RESUMO
The Hippo pathway plays a central role in tissue development and homeostasis. However, the function of Hippo in pancreatic endocrine development remains obscure. Here, we generated novel conditional genetically engineered mouse models to examine the roles of Hippo pathway-mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation. While YAP1 protein was localized to the nuclei in bipotent progenitor cells, Neurogenin 3 expressing endocrine progenitors completely lost YAP1 expression. Using genetically engineered mouse models, we found that inactivation of YAP1 requires both an intact Hippo pathway and Neurogenin 3 protein. Gene deletion of Lats1 and 2 kinases (Lats1&2) in endocrine progenitor cells of developing mouse pancreas using Neurog3Cre blocked endocrine progenitor cell differentiation and specification, resulting in reduced islets size and a disorganized pancreas at birth. Loss of Lats1&2 in Neurogenin 3 expressing cells activated YAP1/TAZ transcriptional activity and recruited macrophages to the developing pancreas. These defects were rescued by deletion of Yap1/Wwtr1 genes, suggesting that tight regulation of YAP1/TAZ by Hippo signaling is crucial for pancreatic endocrine specification. In contrast, deletion of Lats1&2 using ß-cell-specific Ins1CreER resulted in a phenotypically normal pancreas, indicating that Lats1&2 are indispensable for differentiation of endocrine progenitors but not for that of ß-cells. Our results demonstrate that loss of YAP1/TAZ expression in the pancreatic endocrine compartment is not a passive consequence of endocrine specification. Rather, Hippo pathway-mediated inhibition of YAP1/TAZ in endocrine progenitors is a prerequisite for endocrine specification and differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Proteínas de Sinalização YAP , Animais , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Via de Sinalização Hippo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Transativadores/metabolismo , Transativadores/genética , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/embriologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Aciltransferases , Proteínas Supressoras de TumorRESUMO
Cell type-specific enhancers are critically important for lineage specification. The mechanisms that determine cell-type specificity of enhancer activity, however, are not fully understood. Most current models for how enhancers function invoke physical proximity between enhancer elements and their target genes. Here, we use an imaging-based approach to examine the spatial relationship of cell type-specific enhancers and their target genes with single-cell resolution. Using high-throughput microscopy, we measure the spatial distance from target promoters to their cell type-specific active and inactive enhancers in individual pancreatic cells derived from distinct lineages. We find increased proximity of all promoter-enhancer pairs relative to non-enhancer pairs separated by similar genomic distances. Strikingly, spatial proximity between enhancers and target genes was unrelated to tissue-specific enhancer activity. Furthermore, promoter-enhancer proximity did not correlate with the expression status of target genes. Our results suggest that promoter-enhancer pairs exist in a distinctive chromatin environment but that genome folding is not a universal driver of cell-type specificity in enhancer function.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Transcrição Gênica , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Cromatina/metabolismo , Animais , Camundongos , Linhagem da Célula , Pâncreas/metabolismoRESUMO
BACKGROUND: The human pancreas is composed of specialized cell types producing hormones and enzymes critical to human health. These specialized functions are the result of cell type-specific transcriptional programs which manifest in cell-specific gene expression. Understanding these programs is essential to developing therapies for pancreatic disorders. Transcription in the human pancreas has been widely studied by single-cell RNA technologies, however the diversity of protocols and analysis methods hinders their interpretability in the aggregate. RESULTS: In this work, we perform a meta-analysis of pancreatic single-cell RNA sequencing data. We present a database for reference transcriptome abundances and cell-type specificity metrics. This database facilitates the identification and definition of marker genes within the pancreas. Additionally, we introduce a versatile tool which is freely available as an R package, and should permit integration into existing workflows. Our tool accepts count data files generated by widely-used single-cell gene expression platforms in their original format, eliminating an additional pre-formatting step. Although we designed it to calculate expression specificity of pancreas cell types, our tool is agnostic to the biological source of count data, extending its applicability to other biological systems. CONCLUSIONS: Our findings enhance the current understanding of expression specificity within the pancreas, surpassing previous work in terms of scope and detail. Furthermore, our database and tool enable researchers to perform similar calculations in diverse biological systems, expanding the applicability of marker gene identification and facilitating comparative analyses.
Assuntos
Pancreatopatias , Software , Humanos , Análise de Célula Única/métodos , Transcriptoma , Pâncreas , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodosRESUMO
Histone deacetylase inhibitors (HDACi) are part of a growing class of epigenetic therapies used for the treatment of cancer. Although HDACis are effective in the treatment of T-cell lymphomas, treatment of solid tumors with this class of drugs has not been successful. Overexpression of the multidrug resistance protein P-glycoprotein (P-gp), encoded by ABCB1, is known to confer resistance to the HDACi romidepsin in vitro, yet increased ABCB1 expression has not been associated with resistance in patients, suggesting that other mechanisms of resistance arise in the clinic. To identify alternative mechanisms of resistance to romidepsin, we selected MCF-7 breast cancer cells with romidepsin in the presence of the P-gp inhibitor verapamil to reduce the likelihood of P-gp-mediated resistance. The resulting cell line, MCF-7 DpVp300, does not express P-gp and was found to be selectively resistant to romidepsin but not to other HDACis such as belinostat, panobinostat, or vorinostat. RNA-sequencing analysis revealed upregulation of the mRNA coding for the putative methyltransferase, METTL7A, whose paralog, METTL7B, was previously shown to methylate thiol groups on hydrogen sulfide and captopril. As romidepsin has a thiol as the zinc-binding moiety, we hypothesized that METTL7A could inactivate romidepsin and other thiol-based HDACis via methylation of the thiol group. We demonstrate that expression of METTL7A or METTL7B confers resistance to thiol-based HDACis and that both enzymes are capable of methylating thiol-containing HDACis. We thus propose that METTL7A and METTL7B confer resistance to thiol-based HDACis by methylating and inactivating the zinc-binding thiol.