RESUMO
Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Epigênese Genética , Interação Gene-Ambiente , Estudo de Associação Genômica Ampla , Núcleo Celular , Senescência Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Especificidade de ÓrgãosRESUMO
Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Paclitaxel/farmacologia , Piranos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica/tratamento farmacológico , Transplante de Neoplasias , Paclitaxel/uso terapêutico , Piranos/uso terapêuticoRESUMO
BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.
Assuntos
Glioblastoma , Proteínas Nucleares , Humanos , Sobrevivência Celular , Proteínas Nucleares/metabolismo , Glioblastoma/genética , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cinesinas/genética , Cinesinas/metabolismoRESUMO
Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.
Assuntos
Benzimidazóis/farmacologia , Proteína de Ligação a CREB/antagonistas & inibidores , Reprogramação Celular/efeitos dos fármacos , Proteína p300 Associada a E1A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Isoxazóis/farmacologia , Oxazepinas/farmacologia , Piperidinas/farmacologia , Benzimidazóis/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Inibidores Enzimáticos/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isoxazóis/química , Estrutura Molecular , Oxazepinas/química , Piperidinas/química , Domínios Proteicos/efeitos dos fármacosRESUMO
Epithelial-mesenchymal transition (EMT) is driven by complex signaling events that induce dramatic biochemical and morphological changes whereby epithelial cells are converted into cancer cells. However, the underlying molecular mechanisms remain elusive. Here, we used mass spectrometry based quantitative proteomics approach to systematically analyze the post-translational biochemical changes that drive differentiation of human mammary epithelial (HMLE) cells into mesenchymal. We identified 314 proteins out of more than 6,000 unique proteins and 871 phosphopeptides out of more than 7,000 unique phosphopeptides as differentially regulated. We found that phosphoproteome is more unstable and prone to changes during EMT compared with the proteome and multiple alterations at proteome level are not thoroughly represented by transcriptional data highlighting the necessity of proteome level analysis. We discovered cell state specific signaling pathways, such as Hippo, sphingolipid signaling, and unfolded protein response (UPR) by modeling the networks of regulated proteins and potential kinase-substrate groups. We identified two novel factors for EMT whose expression increased on EMT induction: DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) and cluster of differentiation 81 (CD81). Suppression of DNAJB4 or CD81 in mesenchymal breast cancer cells resulted in decreased cell migration in vitro and led to reduced primary tumor growth, extravasation, and lung metastasis in vivo Overall, we performed the global proteomic and phosphoproteomic analyses of EMT, identified and validated new mRNA and/or protein level modulators of EMT. This work also provides a unique platform and resource for future studies focusing on metastasis and drug resistance.
Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Choque Térmico HSP40/metabolismo , Fosfoproteínas/metabolismo , Tetraspanina 28/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal/genética , Feminino , Proteínas de Choque Térmico HSP40/genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Mamárias Experimentais/patologia , Camundongos Nus , Reprodutibilidade dos Testes , Tetraspanina 28/genéticaRESUMO
Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.
Assuntos
Reprogramação Celular , Cromatina/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Reprogramação Celular/genética , Cromatina/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Fibroblastos/citologia , Fibroblastos/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Metilação , Metiltransferases/antagonistas & inibidores , Metiltransferases/biossíntese , Metiltransferases/genética , Metiltransferases/metabolismo , Proteína Homeobox Nanog , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/antagonistas & inibidores , Fator de Transcrição YY1/metabolismoRESUMO
Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-ß and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-ß and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.
Assuntos
Comunicação Autócrina , Neoplasias da Mama/patologia , Quimiocina CXCL12/metabolismo , Glândulas Mamárias Humanas/patologia , Miofibroblastos/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Neoplasias da Mama/metabolismo , Diferenciação Celular , Feminino , Humanos , Glândulas Mamárias Humanas/metabolismo , Camundongos , Invasividade Neoplásica , Receptores CXCR4/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-beta1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-beta1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-beta1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-beta1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.
Assuntos
Neoplasias da Mama/genética , Claudinas/genética , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Mesoderma/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteína Goosecoid/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
BACKGROUND: Lung cancer is known as the most common and highly metastatic form of cancer worldwide. Tumour node metastasis (TNM) staging is the gold standard classification system for the decision-making process for appropriate treatment. Particularly N status has the most important prognostic value in the absence of distant metastasis. Traditional diagnostic methods are capable of detecting metastasis; however, they may fail to detect micrometastasis, which plays a role in disease recurrence and patients' long-term survival. Occult micrometastasis can change the tumour's TNM staging and, consequently, the patient's treatment regimen. METHODS: The median number of three lymph node tissues were collected from 30 patients who underwent surgery for non-small cell lung cancer. Lymph node tissues were collected from different lymph node stations according to the location of the patient's tumour. CK19, EpCAM and CEACAM5 gene expressions were analysed in tissues using quantitative real-time polymerase chain reaction to detect micrometastasis in distant lymph nodes. RESULTS: Triple positivity was seen in 26 out of 30 patients which 19 patients were upstaged from N0 to N2. While survival was not significantly affected between upstaged and non-upstaged patients, patients upstaged with multiple-station N2 had a significantly higher recurrence and lower survival compared to single-station N2. CONCLUSION: A combination of CK19, EpCAM and CEACAM5 gene expressions in lymph nodes can be used to identify micrometastasis which postoperatively may be used as a tool to predict patients' recurrence and survival.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Antígeno Carcinoembrionário , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Molécula de Adesão da Célula Epitelial/genética , Expressão Gênica , Proteínas Ligadas por GPI , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Linfonodos , Micrometástase de Neoplasia/genética , PrognósticoRESUMO
Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of p21(Cip1), p53, and p16(INK4A) tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.
Assuntos
Senescência Celular , Nucleotídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Fibroblastos/citologia , Guanina/análogos & derivados , Humanos , Oxigênio/farmacologia , Monoéster Fosfórico Hidrolases/genéticaRESUMO
Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.
Assuntos
Células-Tronco Pluripotentes Induzidas , Transcriptoma , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Reprogramação Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismoRESUMO
Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.
Assuntos
Sistemas CRISPR-Cas , Neoplasias de Mama Triplo Negativas , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Cromatina , Detecção Precoce de Câncer , Humanos , Masculino , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Epithelial-mesenchymal transition (EMT) programs operate within carcinoma cells, where they generate phenotypes associated with malignant progression. In their various manifestations, EMT programs enable epithelial cells to enter into a series of intermediate states arrayed along the E-M phenotypic spectrum. At present, we lack a coherent understanding of how carcinoma cells control their entrance into and continued residence in these various states, and which of these states favour the process of metastasis. Here we characterize a layer of EMT-regulating machinery that governs E-M plasticity (EMP). This machinery consists of two chromatin-modifying complexes, PRC2 and KMT2D-COMPASS, which operate as critical regulators to maintain a stable epithelial state. Interestingly, loss of these two complexes unlocks two distinct EMT trajectories. Dysfunction of PRC2, but not KMT2D-COMPASS, yields a quasi-mesenchymal state that is associated with highly metastatic capabilities and poor survival of patients with breast cancer, suggesting that great caution should be applied when PRC2 inhibitors are evaluated clinically in certain patient cohorts. These observations identify epigenetic factors that regulate EMP, determine specific intermediate EMT states and, as a direct consequence, govern the metastatic ability of carcinoma cells.
Assuntos
Neoplasias da Mama , Carcinoma , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Metástase Neoplásica/patologiaRESUMO
The establishment and maintenance of cellular identity are crucial during development and tissue homeostasis. Epigenetic mechanisms based largely on DNA methylation and histone modifications serve to reinforce and safeguard differentiated cell states. Somatic cell nuclear transfer (SCNT) or transcription factors such as Oct4, Sox2, Klf4, c-MYC (OSKM) can erase somatic cell identity and reprogram the cells to a pluripotent state. In doing so, reprogramming must reset the chromatin landscape, silence somatic-specific gene expression programs, and, in their place, activate the pluripotency network. In this viewpoint, we consider the major chromatin-based barriers for reprogramming of somatic cells to pluripotency. Among these, repressive chromatin modifications such as DNA methylation, H3K9 methylation, variant histone deposition, and histone deacetylation generally block the activation of pluripotency genes. In contrast, active transcription-associated chromatin marks such as DOT1L-catalyzed H3K79 methylation, FACT-mediated histone turnover, active enhancer SUMOylation, and EP300/CBP bromodomain-mediated interactions act to maintain somatic-specific gene expression programs. We highlight how genetic or chemical inhibition of both types of barriers can enhance the kinetics and/or efficiency of reprogramming. Understanding the mechanisms by which these barriers function provides insight into how chromatin marks help maintain cell identity.
Assuntos
Cromatina/genética , Epigênese Genética/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Reprogramação Celular , Cromatina/metabolismo , Metilação de DNA , Humanos , Fator 4 Semelhante a KruppelRESUMO
Friedreich's ataxia (FRDA) is a rare neurodegenerative disorder which is caused by triplet repeat expansion (GAA) in the first intron of FXN gene. In this present study, we generated induced pluripotent stem cells (iPSC) lines from fibroblasts of three unrelated FRDA patients using integration-free episomal vectors. All iPSC lines express the pluripotency markers such as OCT4 and SSEA4, display normal karyotypes and can differentiate into all three germ layers via in vivo teratoma formation assay.
Assuntos
Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Proteínas de Ligação ao Ferro , Ataxia de Friedreich/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons/genética , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Expansão das Repetições de Trinucleotídeos , FrataxinaRESUMO
BACKGROUND: The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. RESULTS: We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. CONCLUSIONS: Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.
Assuntos
Reprogramação Celular , Histona-Lisina N-Metiltransferase , Fatores de Transcrição , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Proteômica , Fatores de Transcrição/metabolismoRESUMO
Complete hydatidiform mole (HM) is a gestational trophoblastic disease resulting in hyperproliferation of trophoblast cells and absence of embryo development. Mutations in the maternal-effect gene NLRP7 are the major cause of familial recurrent complete HM. Here, we established an in vitro model of HM using patient-specific induced pluripotent stem cells (iPSCs) derived trophoblasts harboring NLRP7 mutations. Using whole transcriptome profiling during trophoblast differentiation, we showed that impaired NLRP7 expression results in precocious downregulation of pluripotency factors, activation of trophoblast lineage markers, and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. Interestingly, we found that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition corrected the excessive trophoblast differentiation of patient-derived iPSCs. Our human iPSC model of a genetic placental disease recapitulates aspects of trophoblast biology, highlights the broad utility of iPSC-derived trophoblasts for modeling human placental diseases and identifies NLRP7 as an essential modulator of key developmental cell fate regulators.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Trofoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteína Morfogenética Óssea 4/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Mola Hidatiforme/genética , Mola Hidatiforme/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Biológicos , Placenta/metabolismo , Gravidez , Transdução de Sinais/fisiologia , Transcriptoma/genéticaRESUMO
Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.
Assuntos
Blastocisto/citologia , Proliferação de Células/efeitos dos fármacos , Leptina/farmacologia , Células-Tronco Embrionárias Murinas/citologia , Teratoma/metabolismo , Animais , Fator de Transcrição CDX2/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária , Corpos Embrioides/citologia , Antígenos CD15/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína Homeobox Nanog/biossíntese , Fator 3 de Transcrição de Octâmero/biossíntese , Teratoma/induzido quimicamenteRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas F-Box/genética , Glioblastoma/genética , Histona Desmetilases com o Domínio Jumonji/genética , RNA Interferente Pequeno/genética , Apoptose/genética , Caspase 7/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Código das Histonas/genética , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Induced pluripotent stem cells (iPSCs) offer great promise as tools for basic biomedical research, disease modeling, and drug screening. In this chapter, we describe the generation of patient-specific, transgene-free iPSCs from skin biopsies and peripheral blood mononuclear cells through electroporation of episomal vectors and growth under two different culture conditions. The resulting iPSC lines are characterized with respect to pluripotency marker expression through immunostaining, tested for transgene integration by PCR, and assayed for differentiation capacity via teratoma formation.