RESUMEN
We combined classical salt fractionation with chromatin immunoprecipitation to recover human centromeric chromatin under native conditions. We found that >85% of the total centromeric chromatin is insoluble under conditions typically used for native chromatin extraction. To map both soluble and insoluble chromatin in situ, we combined CUT&RUN (cleavage under targets and release using nuclease), a targeted nuclease method, with salt fractionation. Using this approach, we observed unexpected structural and conformational variations of centromere protein A (CENP-A)-containing complexes on different α-satellite dimeric units within highly homogenous arrays. Our results suggest that slight α-satellite sequence differences control the structure and occupancy of the associated centromeric chromatin complex.
Asunto(s)
Proteína A Centromérica/química , Centrómero/química , Cromatina/química , Proteína A Centromérica/aislamiento & purificación , Proteína A Centromérica/metabolismo , Proteína B del Centrómero/química , Proteína B del Centrómero/metabolismo , Fraccionamiento Químico , Cromatina/aislamiento & purificación , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , ADN Satélite/química , Humanos , Células K562 , SolubilidadRESUMEN
The three-dimensional architecture of chromatin in the nucleus is important for genome regulation and function. Advanced high-throughput sequencing-based methods have been developed for capturing chromatin interactions (Hi-C, genome-wide chromosome conformation capture) or enriching for those involving a specific protein (ChIA-PET, chromatin interaction analysis with paired-end tag sequencing). There is widespread interest in utilizing and interpreting ChIA-PET and Hi-C. We review methods for comparative ChIA-PET and Hi-C data analysis and visualization. The topics reviewed include: downloading ChIA-PET and Hi-C data from the ENCODE and 4DN portals; processing ChIA-PET data using ChIA-PIPE; processing Hi-C data using Juicer or distiller and cooler; viewing 2D contact maps using Juicebox or Higlass; viewing peaks, loops, and domains using BASIC Browser; annotating convergent and tandem CTCF loops.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Análisis de Datos , Genómica/métodos , Línea Celular , Cromatina/genética , Cromatina/aislamiento & purificación , Conjuntos de Datos como Asunto , Humanos , Análisis de Secuencia de ADN , Programas InformáticosRESUMEN
Chromosome Conformation Capture (3C)-based technologies, such as Hi-C, have represented a significant breakthrough in investigating the structure and function of higher-order genome architecture. However, the mapping of global chromatin interactions remains challenging across many biological conditions due to high background noise and financial constraints, especially for small laboratories. Here, we describe the Bridge linker-Alul-Tn5 Hi-C (BAT Hi-C) method, which is a simple and efficient method for delineating chromatin conformational features of mouse embryonic stem (mES) cells and uncover DNA loops. This protocol combines Alul fragmentation and biotinylated linker-mediated proximity ligation to obtain kilobase (kb) resolution with a marked increase in the amount of unique read pairs. The protocol also includes chromatin isolation to reduce background noise and Tn5 tagmentation to cut down on preparation time. Importantly, with only one-third sequencing depth, our method revealed the same spectrum of chromatin contacts as in situ Hi-C. BAT Hi-C is an economical (i.e., approximately $40 for library preparation) and straightforward (total hands-on time of 3â¯days) tool that is ideal for the in-depth analysis of long-range chromatin looping events in a genome-wide fashion.
Asunto(s)
Cromatina/genética , Mapeo Cromosómico/métodos , Genómica/métodos , Animales , Línea Celular , Núcleo Celular/genética , Cromatina/aislamiento & purificación , Cromatina/metabolismo , Mapeo Cromosómico/economía , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Células Madre Embrionarias , Biblioteca de Genes , Genómica/economía , Ratones , Transposasas/metabolismoRESUMEN
The highly dynamic nature of chromosome conformation and three-dimensional (3D) genome organization leads to cell-to-cell variability in chromatin interactions within a cell population, even if the cells of the population appear to be functionally homogeneous. Hence, although Hi-C is a powerful tool for mapping 3D genome organization, this heterogeneity of chromosome higher order structure among individual cells limits the interpretive power of population based bulk Hi-C assays. Moreover, single-cell studies have the potential to enable the identification and characterization of rare cell populations or cell subtypes in a heterogeneous population. However, it may require surveying relatively large numbers of single cells to achieve statistically meaningful observations in single-cell studies. By applying combinatorial cellular indexing to chromosome conformation capture, we developed single-cell combinatorial indexed Hi-C (sci-Hi-C), a high throughput method that enables mapping chromatin interactomes in large number of single cells. We demonstrated the use of sci-Hi-C data to separate cells by karytoypic and cell-cycle state differences and to identify cellular variability in mammalian chromosomal conformation. Here, we provide a detailed description of method design and step-by-step working protocols for sci-Hi-C.
Asunto(s)
Mapeo Cromosómico/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de la Célula Individual/métodos , Animales , Línea Celular , Núcleo Celular/genética , Separación Celular/métodos , Cromatina/genética , Cromatina/aislamiento & purificación , Cromatina/metabolismo , Simulación por Computador , Biblioteca de Genes , Humanos , Ratones , Conformación de Ácido NucleicoRESUMEN
Studies performed using Hi-C and other high-throughput whole-genome C-methods have demonstrated that 3D organization of eukaryotic genomes is functionally relevant. Unfortunately, ultra-deep sequencing of Hi-C libraries necessary to detect loop structures in large vertebrate genomes remains rather expensive. However, many studies are in fact aimed at determining the fine-scale 3D structure of comparatively small genomic regions up to several Mb in length. Such studies typically focus on the spatial structure of domains of coregulated genes, molecular mechanisms of loop formation, and interrogation of functional significance of GWAS-revealed polymorphisms. Therefore, a handful of molecular techniques based on Hi-C have been developed to address such issues. These techniques commonly rely on in-solution hybridization of Hi-C/3C-seq libraries with pools of biotinylated baits covering the region of interest, followed by deep sequencing of the enriched library. Here, we describe a new protocol of this kind, C-TALE (Chromatin TArget Ligation Enrichment). Preparation of hybridization probes from bacterial artificial chromosomes and an additional round of enrichment make C-TALE a cost-effective alternative to existing many-versus-all C-methods.
Asunto(s)
Mapeo Cromosómico/métodos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Biotinilación , Línea Celular , Cromatina/química , Cromatina/genética , Cromatina/aislamiento & purificación , Cromatina/metabolismo , Mapeo Cromosómico/economía , Cromosomas Artificiales Bacterianos/genética , ADN/genética , ADN/aislamiento & purificación , ADN/metabolismo , Biblioteca de Genes , Genómica/economía , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Humanos , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico/métodosRESUMEN
Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy, called "CLASP" (Cas9 locus-associated proteome), to identify and functionally test associated gene-regulatory factors. Our CLASP method, coupled to mass spectrometry and functional screens, can be efficiently adapted for isolating associated regulatory factors in an unbiased manner targeting multiple genomic loci across different cell types. Here, we applied our method to isolate the Drosophila melanogaster histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone H2A and H3 mRNA via interaction with their 3' UTRs.
Asunto(s)
Proteínas Bacterianas/genética , Cromatina/aislamiento & purificación , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Endonucleasas/genética , Genes Reguladores/genética , Histonas/genética , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3' , Animales , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Cromatina/genética , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Endonucleasas/metabolismo , Expresión Génica , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Protein A capture chromatography is a critical unit operation in the clearance of host cell protein (HCP) impurities in monoclonal antibody (mAb) purification processes. Though one of the most effective purification steps, variable levels of protein impurities are often observed in the eluate. Coelution of HCP impurities is suggested to be strongly affected by the presence of chromatin complexes (Gagnon et al., 2014; Koehler et al., 2019). We investigated the effect of removal of DNA complex and HCP reduction pre-Protein A on the HCP clearance performance of the Protein A capture step itself. We found that only reduction of DNA in the Protein A load consistently lowered HCP in the Protein A eluate. Reduction of HCP in the Protein A load stream did not produce a significant increase in the chromatography HCP clearance performance. These results are consistent across three different biosimilar therapeutic mAbs expressed by the same Chinese hamster ovary (CHO) cell line (i.e., CHOBC ® of Polpharma Biologics). This result demonstrates that optimization of the mAb purification process utilizing Protein A as the primary capture step depends primarily on being able to effectively clear DNA and associated complexes early in the process, rather than trying to incorporate HCP reduction at the harvest cell culture fluid.
Asunto(s)
Cromatina/aislamiento & purificación , Cromatografía de Afinidad/métodos , Proteínas/aislamiento & purificación , Proteína Estafilocócica A , Animales , Anticuerpos Monoclonales , Células CHO , Cromatina/química , Cricetulus , ADN/química , ADN/aislamiento & purificación , Proteínas/química , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismoRESUMEN
In 1869, Johann Friedrich Miescher discovered a new substance in the nucleus of living cells. The substance, which he called nuclein, is now known as DNA, yet both Miescher's name and his theoretical ideas about nuclein are all but forgotten. This paper traces the trajectory of Miescher's reception in the historiography of genetics. To his critics, Miescher was a "contaminator," whose preparations were impure. Modern historians portrayed him as a "confuser," whose misunderstandings delayed the development of molecular biology. Each of these portrayals reflects the disciplinary context in which Miescher's work was evaluated. Using archival sources to unearth Miescher's unpublished speculations-including an analogy between the hereditary material and language, and a speculation that a series of asymmetric carbon atoms could account for hereditary variation-this paper clarifies the ways in which the past was judged through the lens of contemporary concerns. It also shows how organization, structure, function, and information were already being considered when nuclein was first discovered nearly 150 years ago.
Asunto(s)
ADN/historia , Genética/historia , Historiografía , Biología Molecular/historia , Química/historia , Cromatina/aislamiento & purificación , ADN/aislamiento & purificación , Historia del Siglo XIX , Humanos , Relaciones Interprofesionales , Supuración/historia , SuizaRESUMEN
Heart failure is associated with hypertrophying of cardiomyocytes and changes in transcriptional activity. Studies from rapidly dividing cells in culture have suggested that transcription may be compartmentalized into factories within the nucleus, but this phenomenon has not been tested in vivo and the role of nuclear architecture in cardiac gene regulation is unknown. While alterations to transcription have been linked to disease, little is known about the regulation of the spatial organization of transcription and its properties in the pathological setting. In the present study, we investigate the structural features of endogenous transcription factories in the heart and determine the principles connecting chromatin structure to transcriptional regulation in vivo. Super-resolution imaging of endogenous RNA polymerase II clusters in neonatal and adult cardiomyocytes revealed distinct properties of transcription factories in response to pathological stress: neonatal nuclei demonstrated changes in number of clusters, with parallel increases in nuclear area, while the adult nuclei underwent changes in size and intensity of RNA polymerase II foci. Fluorescence in situ hybridization-based labeling of genes revealed locus-specific relationships between expression change and anatomical localization-with respect to nuclear periphery and heterochromatin regions, both sites associated with gene silencing-in the nuclei of cardiomyocytes in hearts (but not liver hepatocytes) of mice subjected to pathologic stimuli that induce heart failure. These findings demonstrate a role for chromatin organization and rearrangement of nuclear architecture for cell type-specific transcription in vivo during disease. RNA polymerase II ChIP and chromatin conformation capture studies in the same model system demonstrate formation and reorganization of distinct nuclear compartments regulating gene expression. These findings reveal locus-specific compartmentalization of stress-activated, housekeeping and silenced genes in the anatomical context of the endogenous nucleus, revealing basic principles of global chromatin structure and nuclear architecture in the regulation of gene expression in healthy and diseased conditions.
Asunto(s)
Insuficiencia Cardíaca/genética , Corazón/diagnóstico por imagen , ARN Polimerasa II/genética , Transcripción Genética/genética , Animales , Animales Recién Nacidos , Cromatina/genética , Cromatina/aislamiento & purificación , Regulación de la Expresión Génica , Corazón/fisiopatología , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/diagnóstico por imagen , Humanos , Hibridación Fluorescente in Situ , Ratones , Imagen Molecular/métodos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN Polimerasa II/aislamiento & purificación , Activación Transcripcional/genéticaRESUMEN
Biochemical, proteomic, and epigenetic studies of chromatin rely on the ability to efficiently isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state and is applicable for both parallel multisample spin-column purification and large-scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes and features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy. The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies and serve as a mild, acid- and detergent-free sample preparation method for MS analysis.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatina/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/química , Núcleo Celular/química , Cromatina/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
BACKGROUND: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. RESULTS: During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45-60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. CONCLUSIONS: This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Cromatina/aislamiento & purificación , Adhesión en Parafina/métodos , Secuenciación Completa del Genoma/métodos , Animales , Cromatina/genética , Epigénesis Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Temperatura , Fijación del TejidoRESUMEN
The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.
Asunto(s)
Cromatina/aislamiento & purificación , ADN Nucleotidiltransferasas/química , Cromatina/química , Cromatina/metabolismo , ADN Nucleotidiltransferasas/metabolismo , HumanosRESUMEN
Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A-H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape.
Asunto(s)
Cromatina/genética , Daño del ADN/genética , Nucleosomas/genética , Animales , Camélidos del Nuevo Mundo , Cromatina/aislamiento & purificación , Histonas/metabolismo , Ubiquitinación/genéticaRESUMEN
The ends of eukaryotic chromosomes, known as telomeres, consist of repetitive DNA sequences, multiple proteins and noncoding RNAs. Telomeres are dynamic structures that play crucial roles as guardians of genome stability and tumor suppressors. Defects in telomere length or protein composition can accelerate aging and are seen in telomere syndromes, which affect various proliferative tissues such as the bone marrow or the lungs. One of the biggest challenges in the telomere field is to identify the molecular changes at telomeres that occur during normal development, in cancer and in telomere syndromes. To tackle this problem, our laboratory has established a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analyzed by mass spectrometry. QTIP involves stable isotope labeling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Cromatina/aislamiento & purificación , Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Telómero/química , Cromatina/química , Reactivos de Enlaces Cruzados , Células HeLa , HumanosRESUMEN
Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (â¼10,000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.
Asunto(s)
Fraccionamiento Celular/normas , Inmunoprecipitación de Cromatina/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Animales , Fraccionamiento Celular/métodos , Línea Celular , Núcleo Celular/genética , Células Cultivadas , Cromatina/aislamiento & purificación , Femenino , Células Hep G2 , Histonas/metabolismo , Humanos , Masculino , Ratones , Reproducibilidad de los Resultados , Sonicación , Factores de Transcripción/metabolismoRESUMEN
The advent of super-resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super-resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) versus age- and gender-matched healthy, non-caregiver controls. In this super-resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D-SIM). Quantitation of the super-resolution DNA structure revealed that the nuclear super-resolution DNA structure of individuals with AD significantly differs from that of their controls (p < 0.05) with an overall increase in the measured DNA-free/poor spaces. This represents a significant increase in the interchromatin compartment. We also find that the DNA structure of AD significantly differs in mild, moderate, and severe disease with respect to the DNA-containing and DNA-free/poor spaces. We conclude that whole genome remodeling is a feature of buccal cells in AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Núcleo Celular/ultraestructura , Cromatina/genética , Cromatina/ultraestructura , ADN/genética , ADN/ultraestructura , Mucosa Bucal/ultraestructura , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Núcleo Celular/química , Cromatina/aislamiento & purificación , Ensamble y Desensamble de Cromatina , ADN/aislamiento & purificación , Femenino , Humanos , Imagenología Tridimensional , Masculino , Microscopía/métodos , Persona de Mediana Edad , Mucosa Bucal/química , Conformación de Ácido Nucleico , Índice de Severidad de la EnfermedadRESUMEN
Transcriptional regulatory networks direct the development of specialized cell types. The transcription factors signal tranducer and activator of transcription 4 (Stat4) and T-bet are required for the interleukin-12 (IL-12)-stimulated development of T helper 1 (Th1) cells, although the hierarchy of activity by these factors has not been clearly defined. In this report, we show that these factors did not function in a linear pathway and that each factor played a unique role in programming chromatin architecture for Th1 gene expression, with subsets of genes depending on Stat4, T-bet, or both for expression in Th1 cells. T-bet was not able to transactivate expression of Stat4-dependent genes in the absence of endogenous Stat4 expression. Thus, T-bet requires Stat4 to achieve complete IL-12-dependent Th1 cell-fate determination. These data provide a basis for understanding how transiently activated and lineage-specific transcription factors cooperate in promoting cellular differentiation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Regulación de la Expresión Génica , Factor de Transcripción STAT4/metabolismo , Proteínas de Dominio T Box/metabolismo , Células TH1/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Cromatina/genética , Cromatina/aislamiento & purificación , Técnicas de Inactivación de Genes , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos C57BL , Células TH1/metabolismo , Transcripción Genética , Transducción GenéticaRESUMEN
The development of a multicellular organism from a single zygote depends on the differentiation of progenitor cells to specialized cell types. The differentiation of these cell types is associated with changes in gene expression and the underlying chromatin landscape. To understand how these processes are regulated, it is desirable to understand how the chromatin features that constitute the epigenome differ between cell types at any given time during development. INTACT, a method for the cell type-specific purification of nuclei that can be used for the isolation of both RNA and chromatin, has emerged as a powerful tool to simultaneously study gene expression and chromatin profiles specifically in cell types of interest. In this review, we focus on the application of INTACT to different model organisms and discuss its potential for profiling cell types in their developmental context.
Asunto(s)
Cromatina/aislamiento & purificación , Epigenómica/métodos , ARN/aislamiento & purificación , Animales , Perfilación de la Expresión Génica , Modelos Biológicos , Especificidad de Órganos , Células Vegetales/metabolismoRESUMEN
Identification of proteins that are directly or indirectly associated with a specific DNA sequence is often an important goal in molecular biology research. Proteomics of isolated chromatin fragments (PICh) is a technique used to isolate chromatin that contains homologous DNA sequence to a specific nucleic acid probe. All proteins directly and indirectly associated with the DNA sequences that hybridize to the probe are then identified by proteomics.1 We used the PICh technique to isolate chromatin associated with the centromeres of barley (Hordeum vulgare) by using a 2'-deoxy-2'fluoro-ribonucleotides (2'-F RNA) probe that is homologous to the AGGGAG satellite DNA specific to barley centromeres. Proteins associated with the barley centromeric chromatin were then isolated and identified by mass spectrometry. Both alpha-cenH3 and beta-cenH3, the two centromeric histone H3 variants associated with barley centromeres, were positively identified. Interestingly, several different H2A and H2B variants were recovered in the PIChed chromatin. The limitations and future potential of PICh in plant chromatin research are discussed.
Asunto(s)
Centrómero , Cromatina/química , Hordeum/química , Proteómica/métodos , Cromatina/aislamiento & purificación , Sondas de ADN , Histonas/análisis , Hordeum/genética , Espectrometría de Masas , Proteínas de Plantas/análisisRESUMEN
Affinity isolation has been an essential technique for molecular studies of cellular assemblies, such as the switch/sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes. However, even biochemically pure isolates can contain heterogeneous mixtures of complexes and their components. In particular, purification strategies that rely on affinity tags fused to only one component of a complex may be susceptible to this phenomenon. This study demonstrates that fusing purification tags to two different proteins enables the isolation of intact complexes of remodels the structure of chromatin (RSC). A Protein A tag was fused to one of the RSC proteins and a Twin-Strep tag to another protein of the complex. By mass spectrometry, we demonstrate the enrichment of the RSC complexes. The complexes had an apparent Svedberg value of about 20S, as shown by glycerol gradient ultracentrifugation. Additionally, purified complexes were demonstrated to be functional. Electron microscopy and single-particle analyses revealed a conformational rearrangement of RSC upon interaction with acetylated histone H3 peptides. This purification method is useful to purify functionally active, structurally well-defined macromolecular assemblies.