RESUMEN
Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.
RESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of three human malignancies, the endothelial cell cancer Kaposi's sarcoma, and two B cell cancers, Primary Effusion Lymphoma and multicentric Castleman's disease. KSHV has latent and lytic phases of the viral life cycle, and while both contribute to viral pathogenesis, lytic proteins contribute to KSHV-mediated oncogenesis. Reactivation from latency is driven by the KSHV lytic gene transactivator RTA, and RTA transcription is controlled by epigenetic modifications. To identify host chromatin-modifying proteins that are involved in the latent to lytic transition, we screened a panel of inhibitors that target epigenetic regulatory proteins for their ability to stimulate KSHV reactivation. We found several novel regulators of viral reactivation: an inhibitor of Bmi1, PTC-209, two additional histone deacetylase inhibitors, Romidepsin and Panobinostat, and the bromodomain inhibitor (+)-JQ1. All of these compounds stimulate lytic gene expression, viral genome replication, and release of infectious virions. Treatment with Romidepsin, Panobinostat, and PTC-209 induces histone modifications at the RTA promoter, and results in nucleosome depletion at this locus. Finally, silencing Bmi1 induces KSHV reactivation, indicating that Bmi1, a member of the Polycomb repressive complex 1, is critical for maintaining KSHV latency.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Herpesvirus Humano 8/fisiología , Activación Viral/fisiología , Latencia del Virus/fisiología , Línea Celular , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Depsipéptidos/farmacología , Epigénesis Genética/efectos de los fármacos , Genoma Viral/efectos de los fármacos , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidad , Compuestos Heterocíclicos con 2 Anillos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Panobinostat/farmacología , Complejo Represivo Polycomb 1/antagonistas & inhibidores , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/fisiología , Regiones Promotoras Genéticas , Interferencia de ARN , Tiazoles/farmacología , Transactivadores/genética , Transactivadores/fisiología , Activación Viral/efectos de los fármacos , Activación Viral/genética , Latencia del Virus/genéticaRESUMEN
Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.
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Cromatina/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Neoplasias Óseas/patología , Línea Celular Tumoral , Cromatina/ultraestructura , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Inhibidores de Histona Desacetilasas/aislamiento & purificación , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Terapia Molecular Dirigida , Nucleosomas/ultraestructura , Proteínas de Fusión Oncogénica/genética , Panobinostat , Fenilbutiratos/farmacología , Sarcoma de Ewing/patología , Bibliotecas de Moléculas Pequeñas , VorinostatRESUMEN
One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.
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Inmunoprecipitación de Cromatina/métodos , Fragmentación del ADN/efectos de la radiación , ADN/genética , Sonicación/métodos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/efectos de la radiación , ADN/química , ADN/efectos de la radiación , Eucromatina/efectos de la radiación , Heterocromatina/efectos de la radiación , Ratones , Nanopartículas/química , Factor 3 de Transcripción de Unión a Octámeros/química , Factor 3 de Transcripción de Unión a Octámeros/genéticaRESUMEN
We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a K(d) of â¼100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.
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Oligopéptidos/farmacología , Complejo Represivo Polycomb 1/antagonistas & inhibidores , Complejo Represivo Polycomb 1/genética , Animales , Disponibilidad Biológica , Biotinilación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Regulación de la Expresión Génica/genética , Humanos , Isomerismo , Ligasas , Masculino , Metilación , Ratones , Modelos Moleculares , Complejo Represivo Polycomb 1/biosíntesis , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999's on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.
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Antineoplásicos/farmacología , Leucemia Bifenotípica Aguda/enzimología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Animales , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/farmacología , Immunoblotting , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Messenger RNA processing is coupled to RNA polymerase II (RNAPII) transcription through coordinated recruitment of accessory proteins to the Rpb1 C-terminal domain (CTD). Dynamic changes in CTD phosphorylation during transcription elongation are responsible for their recruitment, with serine 5 phosphorylation (S5-P) occurring toward the 5' end of genes and serine 2 phosphorylation (S2-P) occurring toward the 3' end. The proteins responsible for regulation of the transition state between S5-P and S2-P CTD remain elusive. We show that a conserved protein of unknown function, Rtr1, localizes within coding regions, with maximum levels of enrichment occurring between the peaks of S5-P and S2-P RNAPII. Upon deletion of Rtr1, the S5-P form of RNAPII accumulates in both whole-cell extracts and throughout coding regions; additionally, RNAPII transcription is decreased, and termination defects are observed. Functional characterization of Rtr1 reveals its role as a CTD phosphatase essential for the S5-to-S2-P transition.
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Fosfoproteínas Fosfatasas/fisiología , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/fisiología , Inmunoprecipitación de Cromatina , Modelos Genéticos , Sistemas de Lectura Abierta , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the majority of histone H3 lysine 9 mono- and di-methylation (H3K9me1/me2). Widely interspaced zinc finger (WIZ) associates with the G9a-GLP protein complex, but its role in mediating lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels by facilitating the interaction of G9a with chromatin. Disrupting the association of G9a-GLP with chromatin by depleting WIZ resulted in altered gene expression and protein-protein interactions that were distinguishable from that of small molecule-based inhibition of G9a/GLP, supporting discrete functions of the G9a-GLP-WIZ chromatin complex in addition to H3K9me2 methylation.
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Cromatina/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Metiltransferasas/metabolismo , Inmunoprecipitación de Cromatina , Metilación de ADN , Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Unión ProteicaRESUMEN
Polymerized ß-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Núcleo Celular , Cromatina , Células Madre Mesenquimatosas , Actinas/metabolismo , Cromatina/metabolismo , Núcleo Celular/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Animales , Diferenciación Celular , Citocalasina D/farmacología , Histonas/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Ratones , Ensamble y Desensamble de CromatinaRESUMEN
Type II innate lymphoid cells (ILC2s) maintain homeostasis and barrier integrity in mucosal tissues. In both mice and humans, ILC2s poorly reconstitute after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Determining the mechanisms involved in their impaired reconstitution could improve transplant outcomes. By integrating single-cell chromatin and transcriptomic analyses of transplanted ILC2s, we identify a previously unreported population of converted ILC1-like cells in the mouse small intestine post-transplant. Exposure of ILC2s to proinflammatory cytokines resulted in a mixed ILC1-ILC2 phenotype but was able to convert only a small population of ILC2s to ILC1s, which were found post-transplant. Whereas ILC2s protected against acute graft-versus-host disease (aGVHD) mediated mortality, infusion of proinflammatory cytokine-exposed ILC2s accelerated aGvHD. Interestingly, murine ILC2 reconstitution post-HSCT is decreased in the presence of alloreactive T cells. Finally, peripheral blood cells from human patients with aGvHD have an altered ILC2-associated chromatin landscape compared to transplanted controls. These data demonstrate that following transplantation ILC2s convert to a pro-pathogenic population with an ILC1-like chromatin state and provide insights into the contribution of ILC plasticity to the impaired reconstitution of ILC2 cells, which is one of several potential mechanisms for the poor reconstitution of these important cells after allo-HSCT.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Inmunidad Innata , Linfocitos , Ratones Endogámicos C57BL , Trasplante Homólogo , Trasplante de Células Madre Hematopoyéticas/métodos , Animales , Humanos , Enfermedad Injerto contra Huésped/inmunología , Ratones , Linfocitos/inmunología , Citocinas/metabolismo , Plasticidad de la Célula , Femenino , Intestino Delgado/inmunología , Masculino , Ratones Endogámicos BALB C , Cromatina/metabolismoRESUMEN
Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.
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Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/metabolismo , Quinazolinas/farmacología , Animales , Línea Celular , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Ratones , Estructura MolecularRESUMEN
The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.
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ARN Polimerasas Dirigidas por ADN/química , Proteómica/métodos , Análisis por Conglomerados , Espectrometría de Masas/métodos , Modelos Estadísticos , Péptidos/química , Proteínas/química , ARN/química , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Análisis de Secuencia de Proteína/métodosRESUMEN
Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.
RESUMEN
Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein-coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy. IMPLICATIONS: We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.
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Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/genética , Femenino , HumanosRESUMEN
L3MBTL3 recognizes mono- and dimethylated lysine residues on histone tails. The recently reported X-ray cocrystal structures of the chemical probe UNC1215 and inhibitor UNC2533 bound to the methyl-lysine reading MBT domains of L3MBTL3 demonstrate a unique and flexible 2:2 dimer mode of recognition. In this study, we describe our in vitro analysis of L3MBTL3 dimerization via its MBT domains and additionally show that this dimerization occurs within a cellular context in the absence of small molecule ligands. Furthermore, mutations to the first and second MBT domains abrogated L3MBTL3 dimerization both in vitro and in cells. These observations are consistent with the hypothesis that L3MBTL3 engages methylated histone tails as a dimer while carrying out its normal function and provides an explanation for the presence of repeated MBT domains within L3MBTL3.
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Proteínas de Unión al ADN/metabolismo , Benzamidas , Biotina , Sistema Libre de Células , Proteínas de Unión al ADN/genética , Células HeLa , Histonas , Humanos , Ligandos , Estructura Molecular , Mutación , Piperidinas , Dominios Proteicos , Multimerización de ProteínaRESUMEN
A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.
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Fragmentación del ADN/efectos de la radiación , Sonicación/métodos , ADN de Hongos , Microburbujas , Sonicación/instrumentaciónRESUMEN
Endothelial-to-mesenchymal transition (EndMT) occurs during development and underlies the pathophysiology of multiple diseases. In tumors, unscheduled EndMT generates cancer-associated myofibroblasts that fuel inflammation and fibrosis, and may contribute to vascular dysfunction that promotes tumor progression. We report that freshly isolated subpopulations of tumor-specific endothelial cells (TEC) from a spontaneous mammary tumor model undergo distinct forms of EndMT in response to TGFß stimulation. Although some TECs strikingly upregulate α smooth muscle actin (SMA), a principal marker of EndMT and activated myofibroblasts, counterpart normal mammary gland endothelial cells (NEC) showed little change in SMA expression after TGFß treatment. Compared with NECs, SMA(+) TECs were 40% less motile in wound-healing assays and formed more stable vascular-like networks in vitro when challenged with TGFß. Lineage tracing using ZsGreen(Cdh5-Cre) reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA(+) TECs, suggesting that not all endothelial cells (EC) respond identically to TGFß in vivo. Indeed, examination of 84 TGFß-regulated target genes revealed entirely different genetic signatures in TGFß-stimulated NEC and TEC cultures. Finally, we found that basic FGF (bFGF) exerts potent inhibitory effects on many TGFß-regulated genes but operates in tandem with TGFß to upregulate others. ECs challenged with TGFß secrete bFGF, which blocks SMA expression in secondary cultures, suggesting a cell-autonomous or lateral-inhibitory mechanism for impeding mesenchymal differentiation. Together, our results suggest that TGFß-driven EndMT produces a spectrum of EC phenotypes with different functions that could underlie the plasticity and heterogeneity of the tumor vasculature.