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Non-small-cell lung cancer (NSCLC) with concurrent mutations in KRAS and the tumour suppressor LKB1 (KL NSCLC) is refractory to most therapies and has one of the worst predicted outcomes. Here we describe a KL-induced metabolic vulnerability associated with serine-glycine-one-carbon (SGOC) metabolism. Using RNA-seq and metabolomics data from human NSCLC, we uncovered that LKB1 loss enhanced SGOC metabolism via serine hydroxymethyltransferase (SHMT). LKB1 loss, in collaboration with KEAP1 loss, activated SHMT through inactivation of the salt-induced kinase (SIK)-NRF2 axis and satisfied the increased demand for one-carbon units necessary for antioxidant defence. Chemical and genetic SHMT suppression increased cellular sensitivity to oxidative stress and cell death. Further, the SHMT inhibitor enhanced the in vivo therapeutic efficacy of paclitaxel (first-line NSCLC therapy inducing oxidative stress) in KEAP1-mutant KL tumours. The data reveal how this highly aggressive molecular subtype of NSCLC fulfills their metabolic requirements and provides insight into therapeutic strategies.
Assuntos
Quinases Proteína-Quinases Ativadas por AMP , Antioxidantes , Carcinoma Pulmonar de Células não Pequenas , Glicina Hidroximetiltransferase , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Pulmonares , Mutação , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas p21(ras) , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Antioxidantes/metabolismo , Animais , Estresse Oxidativo , Camundongos , Linhagem Celular Tumoral , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genéticaRESUMO
DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO.
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X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.
Assuntos
Genes Ligados ao Cromossomo X , RNA Longo não Codificante , Cromossomo X , Animais , Feminino , Humanos , Masculino , Camundongos , Inativação Gênica , RNA Longo não Codificante/genética , Cromossomo X/genética , Células-Tronco Pluripotentes/metabolismoRESUMO
Lactate was implicated in the activation of hepatic stellate cells (HSCs). However, the mechanism by which lactate exerts its effect remains elusive. Using RNA-seq and CUT&Tag chromatin profiling, we found that induction of hexokinase 2 (HK2) expression in activated HSCs is required for induced gene expression by histone lactylation but not histone acetylation. Inhibiting histone lactylation by Hk2 deletion or pharmacological inhibition of lactate production diminishes HSC activation, whereas exogenous lactate but not acetate supplementation rescues the activation phenotype. Thus, lactate produced by activated HSCs determines the HSC fate via histone lactylation. We found that histone acetylation competes with histone lactylation, which could explain why class I HDAC (histone deacetylase) inhibitors impede HSC activation. Finally, HSC-specific or systemic deletion of HK2 inhibits HSC activation and liver fibrosis in vivo. Therefore, we provide evidence that HK2 may be an effective therapeutic target for liver fibrosis.
Assuntos
Hexoquinase , Histonas , Humanos , Histonas/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Expressão Gênica , Lactatos/farmacologiaRESUMO
Recent advances in our understanding of host immune and cancer cells interactions have made immunotherapy a prominent choice in cancer treatment. Despite such promise, cell-based immunotherapies remain inapplicable to many patients due to severe limitations in the availability and quality of immune cells isolated from donors. Reprogramming technologies that facilitate the engineering of cell types of interest, are emerging as a putative solution to such challenges. Here we focus on the recent progress being made in reprogramming technologies with respect to the immune system and their potential for clinical applications.
Assuntos
Imunoterapia , Neoplasias , Humanos , Imunoterapia/métodos , Neoplasias/terapiaRESUMO
Single-cell RNA sequencing is a powerful method that helps delineate the regulatory mechanisms shaping the diverse cellular populations. Heterogeneous cell populations consist of individual cells, and the expression of distinct sets of genes can differentiate one sub-population of cells from another, as they are responsible for the emergence of distinct cellular phenotypes. Of particular importance are cells at transition states that bridge these different cellular phenotypes. In this study, we develop a method to identify the cells at transition states bridging different cellular phenotypes. Our approach is based on persistent homology, which enabled us to identify the group of cells located on the boundaries between different sub-populations of cells. We applied this method to study the reprogramming of human fibroblasts toward induced pluripotent stem cells using single-cell time-course data. Even though only the data that is representative of the early stages of the reprogramming process are analyzed, we are able to uncover transient cells bridging different cell sub-populations. The most prominent group of transient cells are found to be enriched for NANOG, which is a known stem cell transcription factor that takes part in the maintenance of pluripotency and other stem cell marker genes. Overall, our method can identify cells in transient states bridging major cellular phenotypes, even though they are only a small fraction of the overall cell population. We also discuss how this approach can link the topology of the surface of cellular transcripts and bring order to the transition between cellular states and how it automatically uncovers the underlying time process.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Biomarcadores/metabolismo , Fibroblastos/metabolismo , HumanosRESUMO
The self-renewal and pluripotency of embryonic stem cells (ESCs) are conferred by networks including transcription factors and histone modifiers. The Auxin-inducible degron (AID) system can rapidly and reversibly degrade its target proteins and is becoming a powerful tool to explore novel function of key pluripotent and histone modifier genes in ESCs. However, the low biallelic tagging efficiency and a basal degradation level of the current AID systems deem it unsuitable to target key pluripotent genes with tightly controlled expression levels. Here, we develop a one-step strategy to successfully target and repress the endogenous pluripotent genes in mouse ESCs and replace their expression with AID fused transgenes. Therefore, this work provides an efficient way for employing the AID system to uncover novel function of essential pluripotent and chromatin modifier genes in ESCs.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear1. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus2 and binding diverse proteins3-5 to achieve X-chromosome inactivation (XCI)6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off8. Here we show that the Xist RNA-binding proteins PTBP19, MATR310, TDP-4311 and CELF112 assemble on the multivalent E-repeat element of Xist7 and, via self-aggregation and heterotypic protein-protein interactions, form a condensate1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase8, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.
Assuntos
Inativação Gênica , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas CELF1/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Inativação do Cromossomo X/genéticaRESUMO
Whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale. Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6-12% of the CpGs in the human genome. Here, we introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.
Assuntos
Metilação de DNA/genética , Enzimas de Restrição do DNA/metabolismo , Análise de Sequência de DNA/métodos , Sulfitos/química , Animais , Linhagem Celular , Reprogramação Celular , Cromatina/metabolismo , Ilhas de CpG/genética , Genoma , CamundongosRESUMO
BAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation.
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Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/citologia , Complexo Repressor Polycomb 2/metabolismo , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/fisiologia , Animais , Apoptose , Ciclo Celular , Células-Tronco Embrionárias/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Knockout , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Complexo Repressor Polycomb 2/genética , Subunidades Proteicas , Proteínas com Domínio T/genéticaRESUMO
Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor ß (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes.
Assuntos
Cromossomos de Mamíferos , Elementos Facilitadores Genéticos , Elementos Isolantes , Células-Tronco Embrionárias Murinas/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Bases de Dados Genéticas , Regulação para Baixo , Camundongos , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , CoesinasRESUMO
BACKGROUND: Both human and mouse fibroblasts can be reprogrammed to pluripotency with Oct4, Sox2, Klf4, and c-Myc (OSKM) transcription factors. While both systems generate pluripotency, human reprogramming takes considerably longer than mouse. RESULTS: To assess additional similarities and differences, we sought to compare the binding of the reprogramming factors between the two systems. In human fibroblasts, the OSK factors initially target many more closed chromatin sites compared to mouse. Despite this difference, the intra- and intergenic distribution of target sites, target genes, primary binding motifs, and combinatorial binding patterns between the reprogramming factors are largely shared. However, while many OSKM binding events in early mouse cell reprogramming occur in syntenic regions, only a limited number is conserved in human. CONCLUSIONS: Our findings suggest similar general effects of OSKM binding across these two species, even though the detailed regulatory networks have diverged significantly.
Assuntos
Reprogramação Celular/genética , Cromatina/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Especificidade da EspécieRESUMO
A dramatic difference in global DNA methylation between male and female cells characterizes mouse embryonic stem cells (ESCs), unlike somatic cells. We analyzed DNA methylation changes during reprogramming of male and female somatic cells and in resulting induced pluripotent stem cells (iPSCs). At an intermediate reprogramming stage, somatic and pluripotency enhancers are targeted for partial methylation and demethylation. Demethylation within pluripotency enhancers often occurs at ESC binding sites of pluripotency transcription factors. Late in reprogramming, global hypomethylation is induced in a female-specific manner. Genome-wide hypomethylation in female cells affects many genomic landmarks, including enhancers and imprint control regions, and accompanies the reactivation of the inactive X chromosome. The loss of one of the two X chromosomes in propagating female iPSCs is associated with genome-wide methylation gain. Collectively, our findings highlight the dynamic regulation of DNA methylation at enhancers during reprogramming and reveal that X chromosome dosage dictates global DNA methylation levels in iPSCs.
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Reprogramação Celular/genética , Cromossomos de Mamíferos/genética , Metilação de DNA/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Cromossomo X/genética , Animais , Sítios de Ligação , Ilhas de CpG/genética , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Feminino , Genoma , Impressão Genômica , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.
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Adenosina Trifosfatases/metabolismo , Células-Tronco Embrionárias/enzimologia , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA , Eucromatina/genética , Eucromatina/metabolismo , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Genótipo , Heterocromatina/genética , Heterocromatina/metabolismo , Fenótipo , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID , Fatores de Transcrição/genética , TransfecçãoRESUMO
Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.
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Reprogramação Celular , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Fibroblastos/metabolismo , Código das Histonas , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elementos Reguladores de Transcrição , Fatores de Transcrição SOXB1/metabolismo , Elementos Silenciadores TranscricionaisRESUMO
Applications of embryonic stem cells (ESCs) require faithful chromatin changes during differentiation, but the fate of the X chromosome state in differentiating ESCs is unclear. Female human ESC lines either carry two active X chromosomes (XaXa), an Xa and inactive X chromosome with or without XIST RNA coating (XiXIST+Xa;XiXa), or an Xa and an eroded Xi (XeXa) where the Xi no longer expresses XIST RNA and has partially reactivated. Here, we established XiXa, XeXa, and XaXa ESC lines and followed their X chromosome state during differentiation. Surprisingly, we found that the X state pre-existing in primed ESCs is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells lacked XIST, did not induce X inactivation, and displayed higher X-linked gene expression than XiXa cells. These results demonstrate that X chromosome dosage compensation is not required for ESC differentiation. Our data imply that XiXIST+Xa ESCs are most suited for downstream applications and show that all other X states are abnormal byproducts of our ESC derivation and propagation method.
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Diferenciação Celular/genética , Células-Tronco Embrionárias Humanas/metabolismo , Inativação do Cromossomo X/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Metilação de DNA/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Análise de Sequência de RNA , Tretinoína/farmacologiaRESUMO
Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets Xi abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications.
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Cromossomos Humanos X/genética , Células-Tronco Pluripotentes/metabolismo , Inativação do Cromossomo X/genética , Sequência de Bases , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Metilação de DNA/genética , Feminino , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Células-Tronco Pluripotentes/citologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gradually reestablished during reprogramming. Our data confirm that long-range chromatin interactions are primarily associated with the spatial segregation of open and closed chromatin, defining overall chromosome conformation. Additionally, we identified two further levels of genome organization in ESCs characterized by colocalization of regions with high pluripotency factor occupancy and strong enrichment for Polycomb proteins/H3K27me3, respectively. Underlining the independence of these networks and their functional relevance for genome organization, loss of the Polycomb protein Eed diminishes interactions between Polycomb-regulated regions without altering overarching chromosome conformation. Together, our data highlight a pluripotency-specific genome organization in which pluripotency factors such as Nanog and H3K27me3 occupy distinct nuclear spaces and reveal a role for cell-type-specific gene-regulatory networks in genome organization.
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Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Histonas/metabolismo , Hibridização in Situ Fluorescente , Camundongos , Proteínas do Grupo Polycomb/genética , Ligação ProteicaRESUMO
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.