RESUMO
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Fator Inibidor de Leucemia/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Comunicação Parácrina , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Carcinogênese/genética , Carcinoma Ductal Pancreático/diagnóstico , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Feminino , Humanos , Fator Inibidor de Leucemia/antagonistas & inibidores , Fator Inibidor de Leucemia/sangue , Masculino , Espectrometria de Massas , Camundongos , Neoplasias Pancreáticas/diagnóstico , Comunicação Parácrina/efeitos dos fármacos , Receptores de OSM-LIF/deficiência , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/metabolismo , Microambiente TumoralRESUMO
This study develops a "Skill Talent Ecological Evaluation Model" across cultivation, potential energy, kinetic energy, innovation, and service and support ecologies. AHP-entropy determines indicator weights, Hopfield neural network assesses talent ecology levels, and the PVAR model analyzes digital transformation effects. Findings reveal: Cultivation ecology rates A, potential ecology rates B+, kinetic ecology rates B-, service and support ecology rates B-, and innovation ecology rates C. Digital transformation spurs skill demand, impacting talent and economic contributions. Kinetic ecology sees increased demand, potentially impacting traditional industries positively. Innovation ecology necessitates continuous skill learning. Service and support ecology witnesses growth in digital entrepreneurship, requiring policy incentives and incubation center support.
Assuntos
Ecossistema , Redes Neurais de Computação , China , Humanos , Ecologia/tendências , Aptidão , Modelos TeóricosRESUMO
Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.
RESUMO
Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pâncreas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Fibroblastos/metabolismo , Carcinogênese/patologia , Microambiente TumoralRESUMO
Histone methylation is important in regulating chromatin structure and function. In budding yeast, methylation of histone H3 at Lys4 (H3-K4) is associated with active transcription and is enriched at the 5' regions of transcribed genes. Here we identify a novel budding yeast JmjC domain-containing H3-K4 demethylase, Jhd2p, that antagonizes the trimethyl modification state and contributes to regulation of telomeric silencing.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Inativação Gênica , Histona Desmetilases com o Domínio Jumonji , Saccharomyces cerevisiae/citologia , Telômero/metabolismoRESUMO
Background: Ketogenic diet is a potential means of augmenting cancer therapy. Here, we explore ketone body metabolism and its interplay with chemotherapy in pancreatic cancer. Methods: Metabolism and therapeutic responses of murine pancreatic cancer were studied using KPC primary tumors and tumor chunk allografts. Mice on standard high-carbohydrate diet or ketogenic diet were treated with cytotoxic chemotherapy (nab-paclitaxel, gemcitabine, cisplatin). Metabolic activity was monitored with metabolomics and isotope tracing, including 2H- and 13C-tracers, liquid chromatography-mass spectrometry, and imaging mass spectrometry. Findings: Ketone bodies are unidirectionally oxidized to make NADH. This stands in contrast to the carbohydrate-derived carboxylic acids lactate and pyruvate, which rapidly interconvert, buffering NADH/NAD. In murine pancreatic tumors, ketogenic diet decreases glucose's concentration and tricarboxylic acid cycle contribution, enhances 3-hydroxybutyrate's concentration and tricarboxylic acid contribution, and modestly elevates NADH, but does not impact tumor growth. In contrast, the combination of ketogenic diet and cytotoxic chemotherapy substantially raises tumor NADH and synergistically suppresses tumor growth, tripling the survival benefits of chemotherapy alone. Chemotherapy and ketogenic diet also synergize in immune-deficient mice, although long-term growth suppression was only observed in mice with an intact immune system. Conclusions: Ketogenic diet sensitizes murine pancreatic cancer tumors to cytotoxic chemotherapy. Based on these data, we have initiated a randomized clinical trial of chemotherapy with standard versus ketogenic diet for patients with metastatic pancreatic cancer (NCT04631445).
Assuntos
Dieta Cetogênica , Neoplasias Pancreáticas , Animais , Carboidratos , Dieta Cetogênica/métodos , Humanos , Camundongos , NAD , Neoplasias Pancreáticas/dietoterapia , Neoplasias Pancreáticas/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto , Neoplasias PancreáticasRESUMO
Recent studies have demonstrated that embryonic stem cell-like induced pluripotent stem (iPS) cells can be generated by enforced expression of defined transcription factors. The fact that cell fate change is accompanied by changes in epigenetic modifications prompted us to investigate whether chemicals known to modulate epigenetic regulators are capable of enhancing the efficiency of iPS cell generation. Here, we report that butyrate, a natural small fatty acid and histone deacetylase inhibitor, significantly increases the efficiency of mouse iPS cell generation using the transcription factors Oct4, Sox2, Klf4, and c-Myc. We show that butyrate not only changes the reprogramming dynamics, but also increases the ratio of iPS cell colonies to total colonies by reducing the frequency of partially reprogrammed cells and transformed cells. Detailed analysis reveals that the effect of butyrate on reprogramming appears to be mediated by c-Myc and occurs during an early stage of reprogramming. Genome-wide gene expression analysis reveals up-regulation of ES cell-enriched genes when mouse embryonic fibroblasts are treated with butyrate during reprogramming. Thus, our study identifies butyrate as a chemical factor capable of promoting iPS cell generation.
Assuntos
Butiratos/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady-state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in Saccharomyces cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation-coupled microarray studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Deleção de Genes , Células HeLa , Humanos , Ferro/farmacologia , Histona Desmetilases com o Domínio Jumonji , Ácidos Cetoglutáricos/farmacologia , Metilação/efeitos dos fármacos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/isolamento & purificação , Fenótipo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Histone methylation is an important posttranslational modification that contributes to chromatin-based processes including transcriptional regulation, DNA repair, and epigenetic inheritance. In the budding yeast Saccharomyces cerevisiae, histone lysine methylation occurs on histone H3 lysines 4, 36, and 79, and its deposition is coupled mainly to transcription. Until recently, histone methylation was considered to be irreversible, but the identification of histone demethylase enzymes has revealed that this modification can be dynamically regulated. In budding yeast, there are five proteins that contain the JmjC domain, a signature motif found in a large family of histone demethylases spanning many organisms. One JmjC-domain-containing protein in budding yeast, Jhd1, has recently been identified as being a histone demethylase that targets H3K36 modified in the di- and monomethyl state. Here, we identify a second JmjC-domain-containing histone demethylase, Rph1, which can specifically demethylate H3K36 tri- and dimethyl modification states. Surprisingly, Rph1 can remove H3K9 methylation, a histone modification not found in budding yeast chromatin. The capacity of Rph1 to demethylate H3K9 provides the first indication that S. cerevisiae may have once encoded an H3K9 methylation system and suggests that Rph1 is a functional vestige of this modification system.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Histona Desmetilases , Metilação , Fenótipo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of approximately 10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex. RESULTS: We have found that C-terminal processing of Spt7 to its SLIK form (Spt7SLIK) and to a distinct third form (Spt7Form3) occurs in the absence of the SAGA complex components Gcn5, Spt8, Ada1 and Spt20, the latter two of which are required for the integrity of the complex. In addition, N-terminally truncated derivatives of Spt7, including a derivative lacking the histone fold, are processed, indicating that the C-terminus of Spt7 is sufficient for processing and that processing does not require functional Spt7. Using galactose inducible Spt7 expression, we show that the three forms of Spt7 appear and disappear at approximately the same rate with full-length Spt7 not being chased into Spt7SLIK or Spt7Form3. Interestingly, reduced levels of Spt7SLIK and Spt7Form3 were observed in a strain lacking the SAGA component Ubp8, suggesting a regulatory role for Ubp8 in the truncation of Spt7. CONCLUSION: We conclude that truncation of Spt7 occurs early in the biosynthesis of distinct Spt7 containing complexes rather than being a dynamic process linked to the action of the SAGA complex in transcriptional regulation.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/deficiência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Epitopos/imunologia , Deleção de Genes , Peso Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
The direct conversion of fibroblasts to induced dopaminergic (iDA) neurons and other cell types demonstrates the plasticity of cell fate. The low efficiency of these relatively fast conversions suggests that kinetic barriers exist to safeguard cell-type identity. Here we show that suppression of p53, in conjunction with cell cycle arrest at G1 and appropriate extracellular environment, markedly increase the efficiency in the transdifferentiation of human fibroblasts to iDA neurons by Ascl1, Nurr1, Lmx1a and miR124. The conversion is dependent on Tet1, as G1 arrest, p53 knockdown or expression of the reprogramming factors induces Tet1 synergistically. Tet1 knockdown abolishes the transdifferentiation while its overexpression enhances the conversion. The iDA neurons express markers for midbrain DA neurons and have active dopaminergic transmission. Our results suggest that overcoming these kinetic barriers may enable highly efficient epigenetic reprogramming in general and will generate patient-specific midbrain DA neurons for Parkinson's disease research and therapy.
Assuntos
Transdiferenciação Celular/genética , Neurônios Dopaminérgicos/citologia , Fibroblastos/citologia , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Proteína Supressora de Tumor p53/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Reprogramação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Mesencéfalo , MicroRNAs/genética , Oxigenases de Função Mista , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine. However, recent studies on the genetic and epigenetic variations in iPSCs have raised concerns that these variations may compromise the utility of iPSCs. In this Perspective, we review the current understanding of genetic and epigenetic variations in iPSCs, trace their causes, discuss the implications of these variations for iPSC applications, and propose approaches to cope with these variations.
Assuntos
Epigênese Genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Transplante de Células-Tronco , Animais , Variação Genética , HumanosRESUMO
Pluripotent stem cells, like embryonic stem cells (ESCs), have specialized epigenetic landscapes, which are important for pluripotency maintenance. Transcription factor-mediated generation of induced pluripotent stem cells (iPSCs) requires global change of somatic cell epigenetic status into an ESC-like state. Accumulating evidence indicates that epigenetic mechanisms not only play important roles in the iPSC generation process, but also affect the properties of reprogrammed iPSCs. Understanding the roles of various epigenetic factors in iPSC generation contributes to our knowledge of the reprogramming mechanisms.
Assuntos
Células-Tronco Embrionárias/citologia , Epigenômica , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular , Reprogramação Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismoRESUMO
Transcription-factor-directed reprogramming from somatic cells to induced pluripotent stem cells (iPSCs) is by nature an epigenetic process of cell fate change. Previous studies have demonstrated that this inefficient process can be facilitated by the inclusion of additional factors. To gain insight into the reprogramming mechanism, we aimed to identify epigenetic enzymes capable of promoting iPSC generation. Here we show that Kdm2b, a histone H3 Lys 36 dimethyl (H3K36me2)-specific demethylase, has the capacity to promote iPSC generation. This capacity depends on its demethylase and DNA-binding activities, but is largely independent of its role in antagonizing senescence. Kdm2b functions at the beginning of the reprogramming process and enhances activation of early responsive genes in reprogramming. Kdm2b contributes to gene activation by binding to and demethylating the gene promoters. Our studies not only identify an important epigenetic factor for iPSC generation, but also reveal the molecular mechanism underlying how Kdm2b contributes to reprogramming.
Assuntos
Proteínas F-Box/fisiologia , Regulação da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/fisiologia , Células-Tronco Pluripotentes/citologia , Animais , Proliferação de Células , Epigênese Genética , Camundongos , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex of Saccharomyces cerevisiae contains more than 20 components that acetylate and deubiquitylate nucleosomal histones. Its acetyltransferase, Gcn5, preferentially acetylates histones H3 and H2B and is regulated through interactions with Ada2 and Ngg1/Ada3. Sequence alignments of Ada2 homologs indicate a conserved approximately 120-amino-acid-residue central region. To examine the function of this region, we constructed ada2 alleles with mutations of clustered conserved residues. One of these alleles, ada2-RLR (R211S, L212A, and R215A), resulted in an approximately threefold reduction in transcriptional activation of the PHO5 gene and growth changes that parallel deletion of ada2. Microarray analyses further revealed that ada2-RLR alters expression of a subset of those genes affected by deletion of ada2. Indicative of Ada2-RLR affecting Gcn5 function, Ada2-RLR resulted in a decrease in Gcn5-mediated histone acetylation in vitro to a level approximately 40% that with wild-type Ada2. In addition, in vivo acetylation of K16 of histone H2B was almost totally eliminated at Ada2-regulated promoters in the ada2-RLR strain, while acetylation of K9 and K18 of histone H3 was reduced to approximately 40% of wild-type levels. We also show that the central region of Ada2 interacts with phospholipids. Since phosphatidylserine binding paralleled Ada2 function, we suggest that lipid binding may play a role in the function or regulation of the SAGA complex.
Assuntos
Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Fosfatase Ácida , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Perfilação da Expressão Gênica , Histonas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Fatores de Transcrição/genéticaRESUMO
Increasing evidence suggests that islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors and the necessity for chronic immunosuppression has made it impossible for this treatment to be used for the general diabetic population. Recent success in generating insulin-secreting islet-like cells from human embryonic stem (ES) cells, in combination with the success in deriving human ES cell-like induced pluripotent stem (iPS) cells from human fibroblasts by defined factors, have raised the possibility that patient-specific insulin-secreting islet-like cells might be derived from somatic cells through cell fate reprogramming using defined factors. Here we confirm that human ES-like iPS cells can be derived from human skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free protocol, we successfully generated insulin-producing islet-like clusters (ILCs) from the iPS cells under feeder-free conditions. We demonstrate that, like human ES cells, skin fibroblast-derived iPS cells have the potential to be differentiated into islet-like clusters through definitive and pancreatic endoderm. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Thus, our study provides evidence that insulin-secreting ILCs can be generated from skin fibroblasts, raising the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future.
Assuntos
Células Secretoras de Insulina/metabolismo , Pele/metabolismo , Diferenciação Celular , Linhagem Celular , Fibroblastos , Humanos , Células Secretoras de Insulina/citologia , Fator 4 Semelhante a Kruppel , Pele/citologiaRESUMO
In Saccharomyces cerevisiae histone H2B is ubiquitylated at lysine 123 in a process requiring the E2-ubiquitin conjugase, Rad6. We have analyzed gene expression in a strain containing a variant of histone H2B with lysine 123 converted to arginine to address the mechanisms by which ubiquitylation and deubiquitylation of histone H2B affect gene expression. The SAGA complex component, Ubp8, is one of two proteases that remove the ubiquitin moiety at lysine 123. We show that changes in gene expression observed upon deletion of ubp8 are suppressed by htb1 ( K123R ), which provides genetic evidence that Ubp8 alters gene expression through deubiquitylation of histone H2B. Microarray analyses of the htb1 ( K123R ) strain show that loss of histone ubiquitylation results in a twofold or greater change in expression of approximately 1.5% of the protein coding genes with approximately 75% of these increasing. For genes in which ubiquitylation represses expression, ubiquitylation principally acts through its effects on histone methylation. In contrast, decreased expression of the CWP1 gene was not paralleled by deletions of methyltransferase components and is thus likely independent of methylation. Finally, by comparing gene expression changes in the htb1 ( K123R ) strain with those in a strain deleted for rad6, we conclude that lysine 123 affects transcription primarily because of it being a site of ubiquitylation.