RESUMEN
We have developed a novel method for genomic footprinting of transcription factors (TFs) that detects potential gene regulatory relationships from DNase-seq data at the nucleotide level. We introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. A mild cross-linking step in XL-DNase-seq improves the detection of DNase-based footprints of dynamic TFs. The footprint strengths and detectability depend on an optimal cross-linking procedure. This method may help extract novel gene regulatory circuits involving previously undetectable TFs. The XL-DNase-seq method is illustrated here for activated mouse macrophage-like cells, which share several features with inflammatory macrophages.
Asunto(s)
Huella de ADN , Factores de Transcripción , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Animales , Ratones , Huella de ADN/métodos , Cromatina/genética , Cromatina/metabolismo , Macrófagos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Desoxirribonucleasas/metabolismo , Análisis de Secuencia de ADN/métodosRESUMEN
Nucleotide sequences recognized and bound by DNA-binding proteins (DBPs) are critical to controlling and maintaining gene expression, replication, chromosome segregation, cell division, and nucleoid structure in bacterial cells. Therefore, determination of the binding sequences of DBPs is important not only to study DBP recognition mechanisms but also to understand the fundamentals of cell homeostasis. While ChIP-seq analysis appears to be an effective way to determine DBP binding sites on the genome, the resolution is sometimes not sufficient to identify the sites precisely. Here we introduce a simple and effective method named Genome Footprinting with high-throughput sequencing (GeF-seq) to determine binding sites of DBPs with single base-pair resolution. GeF-seq detects binding sites of DBPs as sharp peaks and thus makes it possible to identify the recognition sequence in each "binding peak" more easily and accurately compared to the common ChIP-seq.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Emparejamiento Base , Unión Proteica , Huella de ADN/métodosRESUMEN
Transcription factors (TFs) are crucial epigenetic regulators, which enable cells to dynamically adjust gene expression in response to environmental signals. Computational procedures like digital genomic footprinting on chromatin accessibility assays such as ATACseq can be used to identify bound TFs in a genome-wide scale. This method utilizes short regions of low accessibility signals due to steric hindrance of DNA bound proteins, called footprints (FPs), which are combined with motif databases for TF identification. However, while over 1600 TFs have been described in the human genome, only ~ 700 of these have a known binding motif. Thus, a substantial number of FPs without overlap to a known DNA motif are normally discarded from FP analysis. In addition, the FP method is restricted to organisms with a substantial number of known TF motifs. Here we present DENIS (DE Novo motIf diScovery), a framework to generate and systematically investigate the potential of de novo TF motif discovery from FPs. DENIS includes functionality (1) to isolate FPs without binding motifs, (2) to perform de novo motif generation and (3) to characterize novel motifs. Here, we show that the framework rediscovers artificially removed TF motifs, quantifies de novo motif usage during an early embryonic development example dataset, and is able to analyze and uncover TF activity in organisms lacking canonical motifs. The latter task is exemplified by an investigation of a scATAC-seq dataset in zebrafish which covers different cell types during hematopoiesis.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Motivos de Nucleótidos , Factores de Transcripción , Pez Cebra , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Humanos , Sitios de Unión , Unión Proteica , Huella de ADN/métodos , Biología Computacional/métodos , Cromatina/metabolismo , Cromatina/genéticaRESUMEN
Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to simultaneously resolve the occupancy of multiple proteins including transcription factors, RNA polymerase II and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve transcription factor binding patterns at cis-regulatory elements. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole-genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4-5 d in any laboratory with access to high-throughput sequencing. Analysis can be performed in 2 d using a dedicated R package and requires access to a high-performance computing system. Our method can be used to analyze how transcription factors cooperate and antagonize to regulate transcription.
Asunto(s)
Huella de ADN/métodos , Metilasas de Modificación del ADN/metabolismo , ADN/metabolismo , Genoma , Imagen Individual de Molécula/métodos , Factores de Transcripción/metabolismo , Animales , Núcleo Celular/metabolismo , ADN/genética , Metilasas de Modificación del ADN/genética , Regulación de la Expresión Génica , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Análisis de Secuencia de ADN/estadística & datos numéricos , Programas Informáticos , Factores de Transcripción/genéticaRESUMEN
Elucidating the transcriptional regulatory networks that underlie growth and development requires robust ways to define the complete set of transcription factor (TF) binding sites. Although TF-binding sites are known to be generally located within accessible chromatin regions (ACRs), pinpointing these DNA regulatory elements globally remains challenging. Current approaches primarily identify binding sites for a single TF (e.g. ChIP-seq), or globally detect ACRs but lack the resolution to consistently define TF-binding sites (e.g. DNAse-seq, ATAC-seq). To address this challenge, we developed MNase-defined cistrome-Occupancy Analysis (MOA-seq), a high-resolution (< 30 bp), high-throughput, and genome-wide strategy to globally identify putative TF-binding sites within ACRs. We used MOA-seq on developing maize ears as a proof of concept, able to define a cistrome of 145,000 MOA footprints (MFs). While a substantial majority (76%) of the known ATAC-seq ACRs intersected with the MFs, only a minority of MFs overlapped with the ATAC peaks, indicating that the majority of MFs were novel and not detected by ATAC-seq. MFs were associated with promoters and significantly enriched for TF-binding and long-range chromatin interaction sites, including for the well-characterized FASCIATED EAR4, KNOTTED1, and TEOSINTE BRANCHED1. Importantly, the MOA-seq strategy improved the spatial resolution of TF-binding prediction and allowed us to identify 215 motif families collectively distributed over more than 100,000 non-overlapping, putatively-occupied binding sites across the genome. Our study presents a simple, efficient, and high-resolution approach to identify putative TF footprints and binding motifs genome-wide, to ultimately define a native cistrome atlas.
Asunto(s)
Huella de ADN/métodos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Zea mays/genética , Sitios de Unión , Secuenciación de Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Elementos Reguladores de la Transcripción , Secuenciación Completa del GenomaRESUMEN
Chromatin accessibility is directly linked with transcription in eukaryotes. Accessible regions associated with regulatory proteins are highly sensitive to DNase I digestion and are termed DNase I hypersensitive sites (DHSs). DHSs can be identified by DNase I digestion, followed by high-throughput DNA sequencing (DNase-seq). The single-base-pair resolution digestion patterns from DNase-seq allows identifying transcription factor (TF) footprints of local DNA protection that predict TF-DNA binding. The identification of differential footprinting between two conditions allows mapping relevant TF regulatory interactions. Here, we provide step-by-step instructions to build gene regulatory networks from DNase-seq data. Our pipeline includes steps for DHSs calling, identification of differential TF footprints between treatment and control conditions, and construction of gene regulatory networks. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be adapted to work with DNase-seq data from any organism with a sequenced genome.
Asunto(s)
Cromatina/metabolismo , Mapeo Cromosómico/métodos , Huella de ADN/métodos , Desoxirribonucleasa I/metabolismo , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina/genética , Genómica , Unión Proteica , Programas Informáticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.
Asunto(s)
Huella de ADN/métodos , Nucleosomas , Análisis de Secuencia de ADN/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , ADN/química , ADN/genética , ADN/metabolismo , Genómica , Nucleasa Microcócica/metabolismo , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.
Asunto(s)
Proteínas Arqueales/química , Huella de ADN/métodos , Ácido Edético/análogos & derivados , Radical Hidroxilo/química , Pliegue del ARN/genética , ARN/química , Ribonucleasa P/metabolismo , Sitios de Unión , Ácido Edético/química , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , Unión Proteica , ARN Catalítico/genética , ARN Catalítico/metabolismo , Transcripción Reversa , Ribonucleasa P/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Análisis de Secuencia de ADNRESUMEN
The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated purine bases at DNA sites which are not bound by proteins or transcription factors. The DNA is cleaved by HCl, and the resulting DNA fragments are 5'-end [6-FAM]-labeled by a linker-mediated PCR (LM-PCR). Fluorescent fragments are separated and analyzed on a capillary sequencer, followed by automated data analysis using the software tool ivFAST.
Asunto(s)
Huella de ADN/métodos , Hypocreales/genética , Secuencia de Bases , ADN de Hongos/aislamiento & purificación , Electroforesis Capilar , Metilación , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasRESUMEN
Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin.
Asunto(s)
Huella de ADN/métodos , ADN/genética , Nucleosomas/química , Elementos Reguladores de la Transcripción , Factores de Transcripción/genética , Animales , Sitios de Unión , ADN/química , ADN/metabolismo , Masculino , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq .
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Huella de ADN/métodos , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/genética , Arabidopsis/genética , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Secuenciación de Inmunoprecipitación de Cromatina/normas , ADN/análisis , ADN/genética , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
Type 1 diabetes (T1D) is a chronic disease caused by the selective destruction of the insulin-producing pancreatic beta cells by infiltrating immune cells. We presently evaluated the transcriptomic signature observed in beta cells in early T1D and compared it with the signatures observed following in vitro exposure of human islets to inflammatory or metabolic stresses, with the aim of identifying "footprints" of the immune assault in the target beta cells. We detected similarities between the beta cell signatures induced by cytokines present at different moments of the disease, i.e., interferon-α (early disease) and interleukin-1ß plus interferon-γ (later stages) and the beta cells from T1D patients, identifying biological process and signaling pathways activated during early and late stages of the disease. Among the first responses triggered on beta cells was an enrichment in antiviral responses, pattern recognition receptors activation, protein modification and MHC class I antigen presentation. During putative later stages of insulitis the processes were dominated by T-cell recruitment and activation and attempts of beta cells to defend themselves through the activation of anti-inflammatory pathways (i.e., IL10, IL4/13) and immune check-point proteins (i.e., PDL1 and HLA-E). Finally, we mined the beta cell signature in islets from T1D patients using the Connectivity Map, a large database of chemical compounds/drugs, and identified interesting candidates to potentially revert the effects of insulitis on beta cells.
Asunto(s)
Huella de ADN/métodos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Inmunidad Celular/fisiología , Células Secretoras de Insulina/metabolismo , Animales , Humanos , Islotes Pancreáticos/metabolismoRESUMEN
CUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.
Asunto(s)
Sitios de Unión , Biología Computacional , Huella de ADN , ARN Polimerasa II/metabolismo , Programas Informáticos , Sitio de Iniciación de la Transcripción , Cromatina/genética , Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Huella de ADN/métodos , Proteínas de Unión al ADN , Humanos , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Transcription is tightly regulated by cis-regulatory DNA elements where transcription factors (TFs) can bind. Thus, identification of TF binding sites (TFBSs) is key to understanding gene expression and whole regulatory networks within a cell. The standard approaches used for TFBS prediction, such as position weight matrices (PWMs) and chromatin immunoprecipitation followed by sequencing (ChIP-seq), are widely used but have their drawbacks, including high false-positive rates and limited antibody availability, respectively. Several computational footprinting algorithms have been developed to detect TFBSs by investigating chromatin accessibility patterns; however, these also have limitations. We have developed a footprinting method to predict TF footprints in active chromatin elements (TRACE) to improve the prediction of TFBS footprints. TRACE incorporates DNase-seq data and PWMs within a multivariate hidden Markov model (HMM) to detect footprint-like regions with matching motifs. TRACE is an unsupervised method that accurately annotates binding sites for specific TFs automatically with no requirement for pregenerated candidate binding sites or ChIP-seq training data. Compared with published footprinting algorithms, TRACE has the best overall performance with the distinct advantage of targeting multiple motifs in a single model.
Asunto(s)
Cromatina/metabolismo , Huella de ADN/métodos , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Sitios de Unión , Línea Celular , Desoxirribonucleasas , Humanos , Células K562 , Cadenas de Markov , Motivos de NucleótidosRESUMEN
The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.
Asunto(s)
Huella de ADN/métodos , Genoma Fúngico , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/metabolismo , Regiones no Traducidas 3' , Anisomicina/farmacología , Codón de Terminación , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Mutación , Fosforilación , Estabilidad del ARN , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5' cap. Consequently, only one ribosome scans a 5' UTR at a time, and 5' UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of â¼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.
Asunto(s)
Regiones no Traducidas 5' , Huella de ADN/métodos , Iniciación de la Cadena Peptídica Traduccional , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Animales , Codón Iniciador , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Sistemas de Lectura Abierta , ARN Mensajero/genética , ARN de Transferencia de Metionina/genética , Subunidades Ribosómicas/genética , Subunidades Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/genéticaRESUMEN
Type VI secretion systems (T6SSs) are highly conserved and complex protein secretion systems that deliver effector proteins into eukaryotic hosts or other bacteria. T6SSs are regulated precisely by a variety of regulatory systems, which enables bacteria to adapt to varied environments. A T6SS within Salmonella pathogenicity island 6 (SPI-6) is activated during infection, and it contributes to the pathogenesis, as well as interbacterial competition, of Salmonella enterica serovar Typhimurium (S. Typhimurium). However, the regulation of the SPI-6 T6SS in S. Typhimurium is not well understood. In this study, we found that the SPI-6 T6SS core gene clpV was significantly upregulated in response to the iron-depleted condition and during infection. The global ferric uptake regulator (Fur) was shown to repress the clpV expression in the iron-replete medium. Moreover, electrophoretic mobility shift and DNase I footprinting assays revealed that Fur binds directly to the clpV promoter region at multiple sites spanning the transcriptional start site. We also observed that the relieving of Fur-mediated repression on clpV contributed to the interbacterial competition activity and pathogenicity of S. Typhimurium. These findings provide insights into the direct regulation of Fur in the expression and functional activity of SPI-6 T6SS in S. Typhimurium and thus help to elucidate the mechanisms of bacterial adaptability and virulence.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Proteínas Represoras/genética , Salmonella typhimurium/genética , Sistemas de Secreción Tipo VI/genética , 2,2'-Dipiridil/farmacología , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Huella de ADN/métodos , Desoxirribonucleasa I/química , Ensayo de Cambio de Movilidad Electroforética , Islas Genómicas , Quelantes del Hierro/farmacología , Ratones , Ratones Endogámicos BALB C , Regiones Promotoras Genéticas , Unión Proteica , Células RAW 264.7 , Proteínas Represoras/metabolismo , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Transcripción Genética , Sistemas de Secreción Tipo VI/metabolismo , VirulenciaRESUMEN
BACKGROUND: As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints. RESULTS: Here we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs. CONCLUSIONS: XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina/métodos , Huella de ADN/métodos , Animales , Cromatina/genética , Cromatina/fisiología , Inmunoprecipitación de Cromatina/métodos , Desoxirribonucleasa I , Desoxirribonucleasas , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Ratones , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) is a simple protocol for detection of open chromatin. Computational footprinting, the search for regions with depletion of cleavage events due to transcription factor binding, is poorly understood for ATAC-seq. We propose the first footprinting method considering ATAC-seq protocol artifacts. HINT-ATAC uses a position dependency model to learn the cleavage preferences of the transposase. We observe strand-specific cleavage patterns around transcription factor binding sites, which are determined by local nucleosome architecture. By incorporating all these biases, HINT-ATAC is able to significantly outperform competing methods in the prediction of transcription factor binding sites with footprints.
Asunto(s)
Huella de ADN/métodos , Genómica/métodos , Modelos Genéticos , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/metabolismo , Animales , Células Dendríticas/metabolismo , Humanos , Células K562 , Ratones , Nucleosomas/química , Transposasas/metabolismoRESUMEN
Transcription factor Nrf2, nuclear factor (erythroid-derived 2)-like 2, is considered a master regulator of redox homeostasis and plays a central role in antioxidant and anti-inflammatory defence. It has been largely reported that oxidative stress is implicated in nanoparticle-induced toxicity with the involvement of Nrf2. Several basic methods for Nrf2 evaluation with exposure to nanoparticles are described in this chapter including real-time reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunofluorescence staining, electrophoretic mobility shift assay, DNase I footprinting, dimethylsulfate footprinting, protein pulse-chase analysis, and tert-butylhydroquinone treatment.