RESUMO
Activin/SMAD signaling in human embryonic stem cells (hESCs) ensures NANOG expression and stem cell pluripotency. In the presence of Wnt ligand, the Activin/SMAD transcription network switches to cooperate with Wnt/ß-catenin and induce mesendodermal (ME) differentiation genes. We show here that the Hippo effector YAP binds to the WNT3 gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data show that YAP impairs SMAD recruitment and the accumulation of P-TEFb-associated RNA polymerase II (RNAPII) C-terminal domain (CTD)-Ser7 phosphorylation at the WNT3 gene. CRISPR/CAS9 knockout of YAP in hESCs enables Activin to induce Wnt3 expression and stabilize ß-catenin, which then synergizes with Activin-induced SMADs to activate a subset of ME genes that is required to form cardiac mesoderm. Interestingly, exposure of YAP-/- hESCs to Activin induces cardiac mesoderm markers (BAF60c and HAND1) without activating Wnt-dependent cardiac inhibitor genes (CDX2 and MSX1). Moreover, canonical Wnt target genes are up-regulated only modestly, if at all, under these conditions. Consequently, YAP-null hESCs exposed to Activin differentiate precisely into beating cardiomyocytes without further treatment. We conclude that YAP maintains hESC pluripotency by preventing WNT3 expression in response to Activin, thereby blocking a direct route to embryonic cardiac mesoderm formation.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Proteína Wnt3/genética , Ativinas/fisiologia , Fator de Transcrição CDX2/genética , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Linhagem da Célula , Células Cultivadas , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos , Coração/embriologia , Humanos , Mesoderma/citologia , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Transdução de Sinais , Proteínas Smad/antagonistas & inibidores , Elongação da Transcrição Genética , Fatores de Transcrição/genética , beta Catenina/metabolismoRESUMO
The Wnt3a/ß-catenin and Activin/SMAD2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells; however, the underlying mechanism is not well understood. Using ChIP-seq and GRO-seq analyses, we show here that Wnt3a-induced ß-catenin:LEF-1 enhancers recruit cohesin to direct enhancer-promoter looping and activate mesendodermal (ME) lineage genes. Moreover, we find that LEF-1 and other hESC enhancers recruit RNAPII complexes (eRNAPII) that are highly phosphorylated at Ser5, but not Ser7. Wnt3a signaling further increases Ser5P-RNAPII at LEF-1 sites and ME gene promoters, indicating that elongation remains limiting. However, subsequent Activin/SMAD2,3 signaling selectively increases transcription elongation, P-TEFb occupancy, and Ser7P-RNAPII levels at these genes. Finally, we show that the Hippo regulator, YAP, functions with TEAD to regulate binding of the NELF negative elongation factor and block SMAD2,3 induction of ME genes. Thus, the Wnt3a/ß-catenin and Activin/SMAD2,3 pathways act in concert to counteract YAP repression and upregulate ME genes during early hESC differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células-Tronco Embrionárias/fisiologia , Fosfoproteínas/fisiologia , RNA Polimerase II/metabolismo , Proteínas Smad/fisiologia , beta Catenina/metabolismo , Ativinas/metabolismo , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Elongação da Transcrição Genética , Fatores de Transcrição , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , Proteínas de Sinalização YAP , beta Catenina/genéticaRESUMO
HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P-RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P-S2P transition at the integrated HIV-1 promoter.
Assuntos
Proteínas de Transporte/metabolismo , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Proteínas de Transporte/genética , Células-Tronco Embrionárias/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Fosfoproteínas Fosfatases , Regiões Promotoras Genéticas , Linfócitos T/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Mutation of the adenomatous polyposis coli (APC) tumor suppressor stabilizes ß-catenin and aberrantly reactivates Wnt/ß-catenin target genes in colon cancer. APC mutants in cancer frequently lack the conserved catenin inhibitory domain (CID), which is essential for ß-catenin proteolysis. Here we show that the APC CID interacts with α-catenin, a Hippo signaling regulator and heterodimeric partner of ß-catenin at cell:cell adherens junctions. Importantly, α-catenin promotes ß-catenin ubiquitylation and proteolysis by stabilizing its association with APC and protecting the phosphodegron. Moreover, ß-catenin ubiquitylation requires binding to α-catenin. Multidimensional protein identification technology (MudPIT) proteomics of multiple Wnt regulatory complexes reveals that α-catenin binds with ß-catenin to LEF-1/TCF DNA-binding proteins in Wnt3a signaling cells and recruits APC in a complex with the CtBP:CoREST:LSD1 histone H3K4 demethylase to regulate transcription and ß-catenin occupancy at Wnt target genes. Interestingly, tyrosine phosphorylation of α-catenin at Y177 disrupts binding to APC but not ß-catenin and prevents repression of Wnt target genes in transformed cells. Chromatin immunoprecipitation studies further show that α-catenin and APC are recruited with ß-catenin to Wnt response elements in human embryonic stem cells (hESCs). Knockdown of α-catenin in hESCs prevents the switch-off of Wnt/ß-catenin transcription and promotes endodermal differentiation. Our findings indicate a role for α-catenin in the APC destruction complex and at Wnt target genes.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Proteínas Wnt/metabolismo , alfa Catenina/metabolismo , beta Catenina/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Técnicas de Silenciamento de Genes , Células HCT116 , Células HEK293 , Humanos , Ligação Proteica , Proteólise , Ubiquitinação , alfa Catenina/genéticaRESUMO
During neurogenesis, dynamic developmental cues, transcription factors and histone modifying enzymes regulate the gene expression programs by modulating the activity of neural-specific enhancers. How transient developmental signals coordinate transcription factor recruitment to enhancers and to which extent chromatin modifiers contribute to enhancer activity is starting to be uncovered. Here, we take advantage of neural stem cells as a model to unravel the mechanisms underlying neural enhancer activation in response to the TGFß signaling. Genome-wide experiments demonstrate that the proneural factor ASCL1 assists SMAD3 in the binding to a subset of enhancers. Once located at the enhancers, SMAD3 recruits the histone demethylase JMJD3 and the remodeling factor CHD8, creating the appropriate chromatin landscape to allow enhancer transcription and posterior gene activation. Finally, to analyze the phenotypical traits owed to cis-regulatory regions, we use CRISPR-Cas9 technology to demonstrate that the TGFß-responsive Neurog2 enhancer is essential for proper neuronal polarization.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Elementos Facilitadores Genéticos/genética , Neurogênese/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem da Célula/genética , Polaridade Celular/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Neural development requires crosstalk between signaling pathways and chromatin. In this study, we demonstrate that neurogenesis is promoted by an interplay between the TGFß pathway and the H3K27me3 histone demethylase (HDM) JMJD3. Genome-wide analysis showed that JMJD3 is targeted to gene promoters by Smad3 in neural stem cells (NSCs) and is essential to activate TGFß-responsive genes. In vivo experiments in chick spinal cord revealed that the generation of neurons promoted by Smad3 is dependent on JMJD3 HDM activity. Overall, these findings indicate that JMJD3 function is required for the TGFß developmental program to proceed.
Assuntos
Biologia do Desenvolvimento/métodos , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neurônios/metabolismo , Proteína Smad3/metabolismo , Animais , Embrião de Galinha , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Medula Espinal/embriologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cell transformation is clearly linked to epigenetic changes. However, the role of the histone-modifying enzymes in this process is still poorly understood. In this study, we investigated the contribution of the histone acetyltransferase (HAT) enzymes to Ras-mediated transformation. Our results demonstrated that lysine acetyltransferase 5, also known as Tip60, facilitates histone acetylation of bulk chromatin in Ras-transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac) increases in Ras-transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels of CREB-binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27Kip1) tumour suppressor gene promoters to facilitate Ras-mediated transformation. In agreement with these data, overexpression of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both local and global levels to facilitate Ras-induced transformation.
Assuntos
Proteína de Ligação a CREB/metabolismo , Transformação Celular Neoplásica/genética , Genes ras , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Proteína de Ligação a CREB/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Histona Acetiltransferases/genética , Lisina Acetiltransferase 5 , Camundongos , Células NIH 3T3/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Transativadores/genéticaRESUMO
Epigenetic mechanisms are fundamental for shaping the activity of the central nervous system (CNS). Methyl-CpG binding protein 2 (MECP2) acts as a bridge between methylated DNA and transcriptional effectors responsible for differentiation programs in neurons. The importance of MECP2 dosage in CNS is evident in Rett Syndrome and MECP2 duplication syndrome, which are neurodevelopmental diseases caused by loss-of-function mutations or duplication of the MECP2 gene, respectively. Although many studies have been performed on Rett syndrome models, little is known about the effects of an increase in MECP2 dosage. Herein, we demonstrate that MECP2 overexpression affects neural tube formation, leading to a decrease in neuroblast proliferation in the neural tube ventricular zone. Furthermore, an increase in MECP2 dose provokes premature differentiation of neural precursors accompanied by greater cell death, resulting in a loss of neuronal populations. Overall, our data indicate that correct MECP2 expression levels are required for proper nervous system development.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Galinhas , Dosagem de Genes , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Dados de Sequência Molecular , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismoRESUMO
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.
Assuntos
Gastrulação , Análise de Célula Única , Animais , Camundongos , Análise de Célula Única/métodos , Gastrulação/genética , Feminino , Embrião de Mamíferos , Camadas Germinativas/citologia , Análise de Sequência de RNA/métodos , GravidezRESUMO
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula. SUMMARY: We establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.
RESUMO
Differentiation of cardiac fibroblasts to myofibroblasts is necessary for matrix remodeling and fibrosis in heart failure. We previously reported that mitochondrial calcium signaling drives α-ketoglutarate-dependent histone demethylation, promoting myofibroblast formation. Here we investigate the role of ATP-citrate lyase (ACLY), a key enzyme for acetyl-CoA biosynthesis, in histone acetylation regulating myofibroblast fate and persistence in cardiac fibrosis. We show that inactivation of ACLY prevents myofibroblast differentiation and reverses myofibroblasts towards quiescence. Genetic deletion of Acly in post-activated myofibroblasts prevents fibrosis and preserves cardiac function in pressure-overload heart failure. TGFß stimulation enhances ACLY nuclear localization and ACLY-SMAD2/3 interaction, and increases H3K27ac at fibrotic gene loci. Pharmacological inhibition of ACLY or forced nuclear expression of a dominant-negative ACLY mutant prevents myofibroblast formation and H3K27ac. Our data indicate that nuclear ACLY activity is necessary for myofibroblast differentiation and persistence by maintaining histone acetylation at TGFß-induced myofibroblast genes. These findings provide targets to prevent and reverse pathological fibrosis.
Assuntos
ATP Citrato (pro-S)-Liase , Diferenciação Celular , Fibrose , Histonas , Miofibroblastos , Proteína Smad2 , Miofibroblastos/metabolismo , Miofibroblastos/efeitos dos fármacos , ATP Citrato (pro-S)-Liase/metabolismo , ATP Citrato (pro-S)-Liase/genética , Animais , Fibrose/metabolismo , Diferenciação Celular/efeitos dos fármacos , Histonas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad2/genética , Acetilação/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Proteína Smad3/metabolismo , Proteína Smad3/genética , Células Cultivadas , Cromatina/metabolismo , Camundongos Knockout , Fator de Crescimento Transformador beta/metabolismo , Modelos Animais de Doenças , Transdução de Sinais , Camundongos Endogâmicos C57BL , Masculino , Camundongos , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.
RESUMO
During spinal cord development, the combination of secreted signaling proteins and transcription factors provides information for each neural type differentiation. Studies using embryonic stem cells show that trimethylation of lysine 27 of histone H3 (H3K27me3) contributes to repression of many genes key for neural development. However, it remains unclear how H3K27me3-mediated mechanisms control neurogenesis in developing spinal cord. Here, we demonstrate that H3K27me3 controls dorsal interneuron generation by regulation of BMP activity. Our study indicates that expression of Noggin, a BMP extracellular inhibitor, is repressed by H3K27me3. Moreover, we show that Noggin expression is induced by BMP pathway signaling, generating a negative-feedback regulatory loop. In response to BMP pathway activation, JMJD3 histone demethylase interacts with the Smad1/Smad4 complex to demethylate and activate the Noggin promoter. Together, our data reveal how the BMP signaling pathway restricts its own activity in developing spinal cord by modulating H3K27me3 levels at the Noggin promoter.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Histonas/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proliferação de Células , Embrião de Galinha , Primers do DNA/genética , Epigênese Genética , Histonas/química , Humanos , Metilação , Modelos Neurológicos , Neurogênese , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Medula Espinal/citologiaRESUMO
Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.
Assuntos
Transtornos do Neurodesenvolvimento , Neurogênese , Complexo Repressor Polycomb 2 , Animais , Embrião de Galinha , Humanos , Diferenciação Celular/genética , Núcleo Celular , Cromatina/genética , Metiltransferases , Transtornos do Neurodesenvolvimento/genética , Neurogênese/genética , Complexo Repressor Polycomb 2/genéticaRESUMO
Promoting cardiomyocyte proliferation is a promising strategy to regenerate the heart. Yet, so far, it is poorly understood how cardiomyocyte proliferation is regulated, and no factor identified to promote mammalian cardiomyocyte proliferation has been translated into medical practice. Therefore, finding a novel factor will be vital. Here, we established a live cell screening based on mouse embryonic stem cell-derived cardiomyocytes expressing a non-functional human geminin deletion mutant fused to Azami Green (CM7/1-hgem-derived cardiomyocytes). We screened for a subset of compounds of the small molecule library Spectrum Collection and identified 19 potential inducers of stem cell-derived cardiomyocyte proliferation. Furthermore, the pro-proliferative potential of identified candidate compounds was validated in neonatal and adult rat cardiomyocytes as well as human induced pluripotent stem cell-derived cardiomyocytes. 18 of these compounds promoted mitosis and cytokinesis in neonatal rat cardiomyocytes. Among the top four candidates were two cardiac glycosides, peruvoside and convallatoxin, the flavonoid osajin, and the selective α-adrenoceptor antagonist and imidazoline I1 receptor ligand efaroxan hydrochloride. Inhibition of PTEN and GSK-3ß enhanced cell cycle re-entry and progression upon stimulation with cardiac glycosides and osajin, while inhibition of IP3 receptors inhibited the cell cycle-promoting effect of cardiac glycosides. Collectively, we established a screening system and identified potential compounds to promote cardiomyocyte proliferation. Our data suggest that modulation of calcium handling and metabolism promotes cardiomyocyte proliferation, and cardiac glycosides might, besides increasing myocardial contraction force, contribute to cardiac repair by inducing cardiomyocyte proliferation.
RESUMO
The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.
Assuntos
Estômago/citologia , Proteínas de Sinalização YAP/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Ectoderma/citologia , Ectoderma/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Proteína Nodal/antagonistas & inibidores , Proteína Nodal/genética , Proteína Nodal/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Estômago/metabolismo , Proteínas de Sinalização YAP/deficiência , Proteínas de Sinalização YAP/genéticaRESUMO
Simian Virus 40 (SV40) large T antigen (T Ag) is a multifunctional viral oncoprotein that regulates viral and cellular transcriptional activity. However, the mechanisms by which such regulation occurs remain unclear. Here we show that T antigen represses CBP-mediated transcriptional activity. This repression is concomitant with histone H3 deacetylation and is TSA sensitive. Moreover, our results demonstrate that T antigen interacts with HDAC1 in vitro in an Rb-independent manner. In addition, the overexpression of HDAC1 cooperates with T antigen to antagonize CBP transactivation function and correlates with chromatin deacetylation of the TK promoter. Finally, decreasing HDAC1 levels with small interfering RNA (siRNA) partially abolishes T antigen-induced repression. These findings highlight the importance of the histone acetylation/deacetylation balance in the cellular transformation mediated by oncoviral proteins.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Proteína de Ligação a CREB/antagonistas & inibidores , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Histonas/metabolismo , Acetilação , Animais , Cromatina/enzimologia , Humanos , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
Specifying the primitive streak (PS) guides stem cell differentiation in vitro; however, much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1-/- human embryonic stem cells (hESCs) in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1-/- hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and induced pluripotent stem cell differentiation into distinct mesendodermal lineages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/metabolismo , Ativinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfoproteínas/antagonistas & inibidores , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
The function of EZH2 as a transcription repressor is well characterized. However, its role during vertebrate development is still poorly understood, particularly in neurogenesis. Here, we uncover the role of EZH2 in controlling the integrity of the neural tube and allowing proper progenitor proliferation. We demonstrate that knocking down the EZH2 in chick embryo neural tubes unexpectedly disrupts the neuroepithelium (NE) structure, correlating with alteration of the Rho pathway, and reduces neural progenitor proliferation. Moreover, we use transcriptional profiling and functional assays to show that EZH2-mediated repression of p21(WAF1/CIP1) contributes to both processes. Accordingly, overexpression of cytoplasmic p21(WAF1/CIP1) induces NE structural alterations and p21(WAF1/CIP1) suppression rescues proliferation defects and partially compensates for the structural alterations and the Rho activity. Overall, our findings describe a new role of EZH2 in controlling the NE integrity in the neural tube to allow proper progenitor proliferation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Proteínas Repressoras/metabolismo , Animais , Polaridade Celular , Proliferação de Células , Embrião de Galinha , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismo , Regiões Promotoras Genéticas/genéticaRESUMO
JMJD3 H3K27me3 demethylase plays an important role in the transcriptional response to different signaling pathways; however, the mechanism by which it facilitates transcription has been unclear. Here we show that JMJD3 regulates transcription of transforming growth factor ß (TGFß)-responsive genes by promoting RNA polymerase II (RNAPII) progression along the gene bodies. Using chromatin immunoprecipitation followed by sequencing experiments, we show that, upon TGFß treatment, JMJD3 and elongating RNAPII colocalize extensively along the intragenic regions of TGFß target genes. According to these data, genome-wide analysis shows that JMJD3-dependent TGFß target genes are enriched in H3K27me3 before TGFß signaling pathway activation. Further molecular analyses demonstrate that JMJD3 demethylates H3K27me3 along the gene bodies, paving the way for the RNAPII progression. Overall these findings uncover the mechanism by which JMJD3 facilitates transcriptional activation.