RESUMEN
During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.
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Cromatina/genética , Código de Histonas/genética , Histonas/genética , Mitosis/genética , Células Madre Pluripotentes/fisiología , Acetilación , Animales , Línea Celular , Drosophila/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.
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Reprogramación Celular/genética , Fibroblastos/fisiología , Redes Reguladoras de Genes/genética , Hepatocitos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Animales , Células Madre Embrionarias/fisiología , Femenino , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Ratones , Ratones Endogámicos C57BL , Embarazo , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
Genome-wide studies in tumor cells have indicated that chromatin-modifying proteins are commonly mutated in human cancers. The lysine-specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination-mediated double-strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors.
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Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Animales , Secuencia de Bases , Línea Celular Tumoral , Daño del ADN/genética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Recombinación Homóloga/genética , Humanos , Masculino , Ratones SCID , Neoplasias/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
BACKGROUND: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. METHODS: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. RESULTS: This experimental setting has three advantages that are presented and discussed: i) it covers a "gap" in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. CONCLUSIONS: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.
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Senescencia Celular/genética , Neoplasias Glandulares y Epiteliales/genética , Oncogenes , Carcinogénesis , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Genoma , Humanos , MicroARNs/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Glandulares y Epiteliales/ultraestructura , Proteínas Nucleares/metabolismo , Proteínas/metabolismoRESUMEN
Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.
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Regulación del Desarrollo de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Regiones Promotoras Genéticas/genética , Cromatina/genética , Linaje de la Célula/genética , Expresión Génica , Elementos de Facilitación Genéticos/genética , Mamíferos/genéticaRESUMEN
Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.
RESUMEN
Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and eXtraembryonic ENdoderm (XEN) stem cells - in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4, KLF4 and SOX2-induced XEN-to-iPS reprogramming progressed with diminished efficiency and kinetics. The dominant PrE transcriptional program, safeguarded by Gata4, and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping in vitro trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM.
RESUMEN
Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or "hubness" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide in silico perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors.
RESUMEN
The transcription factor NF-κB is a critical regulator of immune responses. To determine how NF-κB builds transcriptional control networks, we need to obtain a topographic map of the factor bound to the genome and correlate it with global gene expression. We used a ChIP cloning technique and identified novel NF-κB target genes in response to virus infection. We discovered that most of the NF-κB-bound genomic sites deviate from the consensus and are located away from conventional promoter regions. Remarkably, we identified a novel abundant NF-κB-binding site residing in specialized Alu-repetitive elements having the potential for long range transcription regulation, thus suggesting that in addition to its known role, NF-κB has a primate-specific function and a role in human evolution. By combining these data with global gene expression profiling of virus-infected cells, we found that most of the sites bound by NF-κB in the human genome do not correlate with changes in gene expression of the nearby genes and they do not appear to function in the context of synthetic promoters. These results demonstrate that repetitive elements interspersed in the human genome function as common target sites for transcription factors and may play an important role in expanding the repertoire of binding sites to engage new genes into regulatory networks.
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Elementos Alu/genética , FN-kappa B/metabolismo , Animales , Sitios de Unión , Cromatina/química , Inmunoprecipitación de Cromatina , ADN/química , ADN/genética , Genoma , Genoma Humano , Células HeLa , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Primates , Unión Proteica , Transcripción GenéticaRESUMEN
The introduction of chromosome conformation capture (3C)-based technologies coupled with next-generation sequencing have significantly advanced our understanding of how the genetic material is organized within the eukaryotic nucleus. Three-dimensional (3D) genomic organization occurs at hierarchical levels, ranging from chromosome territories and subnuclear compartments to smaller self-associated domains and fine-scale chromatin interactions. The latter can be further categorized into different subtypes, such as structural or regulatory, based either on their presumed functionality and/or the factors that mediate their formation. Various enrichment strategies coupled with 3C-based technologies have been developed to prospectively isolate and quantify chromatin interactions around regions occupied by specific proteins or marks of interest. These approaches not only enable high-resolution characterization of the selected chromatin contacts at a cost-effective manner, but also offer important biological insights into their organizational principles and regulatory function. In this chapter, we will focus on the recently developed HiChIP technology with an emphasis on the discovery of putative active enhancers and promoter interactions in cell types of interest. We will describe the specific steps for designing, performing and analyzing successful HiChIP experiments as well as important limitations and considerations.
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Cromatina , Cromosomas , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/métodos , GenomaRESUMEN
INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.
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Neoplasias Glandulares y Epiteliales , Timoma , Neoplasias del Timo , Factores de Transcripción TFIII , Factores de Transcripción TFII , Animales , Humanos , Ratones , Mutación , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Timoma/genética , Timoma/patología , Neoplasias del Timo/genética , Neoplasias del Timo/patología , Factores de Transcripción TFII/genética , Factores de Transcripción TFIII/genéticaRESUMEN
Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.
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Alelos , Proteínas de Unión al Calcio/genética , Metilación de ADN/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Animales , Cromosomas/genética , Impresión Genómica/genética , Yoduro Peroxidasa/genética , Ratones , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/genéticaRESUMEN
The hierarchical three-dimensional folding of the mammalian genome constitutes an important regulatory layer of gene expression and cell fate control during processes such as development and tumorigenesis. Accumulating evidence supports the existence of complex topological assemblies in which multiple genes and regulatory elements are frequently interacting with each other in the 3D nucleus. Here, we will discuss the nature, organizational principles, and potential function of such assemblies, including the recently reported enhancer "hubs," "cliques," and FIREs (frequently interacting regions) as well as multi-contact hubs. We will also review recent studies that investigate the role of transcription factors (TFs) in driving the topological genome reorganization and hub formation in the context of cell fate transitions and cancer. Finally, we will highlight technological advances that enabled these studies, current limitations, and future directions necessary to advance our understating in the field.
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Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Núcleo Celular/genética , Cromatina/genética , Epigénesis Genética , Humanos , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Conformación Proteica , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.
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Reprogramación Celular , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre Pluripotentes Inducidas/enzimología , Animales , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Metilación , RatonesRESUMEN
Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.
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Reprogramación Celular/genética , Ensamble y Desensamble de Cromatina/genética , Elementos de Facilitación Genéticos , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Masculino , Ratones , Células Madre Pluripotentes/metabolismoRESUMEN
Through the progressive accumulation of genetic and epigenetic alterations in cellular physiology, non-small-cell lung cancer (NSCLC) evolves in distinct steps involving mutually exclusive oncogenic mutations in K-Ras or EGFR along with inactivating mutations in the p53 tumor suppressor. Herein, we show two independent in vivo lung cancer models in which CHUK/IKK-α acts as a major NSCLC tumor suppressor. In a novel transgenic mouse strain, wherein IKKα ablation is induced by tamoxifen (Tmx) solely in alveolar type II (AT-II) lung epithelial cells, IKKα loss increases the number and size of lung adenomas in response to the chemical carcinogen urethane, whereas IKK-ß instead acts as a tumor promoter in this same context. IKKα knockdown in three independent human NSCLC lines (independent of K-Ras or p53 status) enhances their growth as tumor xenografts in immune-compromised mice. Bioinformatics analysis of whole transcriptome profiling followed by quantitative protein and targeted gene expression validation experiments reveals that IKKα loss can result in the up-regulation of activated HIF-1-α protein to enhance NSCLC tumor growth under hypoxic conditions in vivo.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Quinasa I-kappa B/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/genética , Femenino , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Quinasa I-kappa B/deficiencia , Neoplasias Pulmonares/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regulación hacia Arriba , Proteínas ras/genéticaRESUMEN
A major challenge in cancer treatment is predicting the clinical response to anti-cancer drugs on a personalized basis. The success of such a task largely depends on the ability to develop computational resources that integrate big "omic" data into effective drug-response models. Machine learning is both an expanding and an evolving computational field that holds promise to cover such needs. Here we provide a focused overview of: 1) the various supervised and unsupervised algorithms used specifically in drug response prediction applications, 2) the strategies employed to develop these algorithms into applicable models, 3) data resources that are fed into these frameworks and 4) pitfalls and challenges to maximize model performance. In this context we also describe a novel in silico screening process, based on Association Rule Mining, for identifying genes as candidate drivers of drug response and compare it with relevant data mining frameworks, for which we generated a web application freely available at: https://compbio.nyumc.org/drugs/. This pipeline explores with high efficiency large sample-spaces, while is able to detect low frequency events and evaluate statistical significance even in the multidimensional space, presenting the results in the form of easily interpretable rules. We conclude with future prospects and challenges of applying machine learning based drug response prediction in precision medicine.
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Minería de Datos , Aprendizaje Automático , Neoplasias/tratamiento farmacológico , Animales , Simulación por Computador , Humanos , Resultado del TratamientoRESUMEN
A major challenge in cancer treatment is predicting clinical response to anti-cancer drugs on a personalized basis. Using a pharmacogenomics database of 1,001 cancer cell lines, we trained deep neural networks for prediction of drug response and assessed their performance on multiple clinical cohorts. We demonstrate that deep neural networks outperform the current state in machine learning frameworks. We provide a proof of concept for the use of deep neural network-based frameworks to aid precision oncology strategies.
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Aprendizaje Profundo , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Medicina de Precisión/métodos , Análisis de SupervivenciaRESUMEN
Transcription factor-induced reprogramming of somatic cells to pluripotency is mediated via profound alterations in the epigenetic landscape. The histone variant macroH2A1 (mH2A1) is a barrier to the cellular reprogramming process. We demonstrate here that mH2A1 blocks reprogramming and contributes to the preservation of cell identity by trapping cells at the very early stages of the process, namely, at the mesenchymal-to-epithelial transition (MET). We provide a comprehensive analysis of the genomic sites occupied by the mH2A1 nucleosomes in human fibroblasts and embryonic stem (ES) cells and how they affect the reprogramming of fibroblasts to pluripotency. We have integrated chromatin immunoprecipitation sequencing (ChIP-seq) data with transcriptome sequencing (RNA-seq) data using cells containing reduced levels of mH2A1 and have inferred mH2A1-centered gene-regulatory networks that support the fibroblast and ES cell fates. We found that the exact positions of mH2A1 nucleosomes in regulatory regions of specific network genes with key regulatory roles guarantee the functional robustness of the regulatory networks. Using the reconstructed networks, we can predict and validate several components and their interactions in the establishment of stable cell types by limiting progression to alternative cell fates.