RESUMO
Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.
Assuntos
Diferenciação Celular/genética , Cromatina/metabolismo , Neurônios Dopaminérgicos/citologia , Células-Tronco Embrionárias Humanas/citologia , Transcriptoma/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Motivos de Nucleotídeos/genética , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA-Seq , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel regulation determines the commitment of stem cells and their differentiation into a specific cell lineage. Cellular metabolism plays a relevant role in modulating the expression of genes, which act as sensors of the extra-and intracellular environment. In this work, we analyzed mRNAs associated with polysomes by focusing on the expression profile of metabolism-related genes during the cardiac differentiation of human embryonic stem cells (hESCs). We compared different time points during cardiac differentiation (pluripotency, embryoid body aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte) and showed the immature cell profile of energy metabolism. Highly regulated canonical pathways are thoroughly discussed, such as those involved in metabolic signaling and lipid homeostasis. We reveal the critical relevance of retinoic X receptor (RXR) heterodimers in upstream retinoic acid metabolism and their relationship with thyroid hormone signaling. Additionally, we highlight the importance of lipid homeostasis and extracellular matrix component biosynthesis during cardiomyogenesis, providing new insights into how hESCs reorganize their metabolism during in vitro cardiac differentiation.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Miócitos Cardíacos/citologia , Transdução de Sinais , Diferenciação Celular , Linhagem Celular , Metabolismo Energético , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Metabolismo dos Lipídeos , Miócitos Cardíacos/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted exclusively to germline cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in pluripotent, neural, cardiac and even cancer cells. However, controversy remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNA transcripts, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences of amplification loop (ping-pong) were found. Genes hosting piRNA transcripts in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that these piRNAs might have a role in fine-tuning the expression of genes involved in differentiation of pluripotent cells to cardiomyocytes.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Miócitos Cardíacos/citologia , RNA Interferente Pequeno/genética , Adulto , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismoRESUMO
Posttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA-binding proteins (RBPs) that orchestrate mRNA expression. A family of RBPs, which is known as the Pumilio-FBF (PUF) family, is highly conserved among different species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first assessed the influence of the silencing of PUM1 and PUM2 on pluripotency genes and found that the knockdown of Pumilio genes significantly decreased the OCT4 and NANOG mRNA levels and reduced the amount of nuclear OCT4, which suggests that Pumilio proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that PUM1-and-PUM2-silenced hESCs exhibited improved efficiency of in vitro cardiomyogenic differentiation. Through an in silico analysis, we identified mRNA targets of PUM1 and PUM2 that are expressed at the early stages of cardiomyogenesis, and further investigation will determine whether these target mRNAs are active and involved in the progression of cardiomyogenesis. Our findings contribute to the understanding of the role of Pumilio proteins in hESC maintenance and differentiation.
Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Proteínas de Ligação a RNA/fisiologia , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Humanos , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/metabolismoRESUMO
Stem cells genome safeguarding requires strict oxidative stress control. Heme oxygenase-1 (HO-1) and p53 are relevant components of the cellular defense system. p53 controls cellular response to multiple types of harmful stimulus, including oxidative stress. Otherwise, besides having a protective role, HO-1 is also involved in embryo development and in embryonic stem (ES) cells differentiation. Although both proteins have been extensively studied, little is known about their relationship in stem cells. The aim of this work is to explore HO-1-p53 interplay in ES cells. We studied HO-1 expression in p53 knockout (KO) ES cells and we found that they have higher HO-1 protein levels but similar HO-1 mRNA levels than the wild type (WT) ES cell line. Furthermore, cycloheximide treatment increased HO-1 abundance in p53 KO cells suggesting that p53 modulates HO-1 protein stability. Notably, H2O2 treatment did not induce HO-1 expression in p53 KO ES cells. Finally, SOD2 protein levels are also increased while Sod2 transcripts are not in KO cells, further suggesting that the p53 null phenotype is associated with a reinforcement of the antioxidant machinery. Our results demonstrate the existence of a connection between p53 and HO-1 in ES cells, highlighting the relationship between these stress defense pathways.
Assuntos
Heme Oxigenase-1/fisiologia , Células-Tronco Embrionárias Humanas , Proteína Supressora de Tumor p53/fisiologia , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Heme Oxigenase-1/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Estresse Oxidativo , Transdução de Sinais , Superóxido Dismutase/metabolismoRESUMO
The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Integrina alfa5/genética , Miócitos Cardíacos/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Nicho de Células-TroncoRESUMO
BACKGROUND: The essentially unlimited expansion potential and the pluripotency of human embryonic stem cells (hESCs) make them attractive for cell-based therapeutic purposes. Although hESCs can indefinitely proliferate in culture, unlike transformed cancer cells, they are endowed with a cell-intrinsic property termed mitochondrial priming that renders them highly sensitive to apoptotic stimuli. Thus, all attempts to broaden the insights into hESCs apoptosis may be helpful for establishing pro-survival strategies valuable for its in vitro culture and further use in clinical applications. Cyclin-dependent kinases (CDKs), a family of serine/threonine protein kinases originally identified as regulators of the eukaryotic cell cycle, can also regulate transcription and differentiation. Moreover, there are compelling data suggesting that its activities are involved in certain apoptotic programs in different cell types. Currently, it is not completely determined whether CDKs regulate apoptotic processes in rapidly proliferating and apoptosis-prone hESCs. In this study, to elucidate the effect of CDKs inhibition in hESCs we used Roscovitine (ROSC), a purine analogue that selectively inhibits the activities of these kinases. RESULTS: Inhibition of CDKs by ROSC triggers programmed cell death in hESCs but not in proliferating somatic cells (human fibroblasts). The apoptotic process encompasses caspase-9 and -3 activation followed by PARP cleavage. ROSC treatment also leads to p53 stabilization, which coincides with site-specific phosphorylation at serine 46 and decreased levels of Mdm2. Additionally, we observed a transcriptional induction of p53AIP1, a repression of pro-survival factor Mcl-1 and an up-regulation of pro-apoptotic BH3-only proteins NOXA and PUMA. Importantly, we found that the role of CDK2 inhibition appears to be at best accessory as an active CDK2 is not required to ensure hESCs survival. CONCLUSION: Our experimental data reveal that hESCs, contrary to fibroblasts, exhibit a pronounced sensitivity to ROSC.
Assuntos
Quinases Ciclina-Dependentes/farmacologia , Células-Tronco Embrionárias Humanas/citologia , Inibidores de Proteínas Quinases/farmacologia , Roscovitina/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fosforilação/efeitos dos fármacos , Domínios Proteicos , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Cardiac cell fate specification occurs through progressive steps, and its gene expression regulation features are still being defined. There has been an increasing interest in understanding the coordination between transcription and post-transcriptional regulation during the differentiation processes. Here, we took advantage of the polysome profiling technique to isolate and high-throughput sequence ribosome-free and polysome-bound RNAs during cardiomyogenesis. RESULTS: We showed that polysome-bound RNAs exhibit the cardiomyogenic commitment gene expression and that mesoderm-to-cardiac progenitor stages are strongly regulated. Additionally, we compared ribosome-free and polysome-bound RNAs and found that the post-transcriptional regulation vastly contributes to cardiac phenotype determination, including RNA recruitment to and dissociation from ribosomes. Moreover, we found that protein synthesis is decreased in cardiomyocytes compared to human embryonic stem-cells (hESCs), possibly due to the down-regulation of translation-related genes. CONCLUSIONS: Our data provided a powerful tool to investigate genes potentially controlled by post-transcriptional mechanisms during the cardiac differentiation of hESC. This work could prospect fundamental tools to develop new therapy and research approaches.
Assuntos
Biomarcadores/análise , Diferenciação Celular , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Miócitos Cardíacos/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Células Cultivadas , Sequenciamento de Nucleotídeos em Larga Escala , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miócitos Cardíacos/citologia , Organogênese , Polirribossomos/genética , RNA Mensageiro/genéticaRESUMO
Although investigation with human embryonic stem cells (HESC) is not decreasing, the derivation of new lines has been diminished. The preeminence of only a few HESC lines in research is accompanied by lack of universal applicability of results as well as by genetic under-representation. We previously reported the derivation of one line with male karyotype from Mexican population. Here, we derived one HESC line (Amicqui-2) with female karyotype from poor-quality embryos. These line comply the pluripotent requirements (normal karyotype, detection of pluripotency-associated markers, mycoplasma test and teratoma formation) and could be a valuable model for studying diseases specific to under-represented population.
Assuntos
Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias Humanas/citologia , Animais , Linhagem Celular , Feminino , Humanos , México , CamundongosRESUMO
The regulation of gene expression acts at numerous complementary levels to control and refine protein abundance. The analysis of mRNAs associated with polysomes, called polysome profiling, has been used to investigate the post-transcriptional mechanisms that are involved in different biological processes. Pluripotent stem cells are able to differentiate into a variety of cell lineages, and the cell commitment progression is carefully orchestrated. Genome-wide expression profiling has provided the possibility to investigate transcriptional changes during cardiomyogenesis; however, a more accurate study regarding post-transcriptional regulation is required. In the present work, we isolated and high-throughput sequenced ribosome-free and polysome-bound RNAs from NKX2-5eGFP/w HES3 undifferentiated pluripotent stem cells at the subsequent differentiation stages of cardiomyogenesis: embryoid body aggregation, mesoderm, cardiac progenitor and cardiomyocyte. The expression of developmental markers was followed by flow cytometry, and quality analyses were performed as technical controls to ensure high quality data. Our dataset provides valuable information about hESC cardiac differentiation and can be used to investigate genes potentially controlled by post-transcriptional mechanisms.