, we report for the first time that jumonji domain containing protein (JMJD)3, a demethylase of the repressive H3K27me3 mark, orchestrates the expression of M. tuberculosis H37Rv-, MDR-JAL2287-, H37Ra- and M. bovis BCG-induced genes essential for FM generation in a TLR2-dependent manner. Further, NOTCH1-responsive RNA-binding protein MUSASHI (MSI), targets a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein, to positively regulate infection-induced JMJD3 expression, FM generation and M2 phenotype. Investigations in in vivo murine models further substantiated these observations. Together, our study has attributed novel roles for JMJD3 and its regulators during mycobacterial infection that assist FM generation and fine-tune associated host immunity.
Assuntos
Histona Desmetilases com o Domínio Jumonji/imunologia , Macrófagos/microbiologia , Infecções por Mycobacterium/imunologia , Mycobacterium tuberculosis/imunologia , Proteínas do Tecido Nervoso/imunologia , Proteínas de Ligação a RNA/imunologia , Animais , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Imunofluorescência , Regulação Bacteriana da Expressão Gênica/imunologia , Granuloma/imunologia , Granuloma/microbiologia , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Histona Desmetilases com o Domínio Jumonji/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Infecções por Mycobacterium/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Tuberculose/imunologia , Tuberculose/metabolismoRESUMO
Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of ß-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1ß, and tumor necrosis factor alpha (TNF-α). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.