RESUMO
Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.
Assuntos
Encéfalo , Organoides , Humanos , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Técnicas de Cultura de Tecidos , Células-Tronco/metabolismo , MorfogêneseRESUMO
To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.
Assuntos
Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Transcriptoma , Deleção de Genes , Técnicas de Inativação de GenesRESUMO
To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.
Assuntos
Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Epistasia Genética , Perfilação da Expressão Gênica , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotransferases/genética , Fosfotransferases/metabolismoRESUMO
Neuroblastoma is the most common extracranial solid tumor and accounts for â¼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.
Assuntos
Glândulas Suprarrenais/patologia , Neuroblastoma/patologia , Células de Schwann/patologia , Análise de Célula Única , Medula Suprarrenal/patologia , Animais , Agregação Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Estadiamento de Neoplasias , Células-Tronco Neurais , Neuroblastoma/genética , FenótipoRESUMO
Transcription by RNA polymerase II (Pol II) is thought to be predominantly regulated by recruitment of Pol II to promoters. Recent genome-wide analyses demonstrate that many genes are in fact regulated after recruitment of Pol II, by mechanisms such as pausing of Pol II proximal to promoters.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Genoma , Humanos , Modelos Genéticos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists. RESULTS: Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre-tubular aggregates, renal vesicles, and in segments of S-shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single-cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long-term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons. CONCLUSIONS: Given the profound early mesenchymal expression and MET signature of LGR6+ cells, together with the lineage tracing of mesenchymal LGR6+ cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability.
Assuntos
Néfrons , Células-Tronco , Diferenciação Celular , Mesoderma , Organogênese/genéticaRESUMO
Cell type identification is essential for single-cell RNA sequencing (scRNA-seq) studies, currently transforming the life sciences. CHETAH (CHaracterization of cEll Types Aided by Hierarchical classification) is an accurate cell type identification algorithm that is rapid and selective, including the possibility of intermediate or unassigned categories. Evidence for assignment is based on a classification tree of previously available scRNA-seq reference data and includes a confidence score based on the variance in gene expression per cell type. For cell types represented in the reference data, CHETAH's accuracy is as good as existing methods. Its specificity is superior when cells of an unknown type are encountered, such as malignant cells in tumor samples which it pinpoints as intermediate or unassigned. Although designed for tumor samples in particular, the use of unassigned and intermediate types is also valuable in other exploratory studies. This is exemplified in pancreas datasets where CHETAH highlights cell populations not well represented in the reference dataset, including cells with profiles that lie on a continuum between that of acinar and ductal cell types. Having the possibility of unassigned and intermediate cell types is pivotal for preventing misclassification and can yield important biological information for previously unexplored tissues.
Assuntos
Algoritmos , Linhagem da Célula/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , RNA Mensageiro/análise , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/métodos , Células Acinares/imunologia , Células Acinares/patologia , Sequência de Bases , Linhagem da Célula/imunologia , Análise por Conglomerados , Conjuntos de Dados como Assunto , Células Dendríticas/imunologia , Células Dendríticas/patologia , Perfilação da Expressão Gênica , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Especificidade de Órgãos , Pâncreas/imunologia , Pâncreas/patologia , RNA Mensageiro/genética , Software , Linfócitos T/imunologia , Linfócitos T/patologia , Células Tumorais CultivadasRESUMO
Histone H2B ubiquitylation is a transcription-dependent modification that not only regulates nucleosome dynamics but also controls the trimethylation of histone H3 on lysine 4 by promoting ubiquitylation of Swd2, a component of both the histone methyltransferase COMPASS complex and the cleavage and polyadenylation factor(CPF). We show that preventing either H2B ubiquitylation or H2B-dependent modification of Swd2 results in nuclear accumulation of poly(A) RNA due to a defect in the integrity and stability of APT, a subcomplex of the CPF. Ubiquitin-regulated APT complex dynamics is required for the correct recruitment of the mRNA export receptor Mex67 to nuclear mRNPs. While H2B ubiquitylation controls the recruitment of the different Mex67 adaptors to mRNPs, the effect of Swd2 ubiquitylation is restricted to Yra1 and Nab2, which, in turn, controls poly(A) tail length. Modification of H2B thus participates in the crosstalk between cotranscriptional events and assembly of mRNPs linking nuclear processing and mRNA export.
Assuntos
Histonas/metabolismo , Ribonucleoproteínas/metabolismo , Ubiquitinação , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.
Assuntos
Cromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Histona Desacetilases/metabolismo , Histonas/metabolismo , Complexo Mediador/metabolismo , Redes e Vias Metabólicas , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Telômero/metabolismo , Transcrição GênicaRESUMO
Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the up-regulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesis mutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.
Assuntos
Redes Reguladoras de Genes , Chaperonas de Histonas/genética , Biogênese de Organelas , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/genética , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Retroalimentação Fisiológica , Chaperonas de Histonas/metabolismo , Mutação , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Mutações Sintéticas Letais , Fatores de Elongação da Transcrição/metabolismo , TranscriptomaRESUMO
To study the in vivo role of TFIID in the transcriptional regulation of hepatic genes, we generated mice with liver-specific disruption of the TAF10 gene. Inactivation of TAF10 in hepatocytes resulted in the dissociation of TFIID into individual components. This correlated with the downregulation of most hepatocyte-specific genes during embryonic life and a defect in liver organogenesis. Unexpectedly, however, the transcription of less than 5% of active genes was affected by TAF10 inactivation and TFIID disassembly in adult liver. The extent of changes in transcription of the affected genes was dependent on the timing of their activation during liver development, relative to that of TAF10 inactivation. Furthermore, TFIID dissociation from promoters leads to the re-expression of several postnatally silenced hepatic genes. Promoter occupancy analyses, combined with expression profiling, demonstrate that TFIID is required for the initial activation or postnatal repression of genes, while it is dispensable for maintaining ongoing transcription.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fígado/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Perfilação da Expressão Gênica , Marcação de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/embriologia , Camundongos , Camundongos Knockout , Modelos Genéticos , Especificidade de Órgãos , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Growth condition perturbation or gene function disruption are commonly used strategies to study cellular systems. Although it is widely appreciated that such experiments may involve indirect effects, these frequently remain uncharacterized. Here, analysis of functionally unrelated Saccharyomyces cerevisiae deletion strains reveals a common gene expression signature. One property shared by these strains is slower growth, with increased presence of the signature in more slowly growing strains. The slow growth signature is highly similar to the environmental stress response (ESR), an expression response common to diverse environmental perturbations. Both environmental and genetic perturbations result in growth rate changes. These are accompanied by a change in the distribution of cells over different cell cycle phases. Rather than representing a direct expression response in single cells, both the slow growth signature and ESR mainly reflect a redistribution of cells over different cell cycle phases, primarily characterized by an increase in the G1 population. The findings have implications for any study of perturbation that is accompanied by growth rate changes. Strategies to counter these effects are presented and discussed.
Assuntos
Deleção de Genes , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Ciclo Celular , Meios de Cultura , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/citologia , Estresse FisiológicoRESUMO
Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms.
Assuntos
Histona-Lisina N-Metiltransferase , Metilação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transcrição Gênica , Cromatina/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Oligorribonucleotídeos Antissenso/biossíntese , Oligorribonucleotídeos Antissenso/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.
Assuntos
Proliferação de Células , Hepatócitos , Metabolismo dos Lipídeos , Organoides , Transcriptoma , Humanos , Hepatócitos/metabolismo , Hepatócitos/citologia , Organoides/metabolismo , Feto/metabolismo , Adulto , Interleucina-6/metabolismo , Interleucina-6/genética , Células CultivadasRESUMO
Because of the low mutational burden and consequently, fewer potential neoantigens, children with acute myeloid leukemia (AML) are thought to have a T cell-depleted or 'cold' tumor microenvironment and may have a low likelihood of response to T cell-directed immunotherapies. Understanding the composition, phenotype, and spatial organization of T cells and other microenvironmental populations in the pediatric AML bone marrow (BM) is essential for informing future immunotherapeutic trials about targetable immune-evasion mechanisms specific to pediatric AML. Here, we conducted a multidimensional analysis of the tumor immune microenvironment in pediatric AML and non-leukemic controls. We demonstrated that nearly one-third of pediatric AML cases has an immune-infiltrated BM, which is characterized by a decreased ratio of M2- to M1-like macrophages. Furthermore, we detected the presence of large T cell networks, both with and without colocalizing B cells, in the BM and dissected the cellular composition of T- and B cell-rich aggregates using spatial transcriptomics. These analyses revealed that these aggregates are hotspots of CD8+ T cells, memory B cells, plasma cells and/or plasmablasts, and M1-like macrophages. Collectively, our study provides a multidimensional characterization of the BM immune microenvironment in pediatric AML and indicates starting points for further investigations into immunomodulatory mechanisms in this devastating disease.
RESUMO
Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.
Assuntos
Antígeno B7-H1 , Neuroblastoma , Humanos , Criança , Recidiva Local de Neoplasia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Receptores Imunológicos/genética , Imunoterapia , Análise de Sequência de RNARESUMO
Novel therapeutic strategies are urgently needed for patients with high-risk Ewing sarcoma and for the reduction of severe side effects for all patients. Immunotherapy may fill this need, but its successful application has been hampered by a lack of knowledge on the composition and function of the Ewing sarcoma immune microenvironment. Here, we explore the immune microenvironment of Ewing sarcoma, by single-cell RNA sequencing of 18 Ewing sarcoma primary tissue samples. Ewing sarcoma is infiltrated by natural killer, T, and B cells, dendritic cells, and immunosuppressive macrophages. Ewing sarcoma-associated T cells show various degrees of dysfunction. The antigen-presenting cells found in Ewing sarcoma lack costimulatory gene expression, implying functional impairment. Interaction analysis reveals a clear role for Ewing sarcoma tumor cells in turning the Ewing sarcoma immune microenvironment into an immunosuppressive niche. These results provide novel insights into the functional state of immune cells in the Ewing sarcoma tumor microenvironment and suggest mechanisms by which Ewing sarcoma tumor cells interact with, and shape, the immune microenvironment. SIGNIFICANCE: This study is the first presenting a detailed analysis of the Ewing sarcoma microenvironment using single-cell RNA sequencing. We provide novel insight into the functional state of immune cells and suggests mechanisms by which Ewing tumor cells interact with, and shape, their immune microenvironment. These insights provide help in understanding the failures and successes of immunotherapy in Ewing sarcoma and may guide novel targeted (immuno) therapeutic approaches.
Assuntos
Tumores Neuroectodérmicos Primitivos Periféricos , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/genética , Análise da Expressão Gênica de Célula Única , Linhagem Celular Tumoral , Células Apresentadoras de Antígenos/metabolismo , Microambiente Tumoral/genéticaRESUMO
Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.
Assuntos
Rabdomiossarcoma Embrionário , Rabdomiossarcoma , Criança , Humanos , Transcriptoma , Proliferação de Células/genética , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Perfilação da Expressão Gênica , Linhagem Celular Tumoral , Microambiente Tumoral/genéticaRESUMO
Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.
Assuntos
Sistemas CRISPR-Cas , Linhagem da Célula , Células Enteroendócrinas , Regulação da Expressão Gênica , Proteínas Repressoras , Dedos de Zinco , Humanos , Diferenciação Celular/genética , Células Enteroendócrinas/citologia , Células Enteroendócrinas/metabolismo , Organoides , Adulto , Linhagem da Célula/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND: Cellular glucose availability is crucial for the functioning of most biological processes. Our understanding of the glucose regulatory system has been greatly advanced by studying the model organism Saccharomyces cerevisiae, but many aspects of this system remain elusive. To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of the different glucose signalling and metabolic pathways in Saccharomyces cerevisiae using DNA microarrays. RESULTS: In general, the mutations do not induce pathway-specific transcriptional responses. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. Detailed analysis uncovers established and new relationships within and between individual pathways and their members. In contrast to signalling components, metabolic components of the glucose regulatory system are transcriptionally more frequently affected. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system. CONCLUSIONS: The tight interconnection between the different pathways of the glucose regulatory system is reflected by the main transcriptional response observed. Tps2 and Tsl1, two enzymes involved in the biosynthesis of the storage carbohydrate trehalose, are predicted to be the most downstream transcriptional components. Epistasis analysis of tps2Δ double mutants supports this prediction. Although based on transcriptional changes only, these results suggest that all changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis.