RESUMEN
Obesity is an emerging global health issue with an increasing risk of disease linked to lifestyle choices. Previously, we reported that the hexane extract of Citrus sphaerocarpa (CSHE) suppressed lipid accumulation in differentiated 3T3-L1 adipocytes. In this study, we conducted in vivo experiments to assess whether CSHE suppressed obesity in zebrafish and mouse models. We administered 10 and 20 µg/mL CSHE to obese zebrafish juveniles. CSHE significantly inhibited visceral fat accumulation compared to untreated obese fish. Moreover, the oral administration (100 µg/g body weight/day) of CSHE to high-fat-diet-induced obese mice significantly reduced their body weight, visceral fat volume, and hepatic lipid accumulation. The expression analyses of key regulatory genes involved in lipid metabolism revealed that CSHE upregulated the mRNA expression of lipolysis-related genes in the mouse liver (Pparα and Acox1) and downregulated lipogenesis-related gene (Fasn) expression in epididymal white adipose tissue (eWAT). Fluorescence immunostaining demonstrated the CSHE-mediated enhanced phosphorylation of AKT, AMPK, ACC, and FoxO1, which are crucial factors regulating adipogenesis. CSHE-treated differentiated 3T3L1 adipocytes also exhibited an increased phosphorylation of ACC. Therefore, we propose that CSHE suppresses adipogenesis and enhances lipolysis by regulating the PI3K/AKT/FoxO1 and AMPK/ACC signaling pathways. These findings suggested that CSHE is a promising novel preventive and therapeutic agent for managing obesity.
Asunto(s)
Fármacos Antiobesidad , Citrus , Animales , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Ratones Obesos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pez Cebra/metabolismo , Adiposidad , Citrus/metabolismo , Fármacos Antiobesidad/farmacología , Hexanos/farmacología , Obesidad/tratamiento farmacológico , Obesidad/etiología , Obesidad/metabolismo , Adipogénesis , Peso Corporal , Transducción de Señal , Lípidos/farmacología , Dieta , Dieta Alta en Grasa/efectos adversos , Células 3T3-L1 , Ratones Endogámicos C57BLRESUMEN
The temporal order of DNA replication along the chromosomes is thought to reflect the transcriptional competence of the genome. During differentiation of mouse 3T3-L1 cells into adipocytes, cells undergo one or two rounds of cell division called mitotic clonal expansion (MCE). MCE is an essential step for adipogenesis; however, little is known about the regulation of DNA replication during this period. Here, we performed genome-wide mapping of replication timing (RT) in mouse 3T3-L1 cells before and during MCE, and identified a number of chromosomal regions shifting toward either earlier or later replication through two rounds of replication. These RT changes were confirmed in individual cells by single-cell DNA-replication sequencing. Coordinate changes between a shift toward earlier replication and transcriptional activation of adipogenesis-associated genes were observed. RT changes occurred before the full expression of these genes, indicating that RT reorganization might contribute to the mature adipocyte phenotype. To support this, cells undergoing two rounds of DNA replication during MCE had a higher potential to differentiate into lipid droplet-accumulating adipocytes, compared with cells undergoing a single round of DNA replication and non-replicating cells.
Asunto(s)
Adipogénesis , Mitosis , Animales , Ratones , Adipogénesis/genética , Mitosis/genética , Diferenciación Celular/genética , Replicación del ADN/genética , Expresión Génica , Células 3T3-L1RESUMEN
E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.
Asunto(s)
Camptotecina/farmacología , Replicación del ADN/efectos de los fármacos , Momento de Replicación del ADN/efectos de los fármacos , Humanos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Inhibidores de Topoisomerasa I/farmacologíaRESUMEN
Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.
Asunto(s)
Momento de Replicación del ADN , ADN/metabolismo , Mamíferos/metabolismo , Metiltransferasas/metabolismo , Animales , Cromosomas de los Mamíferos/metabolismo , Metilación de ADN/genética , Momento de Replicación del ADN/genética , Genoma , Heterocromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/metabolismo , Transcripción GenéticaRESUMEN
Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d.
Asunto(s)
Replicación del ADN , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Análisis de la Célula Individual/métodos , Animales , Línea Celular , Humanos , Fase S/genéticaRESUMEN
BACKGROUND: Environmental and endogenous factors under genetic predisposition are considered to initiate the human intervertebral disc (IVD) degeneration. DNA methylation is an essential mechanism to ensure cell-specific gene expression for normal development and tissue stability. Aberrant epigenetic alterations play a pivotal role in several diseases, including osteoarthritis. However, epigenetic alternations, including DNA methylation, in IVD degeneration have not been evaluated. The purpose of this study was to comprehensively compare the genome-wide DNA methylation profiles of human IVD tissues, specifically nucleus pulpous (NP) tissues, with early and advanced stages of disc degeneration. METHODS: Human NP tissues were used in this study. The samples were divided into two groups: early stage degeneration (n = 8, Pfirrmann's MRI grade: I-III) and advanced stage degeneration (n = 8, grade: IV). Genomic DNA was processed for genome-wide DNA methylation profiling using the Infinium MethylationEPIC BeadChip array. Extraction of raw methylation data, clustering and scatter plot of each group values of each sample were performed using a methylation module in GenomeStudio software. The identification of differentially methylated loci (DMLs) and the Gene Ontology (GO) analysis were performed using R software with the ChAMP package. RESULTS: Unsupervised hierarchical clustering revealed that early and advanced stage degenerated IVD samples segregated into two main clusters by their DNA methylome. A total of 220 DMLs were identified between early and advanced disc degeneration stages. Among these, four loci were hypomethylated and 216 loci were hypermethylated in the advanced disc degeneration stage. The GO enrichment analysis of genes containing DMLs identified two significant GO terms for biological processes, hemophilic cell adhesion and cell-cell adhesion. CONCLUSIONS: We conducted a genome-wide DNA methylation profile comparative study and observed significant differences in DNA methylation profiles between early and advanced stages of human IVD degeneration. These results implicate DNA methylation in the process of human IVD degeneration.
Asunto(s)
Epigénesis Genética/genética , Degeneración del Disco Intervertebral/genética , Núcleo Pulposo/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Islas de CpG/genética , Metilación de ADN/genética , Epigenómica/métodos , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Genoma/genética , Estudio de Asociación del Genoma Completo/métodos , Humanos , Disco Intervertebral/metabolismo , Masculino , Persona de Mediana Edad , Núcleo Pulposo/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0197165.].
RESUMEN
Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.
Asunto(s)
Replicación del ADN/genética , ADN/genética , Mamíferos/genética , Animales , Diferenciación Celular/genética , Línea Celular , Variaciones en el Número de Copia de ADN/genética , Momento de Replicación del ADN/genética , Células Madre Embrionarias/fisiología , Genoma/genética , Estudio de Asociación del Genoma Completo/métodos , Inestabilidad Genómica/genética , Humanos , Ratones , Células Madre Embrionarias de Ratones/fisiología , Fase S/genética , Cromosoma X/genéticaRESUMEN
Here, we show that semiconductor-based sequencing technology can be used to map mammalian replication domains, chromosomal units with similar DNA replication timing. Replicating DNA purified from mammalian cells was successfully sequenced by the Ion Torrent platform. The resultant replication domain map of mouse embryonic stem cells is comparable to those obtained by the conventional microarray-based method.
Asunto(s)
Replicación del ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Semiconductores , Animales , Células Madre Embrionarias/citología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , RatonesRESUMEN
DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNß-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Fase G1 , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ácido Fítico/metabolismo , ARN Mensajero/metabolismo , Fase S , Transporte Biológico Activo/genética , Citoplasma/genética , Citoplasma/metabolismo , ARN Helicasas DEAD-box/genética , Células HeLa , Humanos , Interferón beta/genética , Interferón beta/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Mensajero/genéticaRESUMEN
Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Quinasa 2 Dependiente de la Ciclina/metabolismo , Daño del ADN , Mutágenos/toxicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Estrés Fisiológico , Línea Celular , Humanos , Quinasa Tipo Polo 1RESUMEN
The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.
Asunto(s)
Cromatina/metabolismo , Replicación del ADN , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Origen de Réplica , Transporte Activo de Núcleo Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Componente 2 del Complejo de Mantenimiento de Minicromosoma/química , Componente 7 del Complejo de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/antagonistas & inhibidores , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/genética , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estabilidad Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Mutación Silenciosa , Homología Estructural de ProteínaRESUMEN
Genetic information is faithfully copied by DNA replication through many rounds of cell division. In mammals, DNA is replicated in Mb-sized chromosomal units called "replication domains." While genome-wide maps in multiple cell types and disease states have uncovered both dynamic and static properties of replication domains, we are still in the process of understanding the mechanisms that give rise to these properties. A better understanding of the molecular basis of replication domain regulation will bring new insights into chromosome structure and function.
RESUMEN
Osteoporosis is a debilitating disease caused by decreased bone density. Compounds with anti-osteoclastic activity, such as bisphosphonates, may help in the prevention and treatment of osteoporosis. Herein, we determined the inhibitory effects of ginger hexane extract (GHE) on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. The results showed that GHE (1) suppressed osteoclast differentiation and the formation of actin rings; (2) inhibited the expression of Nfatc1, a master transcriptional factor for osteoclast differentiation, in a dose-dependent manner (10-20 µg/mL); and (3) inhibited other osteoclastogenesis-related genes, such as Oscar, Dc-stamp, Trap, and Mmp9. These findings suggest that GHE may be used to prevent and treat osteoporosis by inhibiting osteoclast differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Hexanos/química , Osteoclastos/efectos de los fármacos , Extractos Vegetales/farmacología , Ligando RANK/fisiología , Zingiber officinale/química , Animales , Línea Celular , Ratones , Osteoclastos/citología , Extractos Vegetales/químicaRESUMEN
We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.
Asunto(s)
Replicación del ADN , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Pez Cebra/embriología , Animales , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Embrión no Mamífero/ultraestructura , Microscopía Fluorescente , Coloración y Etiquetado , Pez Cebra/genéticaRESUMEN
Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were â¼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.
Asunto(s)
Replicación del ADN/fisiología , ADN/fisiología , Pez Cebra/metabolismo , Animales , Línea Celular , Eritrocitos , Humanos , Mitosis/fisiología , Especificidad de la Especie , Coloración y Etiquetado , Factores de TiempoRESUMEN
Pollination is an important early step in sexual plant reproduction. In Arabidopsis thaliana, sequential pollination events, from pollen adhesion onto the stigma surface to pollen tube germination and elongation, occur on the stigmatic papilla cells. Following successful completion of these events, the pollen tube penetrates the stigma and finally fertilizes a female gametophyte. The pollination events are thought to be initiated and regulated by interactions between papilla cells and pollen. Here, we report the characterization of gene expression profiles of unpollinated (UP), compatible pollinated (CP) and incompatible pollinated (IP) papilla cells in A. thaliana. Based on cell type-specific transcriptome analysis from a combination of laser microdissection and RNA sequencing, 15,475, 17,360 and 16,918 genes were identified as expressed in UP, CP and IP papilla cells, respectively, and, of these, 14,392 genes were present in all three data sets. Differentially expressed gene (DEG) analyses identified 147 and 71 genes up-regulated in CP and IP papilla cells, respectively, and 115 and 46 genes down-regulated. Gene Ontology and metabolic pathway analyses revealed that papilla cells play an active role as the female reproductive component in pollination, particularly in information exchange, signal transduction, internal physiological changes and external morphological modification. This study provides fundamental information on the molecular mechanisms involved in pollination in papilla cells, furthering our understanding of the reproductive role of papilla cells.
Asunto(s)
Arabidopsis/citología , Arabidopsis/genética , Flores/citología , Flores/genética , Polinización/genética , Transcripción Genética , Arabidopsis/fisiología , Vías Biosintéticas/genética , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Genes de Plantas , Redes y Vías Metabólicas/genética , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single-molecule approach to measuring the speed of replication fork progression in cells pulse-labelled with thymidine analogues. We constructed a new thymidine-requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA-combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550-750 nt s(-1) and the average speed to be 653 ± 9 nt s(-1) (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s(-1) in a dnaE173-eCOMB strain producing a mutant-type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s(-1) ) much lower than that of the wild-type Pol III (900 nt s(-1) ). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.
Asunto(s)
ADN Polimerasa III/metabolismo , Replicación del ADN , ADN Bacteriano/biosíntesis , Escherichia coli/crecimiento & desarrollo , Bromodesoxiuridina , Cromosomas Bacterianos , ADN Polimerasa III/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación , Timidina/análogos & derivadosRESUMEN
Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.
Asunto(s)
Cromatina/química , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Chaperonas de Histonas/química , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Animales , Ciclo Celular , Pollos , Epigénesis Genética , Citometría de Flujo/métodos , Histonas/química , Humanos , Chaperonas Moleculares/metabolismo , Nucleosomas/metabolismo , Fase SRESUMEN
In mammals, DNA methylation is an important epigenetic mark that is associated with gene silencing, particularly in constitutive heterochromatin. However, the effect of DNA methylation on other epigenetic properties of chromatin is controversial. In this study, we show that inhibition of DNA methylation in mouse fibroblast cells affects histone modification and the subnuclear localization of histone H3.3 in a cell cycle-dependent manner. Using a DNA methyltransferase (Dnmt) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we found that reduced levels of DNA methylation were associated with the activation of transcription from centromeric and pericentromeric satellite repeats. The de-repressed pericentromeric chromatin was enriched in euchromatic histone modifications such as acetylation of histone H4, and di- and tri-methylation of lysine 4 on histone H3. Spatio-temporal analysis showed that the accumulation of these euchromatic histone modifications occurred during the second S phase following 5-aza-dC treatment, corresponding precisely with a shift in replication timing of the pericentromeric satellite repeats from middle/late S phase to early S phase. Moreover, we found that histone H3.3 was deposited on the pericentromeric heterochromatin prior to the accumulation of the euchromatic histone modifications. These results suggest that DNA CpG methylation is essential for the proper organization of pericentromeric heterochromatin in differentiated mouse cells.