RESUMEN
Gene transcription occurs via a cycle of linked events, including initiation, promoter-proximal pausing, and elongation of RNA polymerase II (Pol II). A key question is how transcriptional enhancers influence these events to control gene expression. Here, we present an approach that evaluates the level and change in promoter-proximal transcription (initiation and pausing) in the context of differential gene expression, genome-wide. This combinatorial approach shows that in primary cells, control of gene expression during differentiation is achieved predominantly via changes in transcription initiation rather than via release of Pol II pausing. Using genetically engineered mouse models, deleted for functionally validated enhancers of the α- and ß-globin loci, we confirm that these elements regulate Pol II recruitment and/or initiation to modulate gene expression. Together, our data show that gene expression during differentiation is regulated predominantly at the level of initiation and that enhancers are key effectors of this process.
Asunto(s)
Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Iniciación de la Transcripción Genética , Globinas alfa/genética , Globinas beta/genética , Animales , Diferenciación Celular , Exones , Feto , Regulación de la Expresión Génica , Biblioteca de Genes , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Intrones , Células K562 , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/metabolismo , Transducción de Señal , Globinas alfa/deficiencia , Globinas beta/deficienciaRESUMEN
Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.
Asunto(s)
Eritropoyesis/genética , Redes Reguladoras de Genes/genética , Factores de Transcripción/genética , Bases de Datos Factuales , Regulación de la Expresión Génica/genética , Hematopoyesis/genética , Humanos , Proteómica/métodos , Factores de Transcripción/análisis , Factores de Transcripción/metabolismoRESUMEN
In higher eukaryotes, many genes are regulated by enhancers that are 104-106 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion1 is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
Asunto(s)
Emparejamiento Base/genética , Genoma/genética , Animales , Sitios de Unión , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos/genética , Células Eritroides/citología , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Globinas alfa/genética , CohesinasRESUMEN
Changes in DNA methylation are among the best-documented epigenetic alterations accompanying organismal aging. However, whether and how altered DNA methylation is causally involved in aging have remained elusive. GADD45α (growth arrest and DNA damage protein 45A) and ING1 (inhibitor of growth family member 1) are adapter proteins for site-specific demethylation by TET (ten-eleven translocation) methylcytosine dioxygenases. Here we show that Gadd45a/Ing1 double-knockout mice display segmental progeria and phenocopy impaired energy homeostasis and lipodystrophy characteristic of Cebp (CCAAT/enhancer-binding protein) mutants. Correspondingly, GADD45α occupies C/EBPß/δ-dependent superenhancers and, cooperatively with ING1, promotes local DNA demethylation via long-range chromatin loops to permit C/EBPß recruitment. The results indicate that enhancer methylation can affect aging and imply that C/EBP proteins play an unexpected role in this process. Our study suggests a causal nexus between DNA demethylation, metabolism, and organismal aging.
Asunto(s)
Envejecimiento Prematuro/genética , Envejecimiento/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , Desmetilación del ADN , Proteína Inhibidora del Crecimiento 1/metabolismo , Proteínas Nucleares/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Homeostasis/genética , Proteína Inhibidora del Crecimiento 1/genética , Lipodistrofia/genética , Ratones , Ratones Noqueados , Proteínas Nucleares/genéticaRESUMEN
The successful development and ongoing functioning of complex organisms depend on the faithful execution of the genetic code. A critical step in this process is the correct spatial and temporal expression of genes. The highly orchestrated transcription of genes is controlled primarily by cis-regulatory elements: promoters, enhancers, and insulators. The medical importance of this key biological process can be seen by the frequency with which mutations and inherited variants that alter cis-regulatory elements lead to monogenic and complex diseases and cancer. Here, we provide an overview of the methods available to characterize and perturb gene regulatory circuits. We then highlight mechanisms through which regulatory rewiring contributes to disease, and conclude with a perspective on how our understanding of gene regulation can be used to improve human health.
Asunto(s)
Regulación de la Expresión Génica , Redes Reguladoras de Genes , Elementos de Facilitación Genéticos , Humanos , Mutación , Regiones Promotoras GenéticasRESUMEN
Predicting the impact of noncoding genetic variation requires interpreting it in the context of three-dimensional genome architecture. We have developed deepC, a transfer-learning-based deep neural network that accurately predicts genome folding from megabase-scale DNA sequence. DeepC predicts domain boundaries at high resolution, learns the sequence determinants of genome folding and predicts the impact of both large-scale structural and single base-pair variations.
Asunto(s)
Genoma Humano/genética , Genómica/métodos , Modelos Genéticos , Redes Neurales de la Computación , Secuencia de Bases , Factor de Unión a CCCTC/genética , Cromatina/genética , Simulación por Computador , Variación Estructural del Genoma , HumanosRESUMEN
MOTIVATION: Genome sequencing experiments have revolutionized molecular biology by allowing researchers to identify important DNA-encoded elements genome wide. Regions where these elements are found appear as peaks in the analog signal of an assay's coverage track, and despite the ease with which humans can visually categorize these patterns, the size of many genomes necessitates algorithmic implementations. Commonly used methods focus on statistical tests to classify peaks, discounting that the background signal does not completely follow any known probability distribution and reducing the information-dense peak shapes to simply maximum height. Deep learning has been shown to be highly accurate for many pattern recognition tasks, on par or even exceeding human capabilities, providing an opportunity to reimagine and improve peak calling. RESULTS: We present the peak calling framework LanceOtron, which combines deep learning for recognizing peak shape with multifaceted enrichment calculations for assessing significance. In benchmarking ATAC-seq, ChIP-seq and DNase-seq, LanceOtron outperforms long-standing, gold-standard peak callers through its improved selectivity and near-perfect sensitivity. AVAILABILITY AND IMPLEMENTATION: A fully featured web application is freely available from LanceOtron.molbiol.ox.ac.uk, command line interface via python is pip installable from PyPI at https://pypi.org/project/lanceotron/, and source code and benchmarking tests are available at https://github.com/LHentges/LanceOtron. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Aprendizaje Profundo , Humanos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Secuenciación de Inmunoprecipitación de Cromatina , Secuencia de Bases , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-fos/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Sitios de Unión , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Epigénesis Genética , Antígeno 2 Relacionado con Fos/metabolismo , Humanos , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/fisiología , Factor de Transcripción AP-1/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
Asunto(s)
Anemia Diseritropoyética Congénita/genética , Predisposición Genética a la Enfermedad , Glicoproteínas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Anemia Diseritropoyética Congénita/patología , Femenino , Regulación de la Expresión Génica/genética , Pruebas Genéticas , Genética de Población , Humanos , Masculino , Complejos Multiproteicos/genética , Mutación/genéticaRESUMEN
The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic 'spongy' heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.
Asunto(s)
Anemia Diseritropoyética Congénita , Anemia Diseritropoyética Congénita/diagnóstico , Anemia Diseritropoyética Congénita/genética , Células Eritroides , Eritropoyesis , Glicoproteínas/genética , Humanos , Proteínas Nucleares/genéticaRESUMEN
The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimal Eomes expression in vivo Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500â kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.
Asunto(s)
Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Dominio T Box/genética , Animales , Diferenciación Celular/genética , Cromatina/metabolismo , Endodermo/metabolismo , Elementos de Facilitación Genéticos , Femenino , Factores de Transcripción Forkhead/metabolismo , Gastrulación/genética , Eliminación de Gen , Marcación de Gen , Genes Reporteros , Genotipo , Ratones Endogámicos C57BL , Modelos Biológicos , Proteínas del Grupo Polycomb/metabolismo , Transducción de Señal/genética , Proteína Smad2/metabolismo , Proteínas de Dominio T Box/metabolismoRESUMEN
Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000-20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C technique based on NanoString technology called C-String. By comparing C-String and Capture-C interaction profiles we show that the latter are not skewed by PCR amplification. Furthermore, we demonstrate that chromatin interactions detected by Capture-C do not depend on the degree of cross-linking by performing experiments with varying formaldehyde concentrations.
Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , Técnicas Genéticas , Nanotecnología/métodos , Animales , Recuento de Células , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromosomas/química , Cromosomas/genética , Femenino , Formaldehído/química , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Conformación MolecularAsunto(s)
Elementos de Facilitación Genéticos , Hemoglobina H/genética , Globinas alfa/genética , Talasemia alfa/genética , Adulto , Alelos , Cromatina/genética , Cromatina/ultraestructura , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 16/ultraestructura , Eritroblastos/patología , Eritropoyesis , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Genotipo , Hemoglobina E/genética , Hemoglobina H/análisis , Humanos , Masculino , Linaje , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Eliminación de Secuencia , Suriname/etnología , Globinas alfa/biosíntesis , Talasemia alfa/sangre , Globinas beta/genéticaRESUMEN
Transcription factors containing DNA binding domains generally regulate transcription by direct interaction with DNA. For most transcription factors, including the fungal Zn(II)2Cys6 zinc binuclear cluster transcription factors, the DNA binding motif is essential for function. However, Aspergillus nidulansâ TamA and the related Saccharomyces cerevisiaeâ Dal81p protein contain Zn(II)2Cys6 motifs shown to be dispensable for function. TamA acts at several promoters as a coactivator of the global nitrogen GATA transcription factor AreA. We now show that TamA is the major transcriptional activator of gdhA, encoding the key nitrogen metabolism enzyme NADP-glutamate dehydrogenase. Moreover, activation of gdhA by TamA occurs primarily by a mechanism requiring the TamA DNA binding motif. We show that the TamA DNA binding motif is required for DNA binding of FLAG-epitope-tagged TamA to the gdhA promoter. We identify a conserved promoter element required for TamA activation, and show that TamA and AreA are reciprocally required for full binding at the gdhA promoter under conditions where AreA is inactive at most promoters but active at gdhA. Therefore TamA has dual functions as a DNA-binding transcription factor and a non-DNA-binding coactivator. Dual DNA-binding and coactivator functions provide an additional level of combinatorial control to mediate gene-specific expression.
Asunto(s)
Aspergillus nidulans/enzimología , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Glutamato Deshidrogenasa (NADP+)/biosíntesis , Factores de Transcripción/metabolismo , Aspergillus nidulans/metabolismo , Nitrógeno/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus, yeast, and mammals, indicating an ancient origin.
Asunto(s)
Aspergillus nidulans/genética , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas Recombinantes de Fusión/genética , Factores de Transcripción/genética , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Aspergillus nidulans/metabolismo , Secuencia Conservada , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Mutación , Nitrógeno/metabolismo , Señales de Localización Nuclear , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
NADP-dependent glutamate dehydrogenase (NADP-GDH) is a key enzyme in the assimilation of alternative nitrogen nutrient sources through ammonium in fungi. In Aspergillus nidulans, NADP-GDH is encoded by gdhA. Several transcription factors are known to regulate gdhA expression, including AreA, the major transcription activator of nitrogen metabolic genes, and TamA, a co-activator of AreA. TamA also interacts with LeuB, the regulator of leucine biosynthesis. We have investigated the effects of leucine biosynthesis on gdhA regulation, and found that leucine regulates the levels of NADP-GDH activity and gdhA expression. We show, using mutants with perturbed levels of α-isopropylmalate (α-IPM), that this leucine biosynthesis intermediate affects gdhA regulation. Leucine regulation of gdhA requires a functional LeuB with an intact Zn(II)2Cys6 DNA-binding domain. By analysing the prevalence of putative LeuB DNA-binding sites in promoters of gdhA orthologues we predict broad conservation of leucine regulation of NADP-GDH expression within ascomycetes except in the fusaria and fission yeasts. Using promoter mutations in gdhA-lacZ reporter genes we identified two sites of action for LeuB within the A. nidulans gdhA promoter. These two sites lack sequence identity, with one site conforming to the predicted LeuB DNA-binding site consensus motif, whereas the second site is a novel regulatory sequence element conserved in Aspergillus gdhA promoters. These data suggest that LeuB regulates NADP-GDH expression in response to leucine levels, which may act as an important sensor of nitrogen availability.
Asunto(s)
Aspergillus nidulans/enzimología , Regulación Fúngica de la Expresión Génica , Glutamato Deshidrogenasa (NADP+)/biosíntesis , Factores de Transcripción/metabolismo , Fusión Artificial Génica , Sitios de Unión/genética , Análisis Mutacional de ADN , Genes Reporteros , Leucina/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Micro Capture-C (MCC) is a chromatin conformation capture (3C) method for visualizing reproducible three-dimensional contacts of specified regions of the genome at base pair resolution. These methods are an established family of techniques that use proximity ligation to assay the topology of chromatin. MCC can generate data at substantially higher resolution than previous techniques through multiple refinements of the 3C method. Using a sequence agnostic nuclease, the maintenance of cellular integrity and full sequencing of the ligation junctions, MCC achieves subnucleosomal levels of resolution, which can be used to reveal transcription factor binding sites analogous to DNAse I footprinting. Gene dense regions, close-range enhancer-promoter contacts, individual enhancers within super-enhancers and multiple other types of loci or regulatory regions that were previously challenging to assay with conventional 3C techniques, are readily observed using MCC. MCC requires training in common molecular biology techniques and bioinformatics to perform the experiment and analyze the data. The protocol can be expected to be completed in a 3 week timeframe for experienced molecular biologists.
Asunto(s)
Cromatina , Cromosomas , Cromatina/genética , Genoma , Biología Computacional/métodos , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Background: Resolving causal genes for type 2 diabetes at loci implicated by genome-wide association studies (GWAS) requires integrating functional genomic data from relevant cell types. Chromatin features in endocrine cells of the pancreatic islet are particularly informative and recent studies leveraging chromosome conformation capture (3C) with Hi-C based methods have elucidated regulatory mechanisms in human islets. However, these genome-wide approaches are less sensitive and afford lower resolution than methods that target specific loci. Methods: To gauge the extent to which targeted 3C further resolves chromatin-mediated regulatory mechanisms at GWAS loci, we generated interaction profiles at 23 loci using next-generation (NG) capture-C in a human beta cell model (EndoC-ßH1) and contrasted these maps with Hi-C maps in EndoC-ßH1 cells and human islets and a promoter capture Hi-C map in human islets. Results: We found improvements in assay sensitivity of up to 33-fold and resolved ~3.6X more chromatin interactions. At a subset of 18 loci with 25 co-localised GWAS and eQTL signals, NG Capture-C interactions implicated effector transcripts at five additional genetic signals relative to promoter capture Hi-C through physical contact with gene promoters. Conclusions: High resolution chromatin interaction profiles at selectively targeted loci can complement genome- and promoter-wide maps.