RESUMEN
The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2, a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the non-displaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.
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There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, 'a site that binds Mcm in G1' might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here, we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least three orders of magnitude. Published data quantifying single-stranded DNA (ssDNA) during S phase revealed replication initiation among the most abundant 1600 of these sites, with replication activity decreasing with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5500 sites. Specifically, these sites: (1) appeared in intergenic nucleosome-free regions flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. We conclude that, if sites at which Mcm double hexamers are loaded can function as replication origins, then DNA replication origins are at least threefold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than widely assumed.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Origen de Réplica , Replicación del ADN , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN Intergénico/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMEN
Low-dose cytarabine (LDAC) is a standard therapy for elderly acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. While high doses of cytarabine induce cytotoxicity, the precise mechanism of action of LDAC in AML remains elusive. In vitro studies have demonstrated LDAC-induced differentiation; however, such differentiation is seldom observed in vivo. We hypothesize that this discrepancy may be attributed to the influence of bone marrow (BM) stromal cells on AML cells. Thus, this study aimed to investigate the impact of BM stromal cells on LDAC-induced differentiation of AML cell lines and primary samples. Our results demonstrate that the presence of MS-5 stromal cells prevented LDAC-induced cell cycle arrest, DNA damage signaling and differentiation of U937 and MOLM-13 cell lines. Although transcriptomic analysis revealed that the stroma reduces the expression of genes involved in cytokine signaling and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role for CXCL12, TGF-ß1 or reactive oxygen species. The presence of stromal cells reduces LDAC-induced differentiation in primary samples from AML-M4 and myelodysplastic syndrome/AML patients. In conclusion, our study demonstrates that BM stroma reduces differentiation of AML induced by LDAC. These findings provide insights into the limited occurrence of terminal differentiation observed in AML patients, and suggest a potential explanation for this observation.
RESUMEN
There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, "a site to which MCM is bound in G1" might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as "Chromatin Endogenous Cleavage", we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed in vitro. We conclude that, if sites at which Mcm double-hexamers are loaded can function as replication origins, then DNA replication origins are at least 3-fold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than is widely assumed.
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Hematopoietic stem cell transplantation is a well-known treatment for hematologic malignancies, wherein nascent stem cells provide regenerating marrow and immunotherapy against the tumor. The progeny of hematopoietic stem cells also populate a wide spectrum of tissues, including the brain, as bone marrow-derived macrophages similar to microglial cells. We developed a sensitive and novel combined immunohistochemistry (IHC) and XY fluorescence in situ hybridization assay to detect, quantify, and characterize donor cells in the cerebral cortices of 19 female patients who underwent allogeneic stem cell transplantation. We showed that the number of male donor cells ranged from 0.14% to 3.0% of the total cells or from 1.2% to 25% of microglial cells. Using tyramide-based fluorescent IHC, we found that at least 80% of the donor cells expressed the microglial marker ionized calcium-binding adapter molecule-1, consistent with bone marrow-derived macrophages. The percentage of donor cells was related to pretransplantation conditioning; donor cells from radiation-based myeloablative cases averaged 8.1% of microglial cells, whereas those from nonmyeloablative cases averaged only 1.3%. The number of donor cells in patients conditioned with busulfan- or treosulfan-based myeloablation was similar to that in total body irradiation-based conditioning; donor cells averaged 6.8% of the microglial cells. Notably, patients who received multiple transplantations and those with the longest posttransplantation survival had the highest level of donor engraftment, with donor cells averaging 16.3% of the microglial cells. Our work represents the largest study characterizing bone marrow-derived macrophages in patients after transplantation. The efficiency of engraftment observed in our study warrants future research on microglial replacement as a therapeutic option for disorders of the central nervous system.
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Trasplante de Células Madre Hematopoyéticas , Humanos , Masculino , Femenino , Hibridación Fluorescente in Situ , Trasplante de Médula Ósea , Sistema Nervioso Central , MacrófagosRESUMEN
Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anafase , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Ribosomas/metabolismo , Replicación del ADN/genética , Replicación ViralRESUMEN
Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.
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Citarabina , Leucemia Mieloide Aguda , Diferenciación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Citarabina/farmacología , Citarabina/uso terapéutico , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/farmacología , Humanos , Leucemia Mieloide Aguda/genética , Pirimidinas/uso terapéuticoRESUMEN
All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.
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Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Crisis Blástica/genética , Crisis Blástica/patología , Inversión Cromosómica/genética , Cromosomas Humanos Par 16/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Tretinoina/farmacología , Tretinoina/uso terapéutico , Antígeno CD11b/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Subunidad beta del Factor de Unión al Sitio Principal/genética , Fusión Génica/efectos de los fármacos , Reordenamiento Génico/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Cadenas Pesadas de Miosina/genéticaRESUMEN
The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.
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Cromosomas Fúngicos , Cromosomas Humanos , Replicación del ADN/genética , Genoma Fúngico , Proteínas de Mantenimiento de Minicromosoma/genética , Saccharomyces cerevisiae/genética , HumanosRESUMEN
BACKGROUND: All-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) is the most successful pharmacological treatment of acute myeloid leukemia (AML). Recent development of inhibitors of mutated isocitrate dehydrogenase and dihydroorotate dehydrogenase (DHODH) has revived interest in differentiation therapy of non-APL AML. Our previous studies demonstrated that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induced differentiation of monocytic cell lines by activating the ATR/Chk1 via pyrimidine depletion. In the present study, the effects of AICAr on the viability and differentiation of primary AML blasts isolated from bone marrow of patients with non-APL AML were tested and compared with the effects of DHODH inhibitor brequinar and ATRA. METHODS: Bone marrow samples were obtained from 35 patients and leukemia blasts were cultured ex vivo. The cell viability was assessed by MTT assay and AML cell differentiation was determined by flow cytometry and morphological analyses. RNA sequencing and partial data analysis were conducted using ClusterProfiler package. Statistical analysis was performed using GraphPad Prism 6.0. RESULTS: AICAr is capable of triggering differentiation in samples of bone marrow blasts cultured ex vivo that were resistant to ATRA. AICAr-induced differentiation correlates with proliferation and sensitivity to DHODH inhibition. RNA-seq data obtained in primary AML blasts confirmed that AICAr treatment induced downregulation of pyrimidine metabolism pathways together with an upregulation of gene set involved in hematopoietic cell lineage. CONCLUSION: AICAr induces differentiation in a subset of primary non-APL AML blasts, and these effects correlate with sensitivity to a well-known, potent DHODH inhibitor.
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Aminoimidazol Carboxamida/análogos & derivados , Biomarcadores de Tumor/metabolismo , Crisis Blástica/tratamiento farmacológico , Médula Ósea/efectos de los fármacos , Diferenciación Celular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ribonucleósidos/farmacología , Aminoimidazol Carboxamida/farmacología , Biomarcadores de Tumor/genética , Crisis Blástica/genética , Crisis Blástica/metabolismo , Crisis Blástica/patología , Médula Ósea/metabolismo , Médula Ósea/patología , Estudios de Casos y Controles , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , RNA-Seq , Células Tumorales CultivadasRESUMEN
Beneficial mutations that arise in an evolving asexual population may compete or interact in ways that alter the overall rate of adaptation through mechanisms such as clonal or functional interference. The application of multiple selective pressures simultaneously may allow for a greater number of adaptive mutations, increasing the opportunities for competition between selectively advantageous alterations, and thereby reducing the rate of adaptation. We evolved a strain of Saccharomyces cerevisiae that could not produce its own histidine or uracil for ~500 generations under one or three selective pressures: limitation of the concentration of glucose, histidine, and/or uracil in the media. The rate of adaptation was obtained by measuring evolved relative fitness using competition assays. Populations evolved under a single selective pressure showed a statistically significant increase in fitness on those pressures relative to the ancestral strain, but the populations evolved on all three pressures did not show a statistically significant increase in fitness over the ancestral strain on any single pressure. Simultaneously limiting three essential nutrients for a population of S. cerevisiae effectively slows the rate of evolution on any one of the three selective pressures applied, relative to the single selective pressure cases. We identify possible mechanisms for fitness changes seen between populations evolved on one or three limiting nutrient pressures by high-throughput sequencing. Adding multiple selective pressures to evolving disease like cancer and infectious diseases could reduce the rate of adaptation and thereby may slow disease progression, prolong drug efficacy and prevent deaths.
RESUMEN
Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.
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Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Linfoma de Células B/tratamiento farmacológico , Sirtuina 2/antagonistas & inhibidores , Acetilación , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Sirtuina 2/metabolismo , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismoRESUMEN
Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage-associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.
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Diferenciación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Pirimidinas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Dihidroorotato Deshidrogenasa , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/fisiopatología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Ribonucleósidos/genética , Ribonucleósidos/metabolismo , Puntos de Control de la Fase S del Ciclo CelularRESUMEN
Repetitive DNA sequences within eukaryotic heterochromatin are poorly transcribed and replicate late in S-phase. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). Despite the widespread association between transcription and replication timing, it remains unclear how transcription might impinge on replication, or vice versa. Here we show that, when silencing of an RNA polymerase II (RNA Pol II)-transcribed non-coding RNA at the rDNA is disrupted by SIR2 deletion, RNA polymerase pushes and thereby relocalizes replicative Mcm2-7 helicases away from their loading sites to an adjacent region with low nucleosome occupancy, and this relocalization is associated with increased rDNA origin efficiency. Our results suggest a model in which two of the major defining features of heterochromatin, transcriptional silencing and late replication, are mechanistically linked through suppression of polymerase-mediated displacement of replication initiation complexes.
Asunto(s)
Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/genética , Sirtuina 2/metabolismo , Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN Ribosómico/genética , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica/genética , Silenciador del Gen , Proteínas de Mantenimiento de Minicromosoma/genética , ARN Polimerasa I/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
BACKGROUND: The long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo. RESULTS: By using an inactive X-chromosome-linked MeCP2-GFP reporter, which allowed us to enumerate reactivation events in the mouse brain even when they occur in very few cells, we found that deletion of Xist in the brain after establishment of X-chromosome inactivation leads to reactivation in 2-5% of neurons and in a smaller fraction of astrocytes. In contrast to global loss of both H3 lysine 27 trimethylation (H3K27m3) and histone H2A lysine 119 monoubiquitylation (H2AK119ub1) we observed upon Xist deletion, alterations in CpG methylation were subtle, and this was mirrored by only minor alterations in X-chromosome-wide gene expression levels, with highly expressed genes more prone to both derepression and demethylation compared to genes with low expression level. CONCLUSION: Our results demonstrate that Xist plays a role in the maintenance of histone repressive marks, DNA methylation and transcriptional repression on the inactive X-chromosome, but that partial loss of X-dosage compensation in the absence of Xist in the brain is well tolerated.
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Encéfalo/metabolismo , Represión Epigenética , ARN Largo no Codificante/genética , Inactivación del Cromosoma X , Animales , Metilación de ADN , Código de Histonas , Ratones , Ratones Endogámicos C57BL , ARN Largo no Codificante/metabolismo , Eliminación de SecuenciaRESUMEN
Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.
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Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Saccharomyces cerevisiae/genética , Citometría de Flujo , HumanosRESUMEN
Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.
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Proteína 2 de Unión a Metil-CpG/genética , ARN Interferente Pequeño/genética , Inactivación del Cromosoma X/genética , Animales , Femenino , Humanos , Proteína 2 de Unión a Metil-CpG/metabolismo , RatonesRESUMEN
Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-ß pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-ß signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.
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Proteína Morfogenética Ósea 2/genética , Proteína 2 de Unión a Metil-CpG/genética , ARN Largo no Codificante/genética , Factor de Crecimiento Transformador beta/genética , Inactivación del Cromosoma X , Animales , Línea Celular , Femenino , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , Ratones , ARN Interferente Pequeño/genética , Síndrome de Rett/genética , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age- and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age- and disease-associated genome instability.
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Replicación del ADN , Inestabilidad Genómica , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Cromosomas Fúngicos/genética , ADN de Hongos/biosíntesis , ADN de Hongos/genética , ADN Ribosómico/biosíntesis , ADN Ribosómico/genética , Eliminación de Gen , Genoma Fúngico , Humanos , Modelos Biológicos , Secuencias Repetitivas de Ácidos Nucleicos , Origen de Réplica , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/deficiencia , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/deficiencia , Sirtuina 2/genéticaRESUMEN
X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened â¼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.