RESUMEN
Chromosomes of eukaryotes adopt highly dynamic and complex hierarchical structures in the nucleus. The three-dimensional (3D) organization of chromosomes profoundly affects DNA replication, transcription and the repair of DNA damage. Thus, a thorough understanding of nuclear architecture is fundamental to the study of nuclear processes in eukaryotic cells. Recent years have seen rapid proliferation of technologies to investigate genome organization and function. Here, we review experimental and computational methodologies for 3D genome analysis, with special focus on recent advances in high-throughput chromatin conformation capture (3C) techniques and data analysis.
Asunto(s)
Cromatina/ultraestructura , Animales , Mapeo Cromosómico , Cromosomas/ultraestructura , Simulación por Computador , Humanos , Modelos MolecularesRESUMEN
After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.
Asunto(s)
Cromosomas Humanos X/genética , Genoma Humano/genética , Telómero/genética , Centrómero/genética , Islas de CpG/genética , Metilación de ADN , ADN Satélite/genética , Femenino , Humanos , Mola Hidatiforme/genética , Masculino , Embarazo , Reproducibilidad de los Resultados , Testículo/metabolismoRESUMEN
Understanding the diversity of human tissues is fundamental to disease and requires linking genetic information, which is identical in most of an individual's cells, with epigenetic mechanisms that could have tissue-specific roles. Surveys of DNA methylation in human tissues have established a complex landscape including both tissue-specific and invariant methylation patterns. Here we report high coverage methylomes that catalogue cytosine methylation in all contexts for the major human organ systems, integrated with matched transcriptomes and genomic sequence. By combining these diverse data types with each individuals' phased genome, we identified widespread tissue-specific differential CG methylation (mCG), partially methylated domains, allele-specific methylation and transcription, and the unexpected presence of non-CG methylation (mCH) in almost all human tissues. mCH correlated with tissue-specific functions, and using this mark, we made novel predictions of genes that escape X-chromosome inactivation in specific tissues. Overall, DNA methylation in several genomic contexts varies substantially among human tissues.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Factores de Edad , Alelos , Mapeo Cromosómico , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Variación Genética , Humanos , Masculino , Especificidad de ÓrganosRESUMEN
A large number of cis-regulatory sequences have been annotated in the human genome, but defining their target genes remains a challenge. One strategy is to identify the long-range looping interactions at these elements with the use of chromosome conformation capture (3C)-based techniques. However, previous studies lack either the resolution or coverage to permit a whole-genome, unbiased view of chromatin interactions. Here we report a comprehensive chromatin interaction map generated in human fibroblasts using a genome-wide 3C analysis method (Hi-C). We determined over one million long-range chromatin interactions at 5-10-kb resolution, and uncovered general principles of chromatin organization at different types of genomic features. We also characterized the dynamics of promoter-enhancer contacts after TNF-α signalling in these cells. Unexpectedly, we found that TNF-α-responsive enhancers are already in contact with their target promoters before signalling. Such pre-existing chromatin looping, which also exists in other cell types with different extracellular signalling, is a strong predictor of gene induction. Our observations suggest that the three-dimensional chromatin landscape, once established in a particular cell type, is relatively stable and could influence the selection or activation of target genes by a ubiquitous transcription activator in a cell-specific manner.
Asunto(s)
Cromatina/metabolismo , Mapeo Cromosómico , Genoma Humano , Línea Celular , Cromatina/química , Cromatina/genética , Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Humanos , Imagenología Tridimensional , Regiones Promotoras Genéticas/fisiología , Unión Proteica , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. METHODS: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. RESULTS: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. CONCLUSIONS: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy.
Asunto(s)
Cardiomegalia/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Epigénesis Genética , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Cromatina/genética , Cromatina/patología , Estudio de Asociación del Genoma Completo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Ratones , Ratones Noqueados , Miocitos Cardíacos/patologíaRESUMEN
BACKGROUND: The MHC and KIR loci are clinically relevant regions of the genome. Typing the sequence of these loci has a wide range of applications including organ transplantation, drug discovery, pharmacogenomics and furthering fundamental research in immune genetics. Rapid advances in biochemical and next-generation sequencing (NGS) technologies have enabled several strategies for precise genotyping and phasing of candidate HLA alleles. Nonetheless, as typing of candidate HLA alleles alone reveals limited aspects of the genetics of MHC region, it is insufficient for the comprehensive utility of the aforementioned applications. For this reason, we believe phasing the entire MHC and KIR locus onto a single locus-spanning haplotype can be a critical improvement for better understanding transplantation biology. RESULTS: Generating long-range (>1 Mb) phase information is traditionally very challenging. As proximity-ligation based methods of DNA sequencing preserves chromosome-span phase information, we have utilized this principle to demonstrate its utility towards generating full-length phasing of MHC and KIR loci in human samples. We accurately (~99%) reconstruct the complete haplotypes for over 90% of sequence variants (coding and non-coding) within these two loci that collectively span 4-megabases. CONCLUSIONS: By haplotyping a majority of coding and non-coding alleles at the MHC and KIR loci in a single assay, this method has the potential to assist transplantation matching and facilitate investigation of the genetic basis of human immunity and disease.
Asunto(s)
Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Complejo Mayor de Histocompatibilidad/genética , Receptores KIR/genética , Genotipo , HumanosRESUMEN
UNLABELLED: Hepatocellular carcinoma (HCC) is the most rapidly increasing cause of cancer-related mortality in the United States. Because of the lack of viable treatment options for HCC, prevention in high-risk patients has been proposed as an alternative strategy. The main risk factor for HCC is cirrhosis and several lines of evidence implicate epidermal growth factor (EGF) in the progression of cirrhosis and development of HCC. We therefore examined the effects of the EGF receptor (EGFR) inhibitor erlotinib on liver fibrogenesis and hepatocellular transformation in three different animal models of progressive cirrhosis: a rat model induced by repeated, low-dose injections of diethylnitrosamine (DEN), a mouse model induced by carbon tetrachloride (CCl4 ), and a rat model induced by bile duct ligation (BDL). Erlotinib reduced EGFR phosphorylation in hepatic stellate cells (HSC) and reduced the total number of activated HSC. Erlotinib also decreased hepatocyte proliferation and liver injury. Consistent with all these findings, pharmacological inhibition of EGFR signaling effectively prevented the progression of cirrhosis and regressed fibrosis in some animals. Moreover, by alleviating the underlying liver disease, erlotinib blocked the development of HCC and its therapeutic efficacy could be monitored with a previously reported gene expression signature predictive of HCC risk in human cirrhosis patients. CONCLUSION: These data suggest that EGFR inhibition using Food and Drug Administration-approved inhibitors provides a promising therapeutic approach for reduction of fibrogenesis and prevention of HCC in high-risk cirrhosis patients who can be identified and monitored by gene expression signatures.
Asunto(s)
Carcinoma Hepatocelular/prevención & control , Progresión de la Enfermedad , Receptores ErbB/antagonistas & inhibidores , Cirrosis Hepática/prevención & control , Neoplasias Hepáticas/prevención & control , Quinazolinas/uso terapéutico , Animales , Conductos Biliares/fisiopatología , Tetracloruro de Carbono/efectos adversos , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dietilnitrosamina/efectos adversos , Modelos Animales de Enfermedad , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Ligadura/efectos adversos , Cirrosis Hepática/etiología , Cirrosis Hepática/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos , Fosforilación/efectos de los fármacos , Pronóstico , Quinazolinas/farmacología , Ratas , Ratas Wistar , TranscriptomaRESUMEN
HiC sequencing is a DNA-based next-generation sequencing method that preserves the 3D conformation of the genome and has shown promise in detecting genomic rearrangements in translational research studies. To evaluate HiC as a potential clinical diagnostic platform, analytical concordance with routine laboratory testing was assessed using primary pediatric leukemia and sarcoma specimens previously positive for clinically significant genomic rearrangements. Archived specimen types tested included viable and nonviable frozen leukemic cells, as well as formalin-fixed paraffin-embedded (FFPE) tumor tissues. Initially, pediatric acute myeloid leukemia (AML) and alveolar rhabdomyosarcoma (A-RMS) specimens with known genomic rearrangements were subjected to HiC analysis to assess analytical concordance. Subsequently, a discovery cohort consisting of AML and acute lymphoblastic leukemia (ALL) cases with no known genomic rearrangements based on prior clinical diagnostic testing were evaluated to determine whether HiC could detect rearrangements. Using a standard sequencing depth of 50 million raw read-pairs per sample, or approximately 5X raw genomic coverage, 100% concordance was observed between HiC and previous clinical cytogenetic and molecular testing. In the discovery cohort, a clinically relevant gene fusion was detected in 45% of leukemia cases (5/11). This study demonstrates the value of HiC sequencing to medical diagnostic testing as it identified several clinically significant rearrangements, including those that might have been missed by current clinical testing workflows. Key points: HiC sequencing is a DNA-based next-generation sequencing method that preserves the 3D conformation of the genome, facilitating detection of genomic rearrangements.HiC was 100% concordant with clinical diagnostic testing workflows for detecting clinically significant genomic rearrangements in pediatric leukemia and rhabdomyosarcoma specimens.HiC detected clinically significant genomic rearrangements not previously detected by prior clinical cytogenetic and molecular testing.HiC performed well with archived non-viable and viable frozen leukemic cell samples, as well as archived formalin-fixed paraffin-embedded tumor tissue specimens.
RESUMEN
Herpes-mediated viral oncolysis alone is not sufficient to completely eradicate tumors. In this study we used a replication conditional, endostatin-expressing herpes simplex virus-1 mutant (HSV-Endo) in a murine lung cancer model. We hypothesized that the anti-angiogenic action of endostatin would improve upon the oncolytic effect of HSV-1. HSV-Endo was evaluated in a pulmonary metastases and orthotopic flank model, where there was significantly less tumor burden and reduced microvessel density compared to a control virus. Endostatin expression appears to improve the anti-tumor effect of HSV-1 in a lung cancer model.
Asunto(s)
Endostatinas/genética , Neoplasias Pulmonares/irrigación sanguínea , Neovascularización Patológica/terapia , Viroterapia Oncolítica/métodos , Simplexvirus/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , TransgenesRESUMEN
BACKGROUND: Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. RESULTS: We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20-25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20-25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. CONCLUSION: We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all â¼70,000 extant vertebrate species.
Asunto(s)
Benchmarking , Dimetilsulfóxido , Animales , ADN/genética , Ácido Edético , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Peso Molecular , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND: The 3-dimensional (3D) conformation of chromatin inside the nucleus is integral to a variety of nuclear processes including transcriptional regulation, DNA replication, and DNA damage repair. Aberrations in 3D chromatin conformation have been implicated in developmental abnormalities and cancer. Despite the importance of 3D chromatin conformation to cellular function and human health, little is known about how 3D chromatin conformation varies in the human population, or whether DNA sequence variation between individuals influences 3D chromatin conformation. RESULTS: To address these questions, we perform Hi-C on lymphoblastoid cell lines from 20 individuals. We identify thousands of regions across the genome where 3D chromatin conformation varies between individuals and find that this variation is often accompanied by variation in gene expression, histone modifications, and transcription factor binding. Moreover, we find that DNA sequence variation influences several features of 3D chromatin conformation including loop strength, contact insulation, contact directionality, and density of local cis contacts. We map hundreds of quantitative trait loci associated with 3D chromatin features and find evidence that some of these same variants are associated at modest levels with other molecular phenotypes as well as complex disease risk. CONCLUSION: Our results demonstrate that common DNA sequence variants can influence 3D chromatin conformation, pointing to a more pervasive role for 3D chromatin conformation in human phenotypic variation than previously recognized.
Asunto(s)
Secuencia de Bases , Variación Genética , Genoma Humano , Conformación de Ácido Nucleico , Epigenoma , Humanos , Sitios de Carácter Cuantitativo , TranscriptomaRESUMEN
There is an established relationship between primary DNA sequence, secondary and tertiary chromatin structure, and transcriptional activity, suggesting that observed differences in one of these properties may reflect changes in the others. Here, we exploit these relationships to show that variations in DNA structure can be used to identify a wide range of genomic alterations in mammalian samples. In this proof-of-concept study we characterized and compared genome-wide histone occupancy by ChIP-Seq, DNA accessibility by ATAC-Seq, and chromosomal conformation by Hi-C for five CRISPR/Cas9-modified mammalian cell lines and their unmodified parent strains, as well as in one modified tissue sample and its parent strain. The results showed that the impact of genomic alterations on each of the levels of DNA organization varied depending on mutation type (insertion or deletion), size, and genomic location. The largest genomic alterations we identified included chromosomal rearrangements and deletions (greater than 200 Kb) in four of the modified cell lines, which can be difficult to identify by standard whole genome sequencing analysis. This multi-level DNA organizational analysis provides a sensitive approach for identifying a wide range of genomic and epigenomic perturbations that can be utilized for biomedical and biosecurity applications.
Asunto(s)
Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Análisis de Secuencia de ADN/métodos , Cromatina/genética , Inmunoprecipitación de Cromatina/métodos , ADN , Epigenómica/métodos , Genoma Humano/genética , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/genética , Humanos , Mutación , Prueba de Estudio Conceptual , Relación Estructura-ActividadRESUMEN
The three-dimensional configuration of DNA is integral to all nuclear processes in eukaryotes, yet our knowledge of the chromosome architecture is still limited. Genome-wide chromosome conformation capture studies have uncovered features of chromatin organization in cultured cells, but genome architecture in human tissues has yet to be explored. Here, we report the most comprehensive survey to date of chromatin organization in human tissues. Through integrative analysis of chromatin contact maps in 21 primary human tissues and cell types, we find topologically associating domains highly conserved in different tissues. We also discover genomic regions that exhibit unusually high levels of local chromatin interactions. These frequently interacting regions (FIREs) are enriched for super-enhancers and are near tissue-specifically expressed genes. They display strong tissue-specificity in local chromatin interactions. Additionally, FIRE formation is partially dependent on CTCF and the Cohesin complex. We further show that FIREs can help annotate the function of non-coding sequence variants.