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2.
Genome Biol ; 20(1): 155, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387612

RESUMO

We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/ß-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.


Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Neoplasias da Mama/genética , Análise Custo-Benefício , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica/economia , Genômica/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA/economia , Análise de Célula Única/economia , Análise de Célula Única/métodos , Via de Sinalização Wnt , Fluxo de Trabalho
3.
Cell Physiol Biochem ; 53(2): 337-354, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31373783

RESUMO

BACKGROUND/AIMS: The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells. METHODS: To address these issues, we have refined existing forward programming protocols by combining forced exogenous overexpression of the early cardiovascular transcription factor Nkx2.5 with a αMHC-promoter-based antibiotic selection step. Additionally, we applied small molecules such as ascorbic acid to enhance cardiomyogenic differentiation efficiency. Subsequently, we evaluated the cell fate of the resulting cardiomyocytes on the mRNA as well as protein levels. The latter was performed using high-resolution confocal microscopy. Furthermore, we examined the response of the cells` beating activities to pharmacological substance administration. RESULTS: Our results reveal an apparent influence of Nkx2.5 on the cell fate of ESC-derived cardiomyocytes. Resulting single cells exhibit characteristics of early ventricular cardiomyocytes, such as sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium channel occurrence. CONCLUSION: Therefore, we demonstrate cardiovascular subtype forward programming of ESCs using a combination of transcription factors along with small molecule administration. However, our findings also underline current assumptions, that a terminal maturation of PSC derived cardiomyocytes in vitro is still an unsolved problem which urgently needs to be addressed in the field.


Assuntos
Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Proteína Homeobox Nkx-2.5/antagonistas & inibidores , Proteína Homeobox Nkx-2.5/genética , Camundongos , Microscopia Confocal , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Verapamil/farmacologia
4.
Plast Reconstr Surg ; 144(2): 372-384, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31348346

RESUMO

BACKGROUND: We investigated expression of prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 by the embryonic stem cell-like population on the endothelium of the microvessels and perivascular cells within keloid-associated lymphoid tissues. METHODS: Immunohistochemical staining for prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 was performed on 11 formalin-fixed, paraffin-embedded sections of keloid tissue samples. Immunofluorescence staining was performed on three keloid tissue samples by co-staining with OCT4, CD34, ERG, and tryptase. Real-time quantitative polymerase chain reaction was performed on five keloid tissue samples and four keloid-derived primary cell lines. Western blotting was performed on the four keloid-derived primary cell lines for mRNA and protein expression of these proteins, respectively. RESULTS: Immunohistochemical and immunofluorescence staining showed expression of prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 in all 11 keloid tissue samples. Prorenin receptor and angiotensin II receptor 1 were expressed on the endothelium and the pericyte layer of the microvessels and perivascular cells, angiotensin II receptor 2 was localized to the endothelium of the microvessels and the tryptase-positive perivascular cells, and angiotensin-converting enzyme was localized to the endothelium of the microvessel, within the keloid-associated lymphoid tissues. Real-time quantitative polymerase chain reaction showed transcripts of prorenin receptor, angiotensin-converting enzyme, and angiotensin II receptor 1 in the keloid tissue samples and keloid-derived primary cell lines, whereas angiotensin II receptor 2 was detected in keloid tissue samples only. Western blotting confirmed the presence of prorenin receptor, angiotensin-converting enzyme, and angiotensin II receptor 1 in the keloid-derived primary cell lines. CONCLUSION: Prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 were expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues, suggesting that this primitive population may be a potential therapeutic target by modulation of the renin-angiotensin system.


Assuntos
Células-Tronco Embrionárias/metabolismo , Queloide/metabolismo , Sistema Renina-Angiotensina/fisiologia , Adolescente , Adulto , Antígenos CD34/metabolismo , Linhagem Celular , Criança , Pré-Escolar , Feminino , Imunofluorescência , Humanos , Queloide/patologia , Masculino , Pessoa de Meia-Idade , Fator 3 de Transcrição de Octâmero/metabolismo , Peptidil Dipeptidase A/metabolismo , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores de Superfície Celular/metabolismo , Regulador Transcricional ERG/metabolismo , Adulto Jovem
5.
Cancer Sci ; 110(10): 3027-3037, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31348591

RESUMO

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS-ML. Human iPS-ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS-ML cells expressing interferon ß (iPS-ML/interferon (IFN)-ß) in xenograft cancer models. However, assessment of host immune system-mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS-ML/IFN, a mouse embryonic stem (ES) cell-derived myeloid cell line producing IFN (ES-ML/IFN). The ES-MLs producing IFN-ß (ß-ML) and IFN-γ (γ-ML) and originating from E14 ES cells derived from the 129 mouse strain (H-2b ) were generated, and the MHC (H-2Kb , Db , and I-Ab ) genes of the ES-ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES-ML/IFN to treat allogeneic BALB/c mice (H-2d ) transplanted with Colon26 cancer cells. Treatment with ß-ML but not with γ-ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES-ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES-ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen-specific CD8+ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8+ T cells were essential for the therapeutic effect, implying that donor MHC-deficient ß-ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA-deficient human iPS-ML/IFN-ß cells for therapy of HLA-mismatched allogeneic cancer patients.


Assuntos
Neoplasias do Colo/terapia , Células-Tronco Embrionárias/citologia , Antígenos de Histocompatibilidade/genética , Interferon beta/metabolismo , Células Mieloides/transplante , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Matadoras Naturais/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/citologia , Células Mieloides/metabolismo , Transplante Homólogo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nat Cell Biol ; 21(7): 835-844, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209294

RESUMO

Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell-embryo-specific elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.


Assuntos
Reprogramação Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Células-Tronco Embrionárias/metabolismo , Genes myc/genética , Animais , Diferenciação Celular/genética , Epigênese Genética/genética , Camundongos , Células-Tronco Embrionárias Murinas , Células-Tronco Pluripotentes/citologia , Células-Tronco Totipotentes/citologia
7.
Int J Mol Sci ; 20(11)2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151253

RESUMO

Ubiquitination regulates nearly every aspect of cellular events in eukaryotes. It modifies intracellular proteins with 76-amino acid polypeptide ubiquitin (Ub) and destines them for proteolysis or activity alteration. Ubiquitination is generally achieved by a tri-enzyme machinery involving ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). E1 activates Ub and transfers it to the active cysteine site of E2 via a transesterification reaction. E3 coordinates with E2 to mediate isopeptide bond formation between Ub and substrate protein. The E1-E2-E3 cascade can create diverse types of Ub modifications, hence effecting distinct outcomes on the substrate proteins. Dysregulation of ubiquitination results in severe consequences and human diseases. There include cancers, developmental defects and immune disorders. In this review, we provide an overview of the ubiquitination machinery and discuss the recent progresses in the ubiquitination-mediated regulation of embryonic stem cell maintenance and cancer biology.


Assuntos
Autorrenovação Celular , Transformação Celular Neoplásica/metabolismo , Células-Tronco Embrionárias/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Autorrenovação Celular/genética , Transformação Celular Neoplásica/genética , Células-Tronco Embrionárias/citologia , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Neoplasias/patologia , Transdução de Sinais , Ubiquitinação
8.
BMC Genomics ; 20(1): 439, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151386

RESUMO

BACKGROUND: The last decade witnessed a number of genome-wide studies on human pre-implantation, which mostly focused on genes and provided only limited information on repeats, excluding the satellites. Considering the fact that repeats constitute a large portion of our genome with reported links to human physiology and disease, a thorough understanding of their spatiotemporal regulation during human embryogenesis will give invaluable clues on chromatin dynamics across time and space. Therefore, we performed a detailed expression analysis of all repetitive DNA elements including the satellites across stages of human pre-implantation and embryonic stem cells. RESULTS: We uncovered stage-specific expressions of more than a thousand repeat elements whose expressions fluctuated with a mild global decrease at the blastocyst stage. Most satellites were highly expressed at the 4-cell level and expressions of ACRO1 and D20S16 specifically peaked at this point. Whereas all members of the SVA elements were highly upregulated at 8-cell and morula stages, other transposons and small RNA repeats exhibited a high level of variation among their specific subtypes. Our repeat enrichment analysis in gene promoters coupled with expression correlations highlighted potential links between repeat expressions and nearby genes, emphasising mostly 8-cell and morula specific genes together with SVA_D, LTR5_Hs and LTR70 transposons. The DNA methylation analysis further complemented the understanding on the mechanistic aspects of the repeatome's regulation per se and revealed critical stages where DNA methylation levels are negatively correlating with repeat expression. CONCLUSIONS: Taken together, our study shows that specific expression patterns are not exclusive to genes and long non-coding RNAs but the repeatome also exhibits an intriguingly dynamic pattern at the global scale. Repeats identified in this study; particularly satellites, which were historically associated with heterochromatin, and those with potential links to nearby gene expression provide valuable insights into the understanding of key events in genomic regulation and warrant further research in epigenetics, genomics and developmental biology.


Assuntos
DNA/química , Desenvolvimento Embrionário/genética , Expressão Gênica , Sequências Repetitivas de Ácido Nucleico , Metilação de DNA , DNA Satélite/química , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , Elementos Nucleotídeos Longos e Dispersos , Elementos Nucleotídeos Curtos e Dispersos
9.
Nat Genet ; 51(6): 941-946, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31152160

RESUMO

The histone variant H3.3 is enriched at enhancers and active genes, as well as repeat regions such as telomeres and retroelements, in mouse embryonic stem cells (mESCs)1-3. Although recent studies demonstrate a role for H3.3 and its chaperones in establishing heterochromatin at repeat regions4-8, the function of H3.3 in transcription regulation has been less clear9-16. Here, we find that H3.3-specific phosphorylation17-19 stimulates activity of the acetyltransferase p300 in trans, suggesting that H3.3 acts as a nucleosomal cofactor for p300. Depletion of H3.3 from mESCs reduces acetylation on histone H3 at lysine 27 (H3K27ac) at enhancers. Compared with wild-type cells, those lacking H3.3 demonstrate reduced capacity to acetylate enhancers that are activated upon differentiation, along with reduced ability to reprogram cell fate. Our study demonstrates that a single amino acid in a histone variant can integrate signaling information and impact genome regulation globally, which may help to better understand how mutations in these proteins contribute to human cancers20,21.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Histonas/metabolismo , Serina/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional
10.
Genome Biol ; 20(1): 119, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174582

RESUMO

BACKGROUND: The uneven use of synonymous codons in the transcriptome regulates the efficiency and fidelity of protein translation rates. Yet, the importance of this codon bias in regulating cell state-specific expression programmes is currently debated. Here, we ask whether different codon usage controls gene expression programmes in self-renewing and differentiating embryonic stem cells. RESULTS: Using ribosome and transcriptome profiling, we identify distinct codon signatures during human embryonic stem cell differentiation. We find that cell state-specific codon bias is determined by the guanine-cytosine (GC) content of differentially expressed genes. By measuring the codon frequencies at the ribosome active sites interacting with transfer RNAs (tRNA), we further discover that self-renewing cells optimize translation of codons that depend on the inosine tRNA modification in the anticodon wobble position. Accordingly, inosine levels are highest in human pluripotent embryonic stem cells. This effect is conserved in mice and is independent of the differentiation stimulus. CONCLUSIONS: We show that GC content influences cell state-specific mRNA levels, and we reveal how translational mechanisms based on tRNA modifications change codon usage in embryonic stem cells.


Assuntos
Códon , Células-Tronco Embrionárias/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Animais , Composição de Bases , Autorrenovação Celular , Humanos
11.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
12.
Nat Genet ; 51(6): 1024-1034, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133748

RESUMO

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Inativação do Cromossomo X , Animais , Diferenciação Celular/genética , Expressão Ectópica do Gene , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Inativação Gênica , Loci Gênicos , Masculino , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Inversão de Sequência , Transcrição Genética
13.
Nat Methods ; 16(6): 489-492, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133759

RESUMO

Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).


Assuntos
Cromatina/química , Cromatina/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , RNA/química , Telomerase/química , Animais , Células Cultivadas , Cromossomos , Células-Tronco Embrionárias/citologia , Genoma , Camundongos , Regiões Promotoras Genéticas , RNA/genética , Telomerase/genética
14.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067653

RESUMO

In recent years, great interest has been devoted to finding alternative sources for human stem cells which can be easily isolated, ideally without raising ethical objections. These stem cells should furthermore have a high proliferation rate and the ability to differentiate into all three germ layers. Amniotic fluid, ordinarily discarded as medical waste, is potentially such a novel source of stem cells, and these amniotic fluid derived stem cells are currently gaining a lot of attention. However, further information will be required about the properties of these cells before they can be used for therapeutic purposes. For example, the risk of tumor formation after cell transplantation needs to be explored. The tumor suppressor protein p53, well known for its activity in controlling Cell Prolif.eration and cell death in differentiated cells, has more recently been found to be also active in amniotic fluid stem cells. In this review, we summarize the major findings about human amniotic fluid stem cells since their discovery, followed by a brief overview of the important role played by p53 in embryonic and adult stem cells. In addition, we explore what is known about p53 in amniotic fluid stem cells to date, and emphasize the need to investigate its role, particularly in the context of cell tumorigenicity.


Assuntos
Líquido Amniótico/citologia , Células-Tronco Embrionárias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Líquido Amniótico/metabolismo , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Humanos , Proteína Supressora de Tumor p53/genética
15.
Nat Commun ; 10(1): 1465, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931944

RESUMO

Embryonic stem cells (ESCs) can propagate in an undifferentiated state indefinitely in culture and retain the potential to differentiate into any somatic lineage as well as germ cells. The catabolic process autophagy has been reported to be involved in ESC identity regulation, but the underlying mechanism is still largely unknown. Here we show that EPG5, a eukaryotic-specific autophagy regulator which mediates autophagosome/lysosome fusion, is highly expressed in ESCs and contributes to ESC identity maintenance. We identify that the deubiquitinating enzyme USP8 binds to the Coiled-coil domain of EPG5. Mechanistically, USP8 directly removes non-classical K63-linked ubiquitin chains from EPG5 at Lysine 252, leading to enhanced interaction between EPG5 and LC3. We propose that deubiquitination of EPG5 by USP8 guards the autophagic flux in ESCs to maintain their stemness. This work uncovers a novel crosstalk pathway between ubiquitination and autophagy through USP8-EPG5 interaction to regulate the stemness of ESCs.


Assuntos
Autofagia/genética , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas/metabolismo , Ubiquitina Tiolesterase/genética , Animais , Autofagossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo , Lisossomos/metabolismo , Fusão de Membrana/genética , Camundongos , Células-Tronco Neurais/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular
16.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965622

RESUMO

Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.


Assuntos
MicroRNAs/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo
17.
BMC Genomics ; 20(Suppl 2): 221, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30967107

RESUMO

BACKGROUND: Epigenome is highly dynamic during the early stages of embryonic development. Epigenetic modifications provide the necessary regulation for lineage specification and enable the maintenance of cellular identity. Given the rapid accumulation of genome-wide epigenomic modification maps across cellular differentiation process, there is an urgent need to characterize epigenetic dynamics and reveal their impacts on differential gene regulation. METHODS: We proposed DiffEM, a computational method for differential analysis of epigenetic modifications and identified highly dynamic modification sites along cellular differentiation process. We applied this approach to investigating 6 epigenetic marks of 20 kinds of human early developmental stages and tissues, including hESCs, 4 hESC-derived lineages and 15 human primary tissues. RESULTS: We identified highly dynamic modification sites where different cell types exhibit distinctive modification patterns, and found that these highly dynamic sites enriched in the genes related to cellular development and differentiation. Further, to evaluate the effectiveness of our method, we correlated the dynamics scores of epigenetic modifications with the variance of gene expression, and compared the results of our method with those of the existing algorithms. The comparison results demonstrate the power of our method in evaluating the epigenetic dynamics and identifying highly dynamic regions along cell differentiation process.


Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Epigenômica , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano , Diferenciação Celular , Histonas/genética , Histonas/metabolismo , Humanos , Especificidade de Órgãos
18.
PLoS Genet ; 15(4): e1008058, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30933982

RESUMO

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Padronização Corporal , Diferenciação Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/citologia , Brânquias/embriologia , Brânquias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte de Íons , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Receptores Notch/genética , Receptores Notch/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
19.
Nature ; 569(7754): 136-140, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30996347

RESUMO

Chromatin remodelling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. These remodellers function in most aspects of genome utilization including transcription-factor binding, DNA replication and repair1,2. Although they are frequently mutated in cancer3, it remains largely unclear how the four mammalian remodeller families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. Here we generated viable embryonic stem cells that lack SNF2H, the ATPase of ISWI complexes, enabling study of SNF2H cellular function, and contrast it to BRG1, the ATPase of SWI/SNF. Loss of SNF2H decreases nucleosomal phasing and increases linker lengths, providing in vivo evidence for an ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription-factor binding reveals that these remodelling activities have specific effects on binding of different transcription factors. One group critically depends on BRG1 and contains the transcriptional repressor REST, whereas a non-overlapping set of transcription factors, including the insulator protein CTCF, relies on SNF2H. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires SNF2H, but not BRG1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodelling pathways for correct genomic binding.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica
20.
J Agric Food Chem ; 67(28): 7977-7985, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30932489

RESUMO

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant heterocyclic aromatic amines (HAAs) found in the human diet, is primarily produced during high-temperature meat or fish cooking. While MeIQx has been investigated as a potential carcinogen, the cytotoxicity and related molecular mechanisms remain unclear. Here, we demonstrate that autophagosome maturation is blocked by MeIQx. Mechanistically, MeIQx inhibits acidification of lysosomes rather than prevents autophagosome-lysosome fusion. Moreover, cellular lipid profiles are altered by MeIQx treatment. Notably, many phospholipids and sphingolipids are significantly upregulated after exposure to MeIQx. Furthermore, MeIQx decreases expression of pluripotency-associated proteins in mouse embryonic stem cells (ESCs). Together, MeIQx blocks autophagosome maturation through inhibiting acidification of lysosomes, alters lipid metabolism, and decreases expression of pluripotent factors. Our studies provide more cytotoxic evidence and elucidate related mechanisms on the risk of HAA exposure and are expected to promote supervision of food safety and human health.


Assuntos
Autofagossomos/efeitos dos fármacos , Lipídeos/química , Quinoxalinas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Transcrição/genética
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