RESUMEN
During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.
Asunto(s)
Cromatina , Histonas , Histonas/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factor 1 de Ensamblaje de la Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Cromatina/genética , Replicación del ADN , ADN/genéticaRESUMEN
Tumor-initiating cells are a subpopulation of cells that have self-renewal capacity to regenerate a tumor. Here, we identify stem cell-like chromatin features in human glioblastoma initiating cells (GICs) and link them to a loss of the repressive histone H3 lysine 9 trimethylation (H3K9me3) mark. Increasing H3K9me3 levels by histone demethylase inhibition led to cell death in GICs but not in their differentiated counterparts. The induction of apoptosis was accompanied by a loss of the activating H3 lysine 9 acetylation (H3K9ac) modification and accumulation of DNA damage and downregulation of DNA damage response genes. Upon knockdown of histone demethylases, KDM4C and KDM7A both differentiation and DNA damage were induced. Thus, the H3K9me3-H3K9ac equilibrium is crucial for GIC viability and represents a chromatin feature that can be exploited to specifically target this tumor subpopulation.
Asunto(s)
Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células Madre Neoplásicas/metabolismo , Acetilación , Animales , Apoptosis/genética , Línea Celular Tumoral , Autorrenovación de las Células/genética , Cromatina/metabolismo , Metilación de ADN , Reparación del ADN/genética , Técnicas de Silenciamiento del Gen , Glioblastoma/patología , Células HEK293 , Histonas , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Lisina/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyperaccessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered.
RESUMEN
The ability to reprogram somatic cells to pluripotency is continuingly attracting increasing amounts of attention, providing both potential opportunities for regenerative medicine, as well as an intriguing model to study basic mechanisms of developmental reversal and epigenetic erasure. Currently, nuclear reprogramming is an inefficient process and a better understanding of its components and the underlying mechanisms will no doubt enable us to increase its robustness and to gain a deeper understanding of its regulation. Here we focus on the reprogramming process from the chromatin and genome organization perspective, describing the chromatin changes that occur both globally and locally. At the global level, chromatin decondenses toward the characteristic 'open' state, while locally, chromatin reorganization supports the silencing of lineage-specific genes and the activation of pluripotency-related genes. Importantly, the proteins that regulate this process are being identified, revealing different layers of chromatin regulation, including histone modifications, histone variants, chromatin remodeling and genomic DNA methylation. The emerging theme is that chromatin and genome organization are not only altered during the transition from a somatic to a pluripotent state, but also play active, regulatory roles during the reprogramming process.
Asunto(s)
Reprogramación Celular/genética , Cromatina/genética , Genoma , Animales , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Histonas/genética , Histonas/metabolismo , Humanos , Células Madre Pluripotentes/fisiologíaRESUMEN
Modified parental histones are segregated symmetrically to daughter DNA strands during replication and can be inherited through mitosis. How this may sustain the epigenome and cell identity remains unknown. Here we show that transmission of histone-based information during DNA replication maintains epigenome fidelity and embryonic stem cell plasticity. Asymmetric segregation of parental histones H3-H4 in MCM2-2A mutants compromised mitotic inheritance of histone modifications and globally altered the epigenome. This included widespread spurious deposition of repressive modifications, suggesting elevated epigenetic noise. Moreover, H3K9me3 loss at repeats caused derepression and H3K27me3 redistribution across bivalent promoters correlated with misexpression of developmental genes. MCM2-2A mutation challenged dynamic transitions in cellular states across the cell cycle, enhancing naïve pluripotency and reducing lineage priming in G1. Furthermore, developmental competence was diminished, correlating with impaired exit from pluripotency. Collectively, this argues that epigenetic inheritance of histone modifications maintains a correctly balanced and dynamic chromatin landscape able to support mammalian cell differentiation.
Asunto(s)
Epigenoma , Histonas , Animales , Histonas/genética , Cromatina/genética , Células Madre Embrionarias , Mitosis , MamíferosRESUMEN
A bacterial genotoxicity reporter strain was constructed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bioreporter responded in a dose-dependent manner to three model DNA-damaging agents-hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)-detected 30-60 min after exposure. Detection thresholds were 0.15 µM for MMC, 7.5 µM for nalidixic acid, and approximately 50 µM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensitivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminoanthracene following metabolic activation with an S9 mammalian liver fraction.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Técnicas Electroquímicas/métodos , Proteínas de Escherichia coli/metabolismo , Genes Reporteros , Peróxido de Hidrógeno/toxicidad , Mitomicina/toxicidad , Ácido Nalidíxico/toxicidad , Regiones Promotoras Genéticas , Respuesta SOS en GenéticaRESUMEN
Single-cell assays have revealed the importance of heterogeneity in many biological systems. However, limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq, combining clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that clonal cells maintain similar transcriptional profiles and cell states. CloneSeq of lung cancer cells revealed cancer-specific subpopulations, including cancer stem-like cells, that were not revealed by scRNA-seq. Clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) even without pluripotent media, and it improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. CloneSeq of ESCs revealed that the differentiation decision is made early during Oct4 downregulation and is maintained during early clonal expansion. Together, we show CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging the enhanced sensitivity within clones.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Linaje de la Célula/genética , Reprogramación Celular/genética , Análisis de la Célula Individual/métodos , Células Madre Embrionarias/citología , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Hidrogeles/química , Células Madre Neoplásicas/citología , Factor 3 de Transcripción de Unión a Octámeros , RNA-Seq/métodos , Transcripción Genética/genéticaRESUMEN
Neuronal stimulation leads to immediate early gene (IEG) expression through calcium-dependent mechanisms. In recent years, considerable attention has been devoted to the transcriptional responses after neuronal stimulation, but relatively little is known about the changes in chromatin dynamics that follow neuronal activation. Here, we use fluorescence recovery after photobleaching, biochemical fractionations, and chromatin immunoprecipitation to show that KCl-induced depolarization in primary cultured cortical neurons causes a rapid release of the linker histone H1 from chromatin, concomitant with IEG expression. H1 release is repressed by PARP inhibition, PARP1 deletion, a non-PARylatable H1, as well as phosphorylation inhibitions and a nonphosphorylatable H1, leading to hindered IEG expression. Further, H1 is replaced by PARP1 on IEG promoters after neuronal stimulation, and PARP inhibition blocks this reciprocal binding response. Our results demonstrate the relationship between neuronal excitation and chromatin plasticity by identifying the roles of polyadenosine diphosphate ribosylation and phosphorylation of H1 in regulating H1 chromatin eviction and IEG expression in stimulated neurons.
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Neuronas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/genética , Cloruro de Potasio/farmacologíaRESUMEN
Embryonic stem cells (ESCs) are regulated by pluripotency-related transcription factors in concert with chromatin regulators. To identify additional stem cell regulators, we screened a library of endogenously labeled fluorescent fusion proteins in mouse ESCs for fluorescence loss during differentiation. We identified SET, which displayed a rapid isoform shift during early differentiation from the predominant isoform in ESCs, SETα, to the primary isoform in differentiated cells, SETß, through alternative promoters. SETα is selectively bound and regulated by pluripotency factors. SET depletion causes proliferation slowdown and perturbed neuronal differentiation in vitro and developmental arrest in vivo, and photobleaching methods demonstrate SET's role in maintaining a dynamic chromatin state in ESCs. This work identifies an important regulator of pluripotency and early differentiation, which is controlled by alternative promoter usage.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histona Acetiltransferasas/genética , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Proliferación Celular , Supervivencia Celular/genética , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Placa Neural/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Isoformas de ProteínasRESUMEN
Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells' long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells.
Asunto(s)
Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Histonas/genética , Neoplasias/genética , Neoplasias/patología , Adenina/química , Línea Celular Tumoral , ADN/química , Metilación de ADN , Elementos de Facilitación Genéticos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias/mortalidad , Nucleosomas/metabolismo , ARN Interferente Pequeño/genética , Timina/químicaRESUMEN
Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Animales , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Factor 4 Similar a Kruppel , RatonesRESUMEN
Embryonic stem cells (ESCs) exhibit unique chromatin features, including a permissive transcriptional program and an open, decondensed chromatin state. Induced pluripotent stem cells (iPSCs), which are very similar to ESCs, hold great promise for therapy and basic research. However, the mechanisms by which reprogramming occurs and the chromatin organization that underlies the reprogramming process are largely unknown. Here we characterize and compare the epigenetic landscapes of partially and fully reprogrammed iPSCs to mouse embryonic fibroblasts (MEFs) and ESCs, which serves as a standard for pluripotency. Using immunofluorescence and biochemical fractionations, we analyzed the levels and distribution of a battery of histone modifications (H3ac, H4ac, H4K5ac, H3K9ac, H3K27ac, H3K4me3, H3K36me2, H3K9me3, H3K27me3, and γH2AX), as well as HP1α and lamin A. We find that fully reprogrammed iPSCs are epigenetically identical to ESCs, and that partially reprogrammed iPSCs are closer to MEFs. Intriguingly, combining both time-course reprogramming experiments and data from the partially reprogrammed iPSCs, we find that heterochromatin reorganization precedes Nanog expression and active histone marking. Together, these data delineate the global epigenetic state of iPSCs in conjunction with their pluripotent state, and demonstrate that heterochromatin precedes euchromatin in reorganization during reprogramming.
Asunto(s)
Reprogramación Celular , Epigénesis Genética , Células Madre Pluripotentes Inducidas/metabolismo , Acetilación , Animales , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Metilación , Ratones , Proteína Homeótica NanogRESUMEN
The pluripotent genome is characterized by unique epigenetic features and a decondensed chromatin conformation. However, the relationship between epigenetic regulation and pluripotency is not altogether clear. Here, using an enhanced MEF/ESC fusion protocol, we compared the reprogramming potency and histone modifications of different embryonic stem cell (ESC) lines (R1, J1, E14, C57BL/6) and found that E14 ESCs are significantly less potent, with significantly reduced H3K9ac levels. Treatment of E14 ESCs with histone deacetylase (HDAC) inhibitors (HDACi) increased H3K9ac levels and restored their reprogramming capacity. Microarray and H3K9ac ChIP-seq analyses, suggested increased extracellular matrix (ECM) activity following HDACi treatment in E14 ESCs. These data suggest that H3K9ac may predict pluripotency and that increasing pluripotency by HDAC inhibition acts through H3K9ac to enhance the activity of target genes involved in ECM production to support pluripotency.
Asunto(s)
Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Acetilación , Animales , Baculoviridae/genética , Línea Celular , Reprogramación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Ever since the introduction of the Salmonella typhimurium mammalian microsome mutagenicity assay (the 'Ames test') over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter-reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induction of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu-test, has been fully validated and ISO- and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual genotoxicity testing devices.
Asunto(s)
Bacterias/efectos de los fármacos , Bacterias/genética , Técnicas Biosensibles/métodos , Mutágenos/toxicidad , Fusión Artificial Génica , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Reparación del ADN , Genes Reporteros/genética , Pruebas de Mutagenicidad/métodos , Supresión GenéticaRESUMEN
Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.