RESUMEN
Most of the genome is transcribed into RNA but only 2% of the sequence codes for proteins. Non-coding RNA transcripts include a very large number of long noncoding RNAs (lncRNAs). A growing number of identified lncRNAs operate in cellular stress responses, for example in response to hypoxia, genotoxic stress, and oxidative stress. Additionally, lncRNA plays important roles in epigenetic mechanisms operating at chromatin and in maintaining chromatin architecture. Here, we address three lncRNA topics that have had significant recent advances. The first is an emerging role for many lncRNAs in cellular stress responses. The second is the development of high throughput screening assays to develop causal relationships between lncRNAs across the genome with cellular functions. Finally, we turn to recent advances in understanding the role of lncRNAs in regulating chromatin architecture and epigenetics, advances that build on some of the earliest work linking RNA to chromatin architecture.
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Cromatina , Epigénesis Genética , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromatina/metabolismo , Cromatina/genética , Humanos , Animales , Estrés Fisiológico/genéticaRESUMEN
Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.
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Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Microglía/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Profilinas/metabolismo , MutaciónRESUMEN
There is a long experimental history supporting the principle that RNA is essential for normal nuclear and chromatin architecture. Most of the genome is transcribed into RNA but only 2% of the sequence codes for proteins. In the nucleus, most non-coding RNA, packaged in proteins, is bound into structures including chromatin and a non-chromatin scaffolding, the nuclear matrix, which was first observed by electron microscopy. Removing nuclear RNA or inhibiting its transcription causes the condensation of chromatin, showing the importance of RNA in spatially and functionally organizing the genome. Today, powerful techniques for the molecular characterization of RNA and for mapping its spatial organization in the nucleus have provided molecular detail to these principles.
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Núcleo Celular , Ribonucleoproteínas , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Matriz Nuclear/química , Matriz Nuclear/genética , Matriz Nuclear/metabolismo , ARN/metabolismo , Ribonucleoproteínas/análisis , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.
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Transporte Activo de Núcleo Celular/fisiología , Neuronas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Humanos , Mutación , Proteína FUS de Unión a ARN/genéticaRESUMEN
Brahma-related gene 1 (BRG1) is one of two mutually exclusive ATPases that function as the catalytic subunit of human SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling enzymes. BRG1 has been identified as a tumor suppressor in some cancer types but has been shown to be expressed at elevated levels, relative to normal tissue, in other cancers. Using TCGA (The Cancer Genome Atlas) prostate cancer database, we determined that BRG1 mRNA and protein expression is elevated in prostate tumors relative to normal prostate tissue. Only 3 of 491 (0.6%) sequenced tumors showed amplification of the locus or mutation in the protein coding sequence, arguing against the idea that elevated expression due to amplification or expression of a mutant BRG1 protein is associated with prostate cancer. Kaplan-Meier survival curves showed that BRG1 expression in prostate tumors inversely correlated with survival. However, BRG1 expression did not correlate with Gleason score/International Society of Urological Pathology (ISUP) Grade Group, indicating it is an independent predictor of tumor progression/patient outcome. To experimentally assess BRG1 as a possible therapeutic target, we treated prostate cancer cells with a biologic inhibitor called ADAADi (active DNA-dependent ATPase A Domain inhibitor) that targets the activity of the SNF2 family of ATPases in biochemical assays but showed specificity for BRG1 in prior tissue culture experiments. The inhibitor decreased prostate cancer cell proliferation and induced apoptosis. When directly injected into xenografts established by injection of prostate cancer cells in mouse flanks, the inhibitor decreased tumor growth and increased survival. These results indicate the efficacy of pursuing BRG1 as both an indicator of patient outcome and as a therapeutic target.
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The RUNX1 transcription factor has recently been shown to be obligatory for normal development. RUNX1 controls the expression of genes essential for proper development in many cell lineages and tissues including blood, bone, cartilage, hair follicles, and mammary glands. Compromised RUNX1 regulation is associated with many cancers. In this review, we highlight evidence for RUNX1 control in both invertebrate and mammalian development and recent novel findings of perturbed RUNX1 control in breast cancer that has implications for other solid tumors. As RUNX1 is essential for definitive hematopoiesis, RUNX1 mutations in hematopoietic lineage cells have been implicated in the etiology of several leukemias. Studies of solid tumors have revealed a context-dependent function for RUNX1 either as an oncogene or a tumor suppressor. These RUNX1 functions have been reported for breast, prostate, lung, and skin cancers that are related to cancer subtypes and different stages of tumor development. Growing evidence suggests that RUNX1 suppresses aggressiveness in most breast cancer subtypes particularly in the early stage of tumorigenesis. Several studies have identified RUNX1 suppression of the breast cancer epithelial-to-mesenchymal transition. Most recently, RUNX1 repression of cancer stem cells and tumorsphere formation was reported for breast cancer. It is anticipated that these new discoveries of the context-dependent diversity of RUNX1 functions will lead to innovative therapeutic strategies for the intervention of cancer and other abnormalities of normal tissues.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Neoplasias/metabolismo , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Neoplasias/genética , Neoplasias/patología , Pronóstico , Transducción de SeñalRESUMEN
Breast cancer is the most common cancer in women, and accounts for ~30% of new cancer cases and 15% of cancer-related deaths. Tumor relapse and metastasis are primary factors contributing to breast cancer-related deaths. Therefore, the challenge for breast cancer treatment is to sustain remission. A driving force behind tumor relapse is breast cancer heterogeneity (both intertumor, between different patients, and intratumor, within the same tumor). Understanding breast cancer heterogeneity is necessary to develop preventive interventions and targeted therapies. A recently emerging concept is that intratumor heterogeneity is driven by cancer stem cells (CSCs) that are capable of giving rise to a multitude of different cells within a tumor. Studies have highlighted linkage of CSC formation with epithelial-to-mesenchymal transition (EMT). In this review, we summarize the current understanding of breast cancer heterogeneity, links between EMT and CSCs, regulation of EMT by Runx transcription factors, and potential therapeutic strategies targeting these processes.
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Neoplasias de la Mama/genética , Carcinogénesis/genética , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama/patología , Femenino , Heterogeneidad Genética , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
Reconfiguration of nuclear structure and function during mitosis presents a significant challenge to resume the next cell cycle in the progeny cells without compromising structural and functional identity of the cells. Equally important is the requirement for cancer cells to retain the transformed phenotype, that is, unrestricted proliferative potential, suppression of cell phenotype, and activation of oncogenic pathways. Mitotic gene bookmarking retention of key regulatory proteins that include sequence-specific transcription factors, chromatin-modifying factors, and components of RNA Pol (RNAP) I and II regulatory machineries at gene loci on mitotic chromosomes plays key roles in coordinate control of cell phenotype, growth, and proliferation postmitotically. There is growing recognition that three distinct protein types, mechanistically, play obligatory roles in mitotic gene bookmarking: (i) Retention of phenotypic transcription factors on mitotic chromosomes is essential to sustain lineage commitment; (ii) Select chromatin modifiers and posttranslational histone modifications/variants retain competency of mitotic chromatin for gene reactivation as cells exit mitosis; and (iii) Functional components of RNAP I and II transcription complexes (e.g., UBF and TBP, respectively) are retained on genes poised for reactivation immediately following mitosis. Importantly, recent findings have identified oncogenes that are associated with target genes on mitotic chromosomes in cancer cells. The current review proposes that mitotic gene bookmarking is an extensively utilized epigenetic mechanism for stringent control of proliferation and identity in normal cells and hypothesizes that bookmarking plays a pivotal role in maintenance of tumor phenotypes, that is, unrestricted proliferation and compromised control of differentiation. Mol Cancer Res; 16(11); 1617-24. ©2018 AACR.
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Mitosis/genética , Diferenciación Celular , Epigénesis Genética , Humanos , Neoplasias/genética , Neoplasias/patología , FenotipoRESUMEN
Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expression for biological control and is modified in cancer. Nuclear organization supports cell growth and phenotypic properties of normal and cancer cells by facilitating physiologically responsive interactions of chromosomes, genes and regulatory complexes at dynamic three-dimensional microenvironments. We will review nuclear structure/function relationships that include: 1. Epigenetic bookmarking of genes by phenotypic transcription factors to control fidelity and plasticity of gene expression as cells enter and exit mitosis; 2. Contributions of chromatin remodeling to breast cancer nuclear morphology, metabolism and effectiveness of chemotherapy; 3. Relationships between fidelity of nuclear organization and metastasis of breast cancer to bone; 4. Dynamic modifications of higher-order inter- and intra-chromosomal interactions in breast cancer cells; 5. Coordinate control of cell growth and phenotype by tissue-specific transcription factors; 6. Oncofetal epigenetic control by bivalent histone modifications that are functionally related to sustaining the stem cell phenotype; and 7. Noncoding RNA-mediated regulation in the onset and progression of breast cancer. The discovery of components to nuclear organization that are functionally related to cancer and compromise gene expression have the potential for translation to innovative cancer diagnosis and targeted therapy.
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Epigénesis Genética/genética , Animales , Neoplasias de la Mama/genética , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Humanos , Mitosis/genética , Mitosis/fisiologíaRESUMEN
Transcriptional regulation is modulated in part by chromatin-remodeling enzymes that control gene accessibility by altering chromatin compaction or nucleosome positioning. Brahma-related gene 1 (Brg1), a catalytic subunit of the mammalian SWI/SNF chromatin-remodeling enzymes, is required for both myoblast proliferation and differentiation, and the control of Brg1 phosphorylation by calcineurin, PKCß1, and p38 regulates the transition to differentiation. However, we hypothesized that Brg1 activity might be regulated by additional kinases. Here, we report that Brg1 is also a target of casein kinase 2 (CK2), a serine/threonine kinase, in proliferating myoblasts. We found that CK2 interacts with Brg1, and mutation of putative phosphorylation sites to non-phosphorylatable (Ser to Ala, SA) or phosphomimetic residues (Ser to Glu, SE) reduced Brg1 phosphorylation by CK2. Although BRG1-deleted myoblasts that ectopically express the SA-Brg1 mutant proliferated similarly to the parental cells or cells ectopically expressing wild-type (WT) Brg1, ectopic expression of the SE-Brg1 mutant reduced proliferation and increased cell death, similar to observations from cells lacking Brg1. Moreover, pharmacological inhibition of CK2 increased myoblast proliferation. Furthermore, the Pax7 promoter, which controls expression of a key transcription factor required for myoblast proliferation, was in an inaccessible chromatin state in the SE-Brg1 mutant, suggesting that hyperphosphorylated Brg1 cannot remodel chromatin. WT-, SA-, and SE-Brg1 exhibited distinct differences in interacting with and affecting expression of the SWI/SNF subunits Baf155 and Baf170 and displayed differential sub-nuclear localization. Our results indicate that CK2-mediated phosphorylation of Brg1 regulates myoblast proliferation and provides insight into one mechanism by which composition of the mammalian SWI/SNF enzyme complex is regulated.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Regulación de la Expresión Génica , Mioblastos Esqueléticos/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Animales , Quinasa de la Caseína II/efectos de los fármacos , Quinasa de la Caseína II/genética , Células Cultivadas , Proteínas Cromosómicas no Histona/química , ADN Helicasas/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , Proteínas Nucleares/genética , Factor de Transcripción PAX7/agonistas , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/efectos de los fármacos , Células Satélite del Músculo Esquelético/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Mammalian SWI/SNF enzymes are ATP-dependent remodelers of chromatin structure. These multisubunit enzymes are heterogeneous in composition; there are two catalytic ATPase subunits, BRM and BRG1, that are mutually exclusive, and additional subunits are incorporated in a combinatorial manner. Recent findings indicate that approximately 20% of human cancers contain mutations in SWI/SNF enzyme subunits, leading to the conclusion that the enzyme subunits are critical tumor suppressors. However, overexpression of specific subunits without apparent mutation is emerging as an alternative mechanism by which cellular transformation may occur. Here we highlight recent evidence linking elevated expression of the BRG1 ATPase to tissue-specific cancers and work suggesting that inhibiting BRG1 may be an effective therapeutic strategy.
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Carcinogénesis/genética , Ensamble y Desensamble de Cromatina , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , ADN Helicasas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Tumor cells reprogram their metabolism to survive and grow in a challenging microenvironment. Some of this reprogramming is performed by epigenetic mechanisms. Epigenetics is in turn affected by metabolism; chromatin modifying enzymes are dependent on substrates that are also key metabolic intermediates. We have shown that the chromatin remodeling enzyme Brahma-related gene 1 (BRG1), an epigenetic regulator, is necessary for rapid breast cancer cell proliferation. The mechanism for this requirement is the BRG1-dependent transcription of key lipogenic enzymes and regulators. Reduction in lipid synthesis lowers proliferation rates, which can be restored by palmitate supplementation. This work has established BRG1 as an attractive target for breast cancer therapy. Unlike genetic alterations, epigenetic mechanisms are reversible, promising gentler therapies without permanent off-target effects at distant sites.
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The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamins A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure-gene expression relationships and for the maintenance of pluripotency. J. Cell. Biochem. 118: 764-774, 2017. © 2016 Wiley Periodicals, Inc.
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Células Madre Embrionarias Humanas/ultraestructura , Línea Celular , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Estructuras del Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Células Madre Embrionarias Humanas/metabolismo , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Poro Nuclear/ultraestructuraRESUMEN
Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix-attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix-associated genome is highly cell-context dependent. J. Cell. Physiol. 232: 1295-1305, 2017. © 2016 Wiley Periodicals, Inc.
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ADN/metabolismo , Genoma Humano , Regiones de Fijación a la Matriz/genética , Matriz Nuclear/metabolismo , Neoplasias de la Mama/genética , Línea Celular , Cromatina/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Sistemas de Lectura Abierta/genética , Reproducibilidad de los ResultadosRESUMEN
Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined with myosin inhibition studies, we identify genes that depend on nuclear tension for their regulation. We also show that LINC disruption does not attenuate nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex facilitates mechano-regulation of expression across the genome.
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Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , ARN Mensajero/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Humanos , Ratones , Células 3T3 NIH , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization.
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Proliferación Celular/genética , Cromatina/genética , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Nucleosomas/genéticaRESUMEN
Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer.
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Neoplasias de la Mama/enzimología , Cromatina/metabolismo , ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , ADN Helicasas/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Lípidos/biosíntesis , Lipogénesis , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.
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Compuestos de Azabiciclo/farmacología , Sondas Moleculares/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Piridinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/química , Cristalografía por Rayos X , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Estructura Molecular , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Especificidad por Sustrato , Factores de Transcripción/metabolismoRESUMEN
Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Ensamble y Desensamble de Cromatina , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Estimación de Kaplan-Meier , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Interferencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Three-dimensional organization of the chromatin has important roles in transcription, replication, DNA repair, and pathologic events such as translocations. There are two fundamental ways to study higher-order chromatin organization: microscopic and molecular approaches. In this review, we briefly introduce the molecular approaches, focusing on chromosome conformation capture or "3C" technology and its derivatives, which can be used to probe chromatin folding at resolutions beyond that provided by microscopy techniques. We further discuss the different types of data generated by the 3C-based methods and how they can be used to answer distinct biological questions.