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1.
Cell ; 159(5): 1126-1139, 2014 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-25416950

RESUMEN

The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins.


Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Modelos Animales de Enfermedad , Neuroblastoma/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Fenilendiaminas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/uso terapéutico , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Proteína Proto-Oncogénica N-Myc , Transcripción Genética/efectos de los fármacos , Quinasa Activadora de Quinasas Ciclina-Dependientes
2.
Cell ; 154(5): 1127-1139, 2013 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-23993100

RESUMEN

Following DNA replication, eukaryotic cells must biorient all sister chromatids prior to cohesion cleavage at anaphase. In animal cells, sister chromatids gradually biorient during prometaphase, but current models of mitosis in S. cerevisiae assume that biorientation is established shortly after S phase. This assumption is based on the observation of a bilobed distribution of yeast kinetochores early in mitosis and suggests fundamental differences between yeast mitosis and mitosis in animal cells. By applying super-resolution imaging methods, we show that yeast and animal cells share the key property of gradual and stochastic chromosome biorientation. The characteristic bilobed distribution of yeast kinetochores, hitherto considered synonymous for biorientation, arises from kinetochores in mixed attachment states to microtubules, the length of which discriminates bioriented from syntelic attachments. Our results offer a revised view of mitotic progression in S. cerevisiae that augments the relevance of mechanistic information obtained in this powerful genetic system for mammalian mitosis.


Asunto(s)
Cromosomas Fúngicos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Anafase , Aurora Quinasas , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Fase S , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático
3.
Nature ; 572(7771): 676-680, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31391581

RESUMEN

The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements1,2. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation3,4 or DNA hypermethylation5 results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)6, is overexpressed in several cancers7-9, but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma10 cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.


Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Animales , Factor de Unión a CCCTC/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Humanos , Ratones , Terapia Molecular Dirigida , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/enzimología , Neuroblastoma/genética , Fenotipo , Unión Proteica
4.
J Immunol ; 206(2): 446-451, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33277386

RESUMEN

Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.


Asunto(s)
Islas de CpG/genética , Enzimas de Restricción del ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T Reguladores/inmunología , Células Cultivadas , Metilación de ADN , Desmetilación , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Especificidad de Órganos , Cultivo Primario de Células
5.
PLoS Genet ; 12(11): e1006429, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27828963

RESUMEN

E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their 'core' cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer.


Asunto(s)
Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/genética , Ciclinas/genética , Proteínas Oncogénicas/genética , Proteómica , Animales , Ciclo Celular/genética , Proliferación Celular/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Humanos , Masculino , Ratones , Proteínas Oncogénicas/metabolismo , Fosforilación , Mapas de Interacción de Proteínas/genética , Fase S/genética , Espermatogénesis/genética , Testículo/metabolismo
6.
BMC Genomics ; 19(1): 212, 2018 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-29562890

RESUMEN

BACKGROUND: Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations. RESULTS: To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes. CONCLUSIONS: UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Reordenamiento Génico , Genoma Humano , Mutación INDEL , Osteosarcoma/diagnóstico , Antígenos de Neoplasias/genética , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/genética , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas del Citoesqueleto , Genómica/métodos , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Osteosarcoma/genética , Eliminación de Secuencia , Linfocitos T/metabolismo , Linfocitos T/patología
7.
Nucleic Acids Res ; 44(14): e122, 2016 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-27207878

RESUMEN

Single-cell gene expression data provide invaluable resources for systematic characterization of cellular hierarchy in multi-cellular organisms. However, cell lineage reconstruction is still often associated with significant uncertainty due to technological constraints. Such uncertainties have not been taken into account in current methods. We present ECLAIR (Ensemble Cell Lineage Analysis with Improved Robustness), a novel computational method for the statistical inference of cell lineage relationships from single-cell gene expression data. ECLAIR uses an ensemble approach to improve the robustness of lineage predictions, and provides a quantitative estimate of the uncertainty of lineage branchings. We show that the application of ECLAIR to published datasets successfully reconstructs known lineage relationships and significantly improves the robustness of predictions. ECLAIR is a powerful bioinformatics tool for single-cell data analysis. It can be used for robust lineage reconstruction with quantitative estimate of prediction accuracy.


Asunto(s)
Linaje de la Célula , Análisis de la Célula Individual/métodos , Algoritmos , Animales , Bases de Datos como Asunto , Embrión de Mamíferos/citología , Hematopoyesis , Ratones , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN
8.
Proc Natl Acad Sci U S A ; 111(52): E5643-50, 2014 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-25512504

RESUMEN

We present single-cell clustering using bifurcation analysis (SCUBA), a novel computational method for extracting lineage relationships from single-cell gene expression data and modeling the dynamic changes associated with cell differentiation. SCUBA draws techniques from nonlinear dynamics and stochastic differential equation theories, providing a systematic framework for modeling complex processes involving multilineage specifications. By applying SCUBA to analyze two complementary, publicly available datasets we successfully reconstructed the cellular hierarchy during early development of mouse embryos, modeled the dynamic changes in gene expression patterns, and predicted the effects of perturbing key transcriptional regulators on inducing lineage biases. The results were robust with respect to experimental platform differences between RT-PCR and RNA sequencing. We selectively tested our predictions in Nanog mutants and found good agreement between SCUBA predictions and the experimental data. We further extended the utility of SCUBA by developing a method to reconstruct missing temporal-order information from a typical single-cell dataset. Analysis of a hematopoietic dataset suggests that our method is effective for reconstructing gene expression dynamics during human B-cell development. In summary, SCUBA provides a useful single-cell data analysis tool that is well-suited for the investigation of developmental processes.


Asunto(s)
Linfocitos B , Diferenciación Celular/fisiología , Embrión de Mamíferos , Epigénesis Genética/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Hematopoyesis/fisiología , Modelos Biológicos , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Proteína Homeótica Nanog , Procesos Estocásticos
10.
Nat Biotechnol ; 42(3): 458-469, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37127662

RESUMEN

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Técnicas de Sustitución del Gen , Transgenes/genética
11.
CRISPR J ; 4(2): 264-274, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33876962

RESUMEN

We describe CALITAS, a CRISPR-Cas-aware aligner and integrated off-target search algorithm. CALITAS uses a modified and CRISPR-tuned version of the Needleman-Wunsch algorithm. It supports an unlimited number of mismatches and gaps and allows protospacer adjacent motif (PAM) mismatches or PAMless searches. CALITAS also includes an exhaustive search routine to scan genomes and genome variants provided with a standard Variant Call Format file. By default, CALITAS returns a single best alignment for a given off-target site, which is a significant improvement compared to other off-target algorithms, and it enables off-targets to be referenced directly using alignment coordinates. We validate and compare CALITAS using a selected set of target sites, as well as experimentally derived specificity data sets. In summary, CALITAS is a new tool for precise and relevant alignments and identification of candidate off-target sites across a genome. We believe it is the state of the art for CRISPR-Cas specificity assessments.


Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Simulación por Computador , Endonucleasas/genética , Algoritmos , Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Proteínas Asociadas a CRISPR , Endodesoxirribonucleasas , Edición Génica , Genoma , ARN Guía de Kinetoplastida/genética , Programas Informáticos
12.
CRISPR J ; 3(3): 177-187, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32584143

RESUMEN

Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Linfocitos T , Translocación Genética , Linfocitos T CD4-Positivos , Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , Endonucleasas/genética , Genoma Humano , Humanos , Hibridación Fluorescente in Situ , Herencia Multifactorial , ARN Guía de Kinetoplastida , Streptococcus pyogenes
13.
Nat Med ; 25(2): 229-233, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30664785

RESUMEN

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.


Asunto(s)
Edición Génica , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/fisiopatología , Animales , Línea Celular , Técnicas de Sustitución del Gen , Humanos , Ratones , Primates , Reproducibilidad de los Resultados , Visión Ocular
14.
Hum Gene Ther ; 29(8): 861-873, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29641323

RESUMEN

Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.


Asunto(s)
Sistemas CRISPR-Cas/genética , Terapia Genética , Deficiencia de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Alelos , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Edición Génica , Vectores Genéticos/uso terapéutico , Hepatocitos/patología , Homocigoto , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Transgénicos/genética , Mutación , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/patología
15.
Nat Commun ; 8: 15011, 2017 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-28387224

RESUMEN

Chromatin-state analysis is widely applied in the studies of development and diseases. However, existing methods operate at a single length scale, and therefore cannot distinguish large domains from isolated elements of the same type. To overcome this limitation, we present a hierarchical hidden Markov model, diHMM, to systematically annotate chromatin states at multiple length scales. We apply diHMM to analyse a public ChIP-seq data set. diHMM not only accurately captures nucleosome-level information, but identifies domain-level states that vary in nucleosome-level state composition, spatial distribution and functionality. The domain-level states recapitulate known patterns such as super-enhancers, bivalent promoters and Polycomb repressed regions, and identify additional patterns whose biological functions are not yet characterized. By integrating chromatin-state information with gene expression and Hi-C data, we identify context-dependent functions of nucleosome-level states. Thus, diHMM provides a powerful tool for investigating the role of higher-order chromatin structure in gene regulation.


Asunto(s)
Algoritmos , Cromatina/genética , Regulación de la Expresión Génica , Cadenas de Markov , Nucleosomas/genética , Línea Celular , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Células K562 , Nucleosomas/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Regiones Promotoras Genéticas/genética
16.
Nat Med ; 22(7): 723-6, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27270588

RESUMEN

Brain metastases represent the greatest clinical challenge in treating HER2-positive breast cancer. We report the development of orthotopic patient-derived xenografts (PDXs) of HER2-expressing breast cancer brain metastases (BCBM), and their use for the identification of targeted combination therapies. Combined inhibition of PI3K and mTOR resulted in durable tumor regressions in three of five PDXs, and therapeutic response was correlated with a reduction in the phosphorylation of 4EBP1, an mTORC1 effector. The two nonresponding PDXs showed hypermutated genomes with enrichment of mutations in DNA-repair genes, which suggests an association of genomic instability with therapeutic resistance. These findings suggest that a biomarker-driven clinical trial of PI3K inhibitor in combination with an mTOR inhibitor should be conducted for patients with HER2-positive BCBM.


Asunto(s)
Aminopiridinas/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Everolimus/farmacología , Morfolinas/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Proteínas de Ciclo Celular , Reparación del ADN/genética , Resistencia a Antineoplásicos/genética , Quimioterapia Combinada , Factores Eucarióticos de Iniciación , Femenino , Perfilación de la Expresión Génica , Inestabilidad Genómica , Humanos , Inmunohistoquímica , Antígeno Ki-67/efectos de los fármacos , Antígeno Ki-67/metabolismo , Imagen por Resonancia Magnética , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones SCID , Terapia Molecular Dirigida , Trasplante de Neoplasias , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación , Receptor ErbB-2/metabolismo , Inducción de Remisión , Ensayos Antitumor por Modelo de Xenoinjerto
17.
Genome Biol ; 16: 162, 2015 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-26272203

RESUMEN

Genome-wide mapping of three dimensional chromatin organization is an important yet technically challenging task. To aid experimental effort and to understand the determinants of long-range chromatin interactions, we have developed a computational model integrating Hi-C and histone mark ChIP-seq data to predict two important features of chromatin organization: chromatin interaction hubs and topologically associated domain (TAD) boundaries. Our model accurately and robustly predicts these features across datasets and cell types. Cell-type specific histone mark information is required for prediction of chromatin interaction hubs but not for TAD boundaries. Our predictions provide a useful guide for the exploration of chromatin organization.


Asunto(s)
Cromatina/metabolismo , Código de Histonas , Línea Celular , Cromatina/química , Inmunoprecipitación de Cromatina , Simulación por Computador , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ADN
18.
Genome Biol ; 16: 257, 2015 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-26596280

RESUMEN

BACKGROUND: CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9. RESULTS: We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq. CONCLUSION: Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.


Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas/genética , Endodesoxirribonucleasas/genética , Técnicas de Transferencia de Gen , Ingeniería Genética , Staphylococcus aureus/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Desoxirribonucleasa I/genética , Dependovirus/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Edición de ARN , Streptococcus pyogenes/genética
19.
Cell Stem Cell ; 13(4): 492-505, 2013 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-24035353

RESUMEN

Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method, we analyzed over 1,500 single cells throughout the mouse hematopoietic system and illustrate its utility for revealing important biological insights. The comprehensive single cell data set permits mapping of the mouse hematopoietic stem cell differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems.


Asunto(s)
Antígenos de Superficie/análisis , Antígenos de Superficie/genética , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Sistema Hematopoyético/citología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Reacción en Cadena de la Polimerasa
20.
PLoS One ; 4(10): e7640, 2009 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-19893618

RESUMEN

BACKGROUND: Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain approximately 70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores. PRINCIPAL FINDINGS: We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells. CONCLUSIONS/SIGNIFICANCE: Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.


Asunto(s)
Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Huso Acromático , Secuencia de Aminoácidos , Anafase , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/química , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Fenotipo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido
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