RESUMEN
Presenilin 1 (PS1) forms, via its large cytosolic loop, a trimeric complex with N-cadherin and ß-catenin, which is a key component of Wnt signaling. PS1 undergoes phosphorylation at 353 and 357 serines upon enhanced activity and elevated levels of the GSK3ß isoform. PS1 mutations surrounding these serines may alter the stability of the ß-catenin complex. Such mutations are found in some cases of familial early-onset Alzheimer's disease (fEOAD), but their functional impact remains obscure. One of such variants of PS1, the A360T substitution, is located close to GSK3ß-targeted serine residues. This variant was recently demonstrated in the French population, but more detail is needed to understand its biological effects. To assess the significance of this variant, we employed functional studies using a fibroblast cell line from an Alzheimer's disease case (a female proband) carrying the A360T mutation. Based on functional transcriptomic, cellular, and biochemical assays, we demonstrated atypically impaired ß-catenin/GSK3ß signaling in the A360T patient's fibroblasts. In detail, this was characterized by a decreased level of active cytosolic ß-catenin and bound by PS1, an increased level of nuclear ß-catenin, an increased level of inhibited GSK3ß phosphorylated on Ser9, and enhanced interaction of GSK3ß(Ser9) with PS1. Based on the transcriptomic profile of the A360T fibroblasts, we proposed a dysregulated transcriptional activity of ß-catenin, exemplified by increased expression of various cyclin-dependent kinases and cyclins, such as cyclin D1, potentially inducing neurons' cell cycle re-entry followed by apoptosis. The A360T cells did not exhibit significant amyloid pathology. Therefore, cell death in this PS1 cytosolic loop mutation may be attributed to impaired ß-catenin/GSK3ß signaling rather than amyloid deposition per se. We further estimated the biological and clinical relevance of the A360T variant by whole exome sequencing (WES). WES was performed on DNA from the blood of an A360T female proband, as well as an unrelated male patient carrying the A360T mutation and his mutation-free daughter (both unavailable for the derivation of the fibroblast cell lines). WES confirmed the highest-priority AD causality of the A360T variant in PS1 and also profiled the pathways and processes involved in the A360T case, highlighting the greatest importance of altered Wnt signaling.
Asunto(s)
Enfermedad de Alzheimer , beta Catenina , Femenino , Masculino , Humanos , beta Catenina/genética , Enfermedad de Alzheimer/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Transactivadores/genética , Presenilina-1/genética , Mutación , Expresión GénicaRESUMEN
Administration of PCSK9-specific monoclonal antibodies, as well as peptide-based PCSK9 vaccines, can lower plasma LDL cholesterol by blocking PCSK9. However, these treatments also cause an increase in plasma PCSK9 levels, presumably due to the formation of immune complexes. Here, we utilize a versatile capsid virus-like particle (cVLP)-based vaccine platform to deliver both full-length (FL) PCSK9 and PCSK9-derived peptide antigens, to investigate whether induction of a broader polyclonal anti-PCSK9 antibody response would mediate more efficient clearance of plasma PCSK9. This head-to-head immunization study reveals a significantly increased capacity of the FL PCSK9 cVLP vaccine to opsonize and clear plasma PCSK9. These findings may have implications for the design of PCSK9 and other vaccines that should effectively mediate opsonization and immune clearance of target antigens.
RESUMEN
Tiwanaku civilization flourished in the Lake Titicaca basin between 500 and 1000 CE and at its apogee influenced wide areas across the southern Andes. Despite a considerable amount of archaeological data, little is known about the Tiwanaku population. We analyzed 17 low-coverage genomes from individuals dated between 300 and 1500 CE and demonstrated genetic continuity in the Lake Titicaca basin throughout this period, which indicates that the substantial cultural and political changes in the region were not accompanied by large-scale population movements. Conversely, the ritual center of Tiwanaku revealed high diversity, including individuals with primarily local genetic ancestry and those with foreign admixture or provenance from as far as the Amazon. Nonetheless, most human offerings associated with the Akapana platform exhibited pure Titicaca basin ancestry and dated to ca. 950 CEthe onset of Tiwanaku's decline as a sociopolitical center. Our results strengthen the view of Tiwanaku as a complex and far-reaching polity.
RESUMEN
The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Vacunas contra la COVID-19/inmunología , Cápside/inmunología , Unión Proteica/inmunología , SARS-CoV-2/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Antivirales/inmunología , COVID-19/prevención & control , Femenino , Humanos , Inmunogenicidad Vacunal , Cinética , Ratones , Ratones Endogámicos BALB C , Unión Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Pruebas Serológicas , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).
Asunto(s)
Roturas del ADN de Doble Cadena , ADN/química , Etiquetado in Situ Primed/métodos , ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Células Eucariotas , Inestabilidad Genómica/genética , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Meiosis/genéticaRESUMEN
Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.
Asunto(s)
Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Estructuras R-Loop/genética , Regiones Terminadoras Genéticas/genética , Transcripción Genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo I/genética , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Fosforilación , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismoRESUMEN
The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.
Asunto(s)
Replicación del ADN/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Desoxirribonucleótidos/genética , Desoxirribonucleótidos/metabolismo , Regulación Fúngica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Mitosis , Proteínas Serina-Treonina Quinasas/genética , Origen de Réplica , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Replicación del ADN/genética , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Nucleosomas/genética , RecQ Helicasas/genética , Saccharomyces cerevisiae/genética , CohesinasRESUMEN
DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.
Asunto(s)
Biología Computacional/métodos , Roturas del ADN de Doble Cadena , Secuenciación Completa del Genoma/métodos , Línea Celular Tumoral , Replicación del ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Genoma Fúngico/genética , Genoma Humano/genética , Humanos , Saccharomycetales/genéticaRESUMEN
Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.
RESUMEN
Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.
Asunto(s)
Cromatina/genética , Reparación del ADN/genética , Histonas/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Inestabilidad Genómica/genética , Recombinación Homóloga/genética , Humanos , Células K562 , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
BACKGROUND: Approximately 90% of colorectal cancer (CRC) deaths are caused by tumors ability to migrate into the adjacent tissues and metastase into distant organs. More than 40 genes have been causally linked to the development of CRC but no mutations have been associated with metastasis yet. To identify molecular basis of CRC metastasis we performed whole-exome and genome-scale transcriptome sequencing of 7 liver metastases along with their matched primary tumours and normal tissue. Multiple, spatially separated fragments of primary tumours were analyzed in each case. Uniformly malignant tissue specimen were selected with macrodissection, for three samples followed with laser microdissection. RESULTS: > 100 sequencing coverage allowed for detection of genetic alterations in subpopulation of tumour cells. Mutations in KRAS, APC, POLE, and PTPRT, previously associated with CRC development, were detected in most patients. Several new associations were identified, including PLXND1, CELSR3, BAHD1 and PNPLA6. CONCLUSIONS: We confirm the essential role of inflammation in CRC progression but question the mechanism of matrix metalloproteinases activation described in other work. Comprehensive sequencing data made it possible to associate genome-scale mutation distribution with gene expression patterns. To our knowledge, this is the first work to report such link in CRC metastasis context.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Hepáticas/genética , Mutación , Metástasis de la Neoplasia/genética , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN , Exoma , Perfilación de la Expresión Génica , Humanos , Neoplasias Hepáticas/secundario , Análisis de Secuencia de ARNRESUMEN
Epigenetic mechanisms play an important role in the development and progression of various neurodegenerative diseases. Abnormal methylation of numerous genes responsible for regulation of transcription, DNA replication, and apoptosis has been linked to Alzheimer's disease (AD) pathology. We have recently performed whole transcriptome profiling of familial early-onset Alzheimer's disease (fEOAD) patient-derived fibroblasts. On this basis, we demonstrated a strong dysregulation of cell cycle checkpoints and DNA damage response (DDR) in both fibroblasts and reprogrammed neurons. Here, we show that the aging-correlated hypermethylation of KLF14 and TRIM59 genes associates with abnormalities in DNA repair and cell cycle control in fEOAD. Based on the resulting transcriptome networks, we found that the hypermethylation of KLF14 might be associated with epigenetic regulation of the chromatin organization and mRNA processing followed by hypermethylation of TRIM59 likely associated with the G2/M cell cycle phase and p53 role in DNA repair with BRCA1 protein as the key player. We propose that the hypermethylation of KLF14 could constitute a superior epigenetic mechanism for TRIM59 hypermethylation. The methylation status of both genes affects genome stability and might contribute to proapoptotic signaling in AD. Since this study combines data obtained from various tissues from AD patients, it reinforces the view that the genetic methylation status in the blood may be a valuable predictor of molecular processes occurring in affected tissues. Further research is necessary to define a detailed role of TRIM59 and KLF4 in neurodegeneration of neurons.
Asunto(s)
Enfermedad de Alzheimer/patología , Metilación de ADN , Proteínas de la Membrana/metabolismo , Metaloproteínas/metabolismo , Transducción de Señal , Factores de Transcripción Sp/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Apoptosis , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Puntos de Control del Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Reparación del ADN , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Redes Reguladoras de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Masculino , Proteínas de la Membrana/genética , Metaloproteínas/genética , Persona de Mediana Edad , Factores de Transcripción Sp/genética , Proteínas de Motivos Tripartitos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-ß. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-ß pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Proteína BRCA1/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Presenilina-1/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Células Cultivadas , Técnicas de Reprogramación Celular , Biología Computacional , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Humanos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Neuronas/patología , Fosforilación , Presenilina-1/genética , Presenilina-2/genética , Presenilina-2/metabolismo , Transducción de Señal , Transcriptoma , Fosfatasas cdc25/metabolismoRESUMEN
Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.
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Proliferación Celular/fisiología , Roturas del ADN de Doble Cadena , Inestabilidad Genómica/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Supervivencia Celular/fisiología , Islas de CpG/fisiología , Células Madre Hematopoyéticas/citología , Ratones , Ratones Noqueados , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The ability of DNA double-strand breaks (DSBs) to cluster in mammalian cells has been a subject of intense debate in recent years. Here we used a high-throughput chromosome conformation capture assay (capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. The results unambiguously demonstrated that DSBs cluster, but only when they are induced within transcriptionally active genes. Clustering of damaged genes occurs primarily during the G1 cell-cycle phase and coincides with delayed repair. Moreover, DSB clustering depends on the MRN complex as well as the Formin 2 (FMN2) nuclear actin organizer and the linker of nuclear and cytoplasmic skeleton (LINC) complex, thus suggesting that active mechanisms promote clustering. This work reveals that, when damaged, active genes, compared with the rest of the genome, exhibit a distinctive behavior, remaining largely unrepaired and clustered in G1, and being repaired via homologous recombination in postreplicative cells.
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Mapeo Cromosómico , Roturas del ADN de Doble Cadena , Genoma Humano , Línea Celular , Análisis por Conglomerados , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , ADN Intergénico/genética , Fase G1/efectos de los fármacos , Fase G1/genética , Histonas/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Dominios Proteicos , ARN Interferente Pequeño/metabolismo , Recombinación Genética/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Initiation of eukaryotic chromosome replication follows a spatiotemporal program. The current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by the Dbf4 C terminus and was successfully reconstituted in vitro. An interaction-defective mutant, dbf4ΔC, phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain (DBD) of Fkh1 to Dbf4 restores the Fkh-dependent origin firing but interferes specifically with the pericentromeric origin activation. Furthermore, Dbf4 interacts directly with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of noncentromeric origins to promote early replication in a manner that is reminiscent of the recruitment of Dbf4 to pericentromeric origins by Ctf19.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Origen de Réplica/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Genoma Fúngico/genética , Mutación , Proteínas Nucleares/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Sequencing microRNA, reduced representation sequencing, Hi-C technology and any method requiring the use of in-house barcodes result in sequencing libraries with low initial sequence diversity. Sequencing such data on the Illumina platform typically produces low quality data due to the limitations of the Illumina cluster calling algorithm. Moreover, even in the case of diverse samples, these limitations are causing substantial inaccuracies in multiplexed sample assignment (sample bleeding). Such inaccuracies are unacceptable in clinical applications, and in some other fields (e.g. detection of rare variants). Here, we discuss how both problems with quality of low-diversity samples and sample bleeding are caused by incorrect detection of clusters on the flowcell during initial sequencing cycles. We propose simple software modifications (Long Template Protocol) that overcome this problem. We present experimental results showing that our Long Template Protocol remarkably increases data quality for low diversity samples, as compared with the standard analysis protocol; it also substantially reduces sample bleeding for all samples. For comprehensiveness, we also discuss and compare experimental results from alternative approaches to sequencing low diversity samples. First, we discuss how the low diversity problem, if caused by barcodes, can be avoided altogether at the barcode design stage. Second and third, we present modified guidelines, which are more stringent than the manufacturer's, for mixing low diversity samples with diverse samples and lowering cluster density, which in our experience consistently produces high quality data from low diversity samples. Fourth and fifth, we present rescue strategies that can be applied when sequencing results in low quality data and when there is no more biological material available. In such cases, we propose that the flowcell be re-hybridized and sequenced again using our Long Template Protocol. Alternatively, we discuss how analysis can be repeated from saved sequencing images using the Long Template Protocol to increase accuracy.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/análisis , Análisis de Secuencia de ARN , Humanos , Reconocimiento de Normas Patrones Automatizadas , Proyectos de Investigación , Programas InformáticosRESUMEN
In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins, whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3Δ cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors.