RESUMO
5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.
Assuntos
5-Metilcitosina , Sulfitos , Transcriptoma , 5-Metilcitosina/metabolismo , 5-Metilcitosina/química , Animais , Humanos , Camundongos , Sulfitos/química , Metiltransferases/metabolismo , Metiltransferases/genética , Perfilação da Expressão Gênica/métodos , Diferenciação CelularRESUMO
Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p < 8.4 × 10-9), and 32,862 CpG sites exhibited ASM in CLL samples (p < 8.5 × 10-9). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.
Assuntos
Metilação de DNA , Leucemia Linfocítica Crônica de Células B , Sulfitos , Humanos , Metilação de DNA/genética , Alelos , Leucemia Linfocítica Crônica de Células B/genética , Funções Verossimilhança , Impressão Genômica/genética , Ilhas de CpG/genéticaRESUMO
Recent studies exploring the impact of methylation in tumor evolution suggest that although the methylation status of many of the CpG sites are preserved across distinct lineages, others are altered as the cancer progresses. Because changes in methylation status of a CpG site may be retained in mitosis, they could be used to infer the progression history of a tumor via single-cell lineage tree reconstruction. In this work, we introduce the first principled distance-based computational method, Sgootr, for inferring a tumor's single-cell methylation lineage tree and for jointly identifying lineage-informative CpG sites that harbor changes in methylation status that are retained along the lineage. We apply Sgootr on single-cell bisulfite-treated whole-genome sequencing data of multiregionally sampled tumor cells from nine metastatic colorectal cancer patients, as well as multiregionally sampled single-cell reduced-representation bisulfite sequencing data from a glioblastoma patient. We show that the tumor lineages constructed reveal a simple model underlying tumor progression and metastatic seeding. A comparison of Sgootr against alternative approaches shows that Sgootr can construct lineage trees with fewer migration events and with more in concordance with the sequential-progression model of tumor evolution, with a running time a fraction of that used in prior studies. Lineage-informative CpG sites identified by Sgootr are in inter-CpG island (CGI) regions, as opposed to intra-CGIs, which have been the main regions of interest in genomic methylation-related analyses.
Assuntos
Metilação de DNA , Neoplasias , Humanos , Metilação de DNA/genética , Sulfitos , Análise de Sequência de DNA/métodos , Genoma , Neoplasias/genética , Ilhas de CpG/genéticaRESUMO
This study describes the development of a resource module that is part of a learning platform named 'NIGMS Sandbox for Cloud-based Learning' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module is designed to facilitate interactive learning of whole-genome bisulfite sequencing (WGBS) data analysis utilizing cloud-based tools in Google Cloud Platform, such as Cloud Storage, Vertex AI notebooks and Google Batch. WGBS is a powerful technique that can provide comprehensive insights into DNA methylation patterns at single cytosine resolution, essential for understanding epigenetic regulation across the genome. The designed learning module first provides step-by-step tutorials that guide learners through two main stages of WGBS data analysis, preprocessing and the identification of differentially methylated regions. And then, it provides a streamlined workflow and demonstrates how to effectively use it for large datasets given the power of cloud infrastructure. The integration of these interconnected submodules progressively deepens the user's understanding of the WGBS analysis process along with the use of cloud resources. Through this module, we can enhance the accessibility and adoption of cloud computing in epigenomic research, speeding up the advancements in the related field and beyond. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.
Assuntos
Computação em Nuvem , Metilação de DNA , Software , Sequenciamento Completo do Genoma , Sequenciamento Completo do Genoma/métodos , Sulfitos/química , Humanos , Epigênese Genética , Biologia Computacional/métodosRESUMO
Data from both bulk and single-cell whole-genome DNA methylation experiments are under-utilized in many ways. This is attributable to inefficient mapping of methylation sequencing reads, routinely discarded genetic information, and neglected read-level epigenetic and genetic linkage information. We introduce the BISulfite-seq Command line User Interface Toolkit (BISCUIT) and its companion R/Bioconductor package, biscuiteer, for simultaneous extraction of genetic and epigenetic information from bulk and single-cell DNA methylation sequencing. BISCUIT's performance, flexibility and standards-compliant output allow large, complex experimental designs to be characterized on clinical timescales. BISCUIT is particularly suited for processing data from single-cell DNA methylation assays, with its excellent scalability, efficiency, and ability to greatly enhance mappability, a key challenge for single-cell studies. We also introduce the epiBED format for single-molecule analysis of coupled epigenetic and genetic information, facilitating the study of cellular and tissue heterogeneity from DNA methylation sequencing.
Assuntos
Metilação de DNA , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Software , Epigenômica , Análise de Sequência de DNA , SulfitosRESUMO
Hemi-methylated cytosine dyads widely occur on mammalian genomic DNA, and can be stably inherited across cell divisions, serving as potential epigenetic marks. Previous identification of hemi-methylation relied on harsh bisulfite treatment, leading to extensive DNA degradation and loss of methylation information. Here we introduce Mhemi-seq, a bisulfite-free strategy, to efficiently resolve methylation status of cytosine dyads into unmethylation, strand-specific hemi-methylation, or full-methylation. Mhemi-seq reproduces methylomes from bisulfite-based sequencing (BS-seq & hpBS-seq), including the asymmetric hemi-methylation enrichment flanking CTCF motifs. By avoiding base conversion, Mhemi-seq resolves allele-specific methylation and associated imprinted gene expression more efficiently than BS-seq. Furthermore, we reveal an inhibitory role of hemi-methylation in gene expression and transcription factor (TF)-DNA binding, and some displays a similar extent of inhibition as full-methylation. Finally, we uncover new hemi-methylation patterns within Alu retrotransposon elements. Collectively, Mhemi-seq can accelerate the identification of DNA hemi-methylation and facilitate its integration into the chromatin environment for future studies.
Assuntos
Metilação de DNA , Análise de Sequência de DNA , Animais , Citosina/metabolismo , Metilação de DNA/genética , Mamíferos/genética , Retroelementos , Análise de Sequência de DNA/métodos , Sulfitos , Regulação da Expressão GênicaRESUMO
Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.
Assuntos
Pseudouridina , Reação em Cadeia da Polimerase em Tempo Real , Sulfitos , Humanos , Pseudouridina/química , Pseudouridina/genética , Pseudouridina/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , RNA/química , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sulfitos/químicaRESUMO
Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, â¼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (â¼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.
Assuntos
5-Metilcitosina , Ácidos Nucleicos Livres , Ilhas de CpG , Metilação de DNA , DNA de Cadeia Simples , Humanos , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/sangue , 5-Metilcitosina/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangue , Sulfitos/química , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodosRESUMO
Existing single-cell bisulfite-based DNA methylation analysis is limited by low DNA recovery, and the measurement of 5hmC at single-base resolution remains challenging. Here, we present a bisulfite-free single-cell whole-genome 5mC and 5hmC profiling technique, named Cabernet, which can characterize 5mC and 5hmC at single-base resolution with high genomic coverage. Cabernet utilizes Tn5 transposome for DNA fragmentation, which enables the discrimination between different alleles for measuring hemi-methylation status. Using Cabernet, we revealed the 5mC, hemi-5mC and 5hmC dynamics during early mouse embryo development, uncovering genomic regions exclusively governed by active or passive demethylation. We show that hemi-methylation status can be used to distinguish between pre- and post-replication cells, enabling more efficient cell grouping when integrated with 5mC profiles. The property of Tn5 naturally enables Cabernet to achieve high-throughput single-cell methylome profiling, where we probed mouse cortical neurons and embryonic day 7.5 (E7.5) embryos, and constructed the library for thousands of single cells at high efficiency, demonstrating its potential for analyzing complex tissues at substantially low cost. Together, we present a way of high-throughput methylome and hydroxymethylome detection at single-cell resolution, enabling efficient analysis of the epigenetic status of biological systems with complicated nature such as neurons and cancer cells.
Assuntos
5-Metilcitosina , Metilação de DNA , Animais , Camundongos , Sulfitos , Análise de Sequência de DNA/métodos , CitosinaRESUMO
Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders.
Assuntos
Blastocisto , Metilação de DNA , Embrião de Mamíferos , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Análise da Expressão Gênica de Célula Única , Sulfitos , Animais , Bovinos , Feminino , Gravidez , Blastocisto/metabolismo , Desenvolvimento Embrionário/genética , Epigênese Genética , Perfilação da Expressão Gênica , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Sulfitos/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/enzimologiaRESUMO
The sulfite-reducing bacterium Bilophila wadsworthia, a common human intestinal pathobiont, is unique in its ability to metabolize a wide variety of sulfonates to generate sulfite as a terminal electron acceptor (TEA). The resulting formation of H2S is implicated in inflammation and colon cancer. l-cysteate, an oxidation product of l-cysteine, is among the sulfonates metabolized by B. wadsworthia, although the enzymes involved remain unknown. Here we report a pathway for l-cysteate dissimilation in B. wadsworthia RZATAU, involving isomerization of l-cysteate to d-cysteate by a cysteate racemase (BwCuyB), followed by cleavage into pyruvate, ammonia and sulfite by a d-cysteate sulfo-lyase (BwCuyA). The strong selectivity of BwCuyA for d-cysteate over l-cysteate was rationalized by protein structural modeling. A homolog of BwCuyA in the marine bacterium Silicibacter pomeroyi (SpCuyA) was previously reported to be a l-cysteate sulfo-lyase, but our experiments confirm that SpCuyA too displays a strong selectivity for d-cysteate. Growth of B. wadsworthia with cysteate as the electron acceptor is accompanied by production of H2S and induction of BwCuyA. Close homologs of BwCuyA and BwCuyB are present in diverse bacteria, including many sulfate- and sulfite-reducing bacteria, suggesting their involvement in cysteate degradation in different biological environments.
Assuntos
Cisteína , Cisteína/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bilophila/metabolismo , Bilophila/enzimologia , Racemases e Epimerases/metabolismo , Oxirredução , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/química , Sulfitos/metabolismo , HumanosRESUMO
Covalent modifications of genomic DNA are crucial for most organisms to survive. Amplicon-based high-throughput sequencing technologies erase all DNA modifications to retain only sequence information for the four canonical nucleobases, necessitating specialized technologies for ascertaining epigenetic information. To also capture base modification information, we developed Methyl-SNP-seq, a technology that takes advantage of the complementarity of the double helix to extract the methylation and original sequence information from a single DNA molecule. More specifically, Methyl-SNP-seq uses bisulfite conversion of one of the strands to identify cytosine methylation while retaining the original four-bases sequence information on the other strand. As both strands are locked together to link the dual readouts on a single paired-end read, Methyl-SNP-seq allows detecting the methylation status of any DNA even without a reference genome. Because one of the strands retains the original four nucleotide composition, Methyl-SNP-seq can also be used in conjunction with standard sequence-specific probes for targeted enrichment and amplification. We show the usefulness of this technology in a broad spectrum of applications ranging from allele-specific methylation analysis in humans to identification of methyltransferase specificity in complex bacterial communities.
Assuntos
Metilação de DNA , Epigenoma , Humanos , Análise de Sequência de DNA , DNA/genética , Alelos , Sequenciamento de Nucleotídeos em Larga Escala , Sulfitos/químicaRESUMO
MOTIVATION: The utilization of single-cell bisulfite sequencing (scBS-seq) methods allows for precise analysis of DNA methylation patterns at the individual cell level, enabling the identification of rare populations, revealing cell-specific epigenetic changes, and improving differential methylation analysis. Nonetheless, the presence of sparse data and an overabundance of zeros and ones, attributed to limited sequencing depth and coverage, frequently results in reduced precision accuracy during the process of differential methylation detection using scBS-seq. Consequently, there is a pressing demand for an innovative differential methylation analysis approach that effectively tackles these data characteristics and enhances recognition accuracy. RESULTS: We propose a novel beta mixture approach called scDMV for analyzing methylation differences in single-cell bisulfite sequencing data, which effectively handles excess zeros and ones and accommodates low-input sequencing. Our extensive simulation studies demonstrate that the scDMV approach outperforms several alternative methods in terms of sensitivity, precision, and controlling the false positive rate. Moreover, in real data applications, we observe that scDMV exhibits higher precision and sensitivity in identifying differentially methylated regions, even with low-input samples. In addition, scDMV reveals important information for GO enrichment analysis with single-cell whole-genome sequencing data that are often overlooked by other methods. AVAILABILITY AND IMPLEMENTATION: The scDMV method, along with a comprehensive tutorial, can be accessed as an R package on the following GitHub repository: https://github.com/PLX-m/scDMV.
Assuntos
Metilação de DNA , Sulfitos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do GenomaRESUMO
MOTIVATION: Local alignments of query sequences in large databases represent a core part of metagenomic studies and facilitate homology search. Following the development of NCBI Blast, many applications aimed to provide faster and equally sensitive local alignment frameworks. Most applications focus on protein alignments, while only few also facilitate DNA-based searches. None of the established programs allow searching DNA sequences from bisulfite sequencing experiments commonly used for DNA methylation profiling, for which specific alignment strategies need to be implemented. RESULTS: Here, we introduce Lambda3, a new version of the local alignment application Lambda. Lambda3 is the first solution that enables the search of protein, nucleotide as well as bisulfite-converted nucleotide query sequences. Its protein mode achieves comparable performance to that of the highly optimized protein alignment application Diamond, while the nucleotide mode consistently outperforms established local nucleotide aligners. Combined, Lambda3 presents a universal local alignment framework that enables fast and sensitive homology searches for a wide range of use-cases. AVAILABILITY AND IMPLEMENTATION: Lambda3 is free and open-source software publicly available at https://github.com/seqan/lambda/.
Assuntos
Algoritmos , Software , Sulfitos , Alinhamento de Sequência , ProteínasRESUMO
Methanogenic archaea are main actors in the carbon cycle but are sensitive to reactive sulfite. Some methanogens use a sulfite detoxification system that combines an F420H2-oxidase with a sulfite reductase, both of which are proposed precursors of modern enzymes. Here, we present snapshots of this coupled system, named coenzyme F420-dependent sulfite reductase (Group I Fsr), obtained from two marine methanogens. Fsr organizes as a homotetramer, harboring an intertwined six-[4Fe-4S] cluster relay characterized by spectroscopy. The wire, spanning 5.4 nm, electronically connects the flavin to the siroheme center. Despite a structural architecture similar to dissimilatory sulfite reductases, Fsr shows a siroheme coordination and a reaction mechanism identical to assimilatory sulfite reductases. Accordingly, the reaction of Fsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide structural insights into this unique fusion, in which a primitive sulfite reductase turns a poison into an elementary block of life.
Assuntos
Euryarchaeota , Methanococcales , Methanococcales/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Riboflavina/química , Riboflavina/metabolismo , Sulfitos , OxirreduçãoRESUMO
Pseudouridine (Ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, stoichiometric measurement of individual Ψ sites in human transcriptome remains unaddressed. Here we develop 'PRAISE', via selective chemical labeling of Ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the Ψ landscape in the human transcriptome. Unlike traditional bisulfite treatment, our approach is based on quaternary base mapping and revealed an ~10% median modification level for 2,209 confident Ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7, TRUB1 and DKC1, with TRUB1 targets showing the highest modification stoichiometry. In addition, we quantified known and new Ψ sites in mitochondrial mRNA catalyzed by PUS1. Collectively, we provide a sensitive and convenient method to measure transcriptome-wide Ψ; we envision this quantitative approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.
Assuntos
Sulfitos , Transcriptoma , Humanos , Células HEK293 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Processamento Pós-Transcricional do RNA , Pseudouridina/genética , Pseudouridina/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/genéticaRESUMO
The development of reliable probe technology for the detection of bisulfite (HSO3-) in situ in food and biological samples is contributing significantly to food quality and safety assurance as well as community health. In this work, a responsive probe, EHDI, is developed for ratiometric fluorescence detection of HSO3- in aqueous solution, meat samples, and living cells. The probe is designed based on the HSO3- triggered 1,4-addition of electron deficit C = C bond of EHDI. As a result of this specific 1,4-addition, the π-conjugation system was destructed, resulting in blue shifts of the emission from 687 to 440 nm and absorption from 577 to 355 nm. The probe has good water solubility, high sensitivity and selectivity, allowing it to be used for imaging of HSO3- internalization and production endogenously. The capability of probe EHDI for HSO3- was then validated by traditional HPLC technology, enabling accurately detect HSO3- in beef samples. The successful development of this probe thus offers a new tool for investigating HSO3- in situ in food and biological conditions.
Assuntos
Corantes Fluorescentes , Carne , Sulfitos , Sulfitos/análise , Sulfitos/química , Corantes Fluorescentes/química , Animais , Humanos , Carne/análise , Espectrometria de Fluorescência/métodos , Bovinos , Carne Vermelha/análiseRESUMO
In recent decades, study of DNA structure has largely been focused on the interrelationships between nucleotides at the level of nearest neighbours. A little-utilized approach to probing structure on a larger scale is non-denaturing bisulfite modification of genomic DNA in conjunction with high-throughput sequencing. This technique revealed a marked gradient in reactivity increasing towards the 5' end of poly-dC:dG mononucleotide repeats as short as two base pairs, suggesting that access of the anion may be greater at these points due to positive-roll bending not predicted by existing models. Consistent with this, the 5' ends of these repeats are strikingly enriched at positions relative to the nucleosome dyad that bend towards the major groove, while their 3' ends tend to sit outside these areas. Mutation rates are also higher at the 5' ends of poly-dC:dG when CpG dinucleotides are excluded. These findings shed light on the mechanisms underlying bending/flexibility of the DNA double helix as well as the sequences that facilitate DNA packaging.
Assuntos
DNA , Sulfitos , Modelos Moleculares , Conformação de Ácido Nucleico , DNA/genética , DNA/químicaRESUMO
SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.
Assuntos
Proteínas de Bactérias , Cronobacter sakazakii , Estresse Fisiológico , Sulfitos , Fatores de Transcrição , Fatores de Virulência , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cronobacter sakazakii/genética , Cronobacter sakazakii/metabolismo , Cronobacter sakazakii/patogenicidade , Cristalização , Ligantes , Domínios Proteicos , Sulfitos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Bisulfite sequencing detects and quantifies DNA methylation patterns, contributing to our understanding of gene expression regulation, genome stability maintenance, conservation of epigenetic mechanisms across divergent taxa, epigenetic inheritance and, eventually, phenotypic variation. Graphical representation of methylation data is crucial in exploring epigenetic regulation on a genome-wide scale in both plants and animals. This is especially relevant for non-model organisms with poorly annotated genomes and/or organisms where genome sequences are not yet assembled on chromosome level. Despite being a technology of choice to profile DNA methylation for many years now there are surprisingly few lightweight and robust standalone tools available for efficient graphical analysis of data in non-model systems. This significantly limits evolutionary studies and agrigenomics research. BSXplorer is a tool specifically developed to fill this gap and assist researchers in explorative data analysis and in visualising and interpreting bisulfite sequencing data more easily. RESULTS: BSXplorer provides in-depth graphical analysis of sequencing data encompassing (a) profiling of methylation levels in metagenes or in user-defined regions using line plots and heatmaps, generation of summary statistics charts, (b) enabling comparative analyses of methylation patterns across experimental samples, methylation contexts and species, and (c) identification of modules sharing similar methylation signatures at functional genomic elements. The tool processes methylation data quickly and offers API and CLI capabilities, along with the ability to create high-quality figures suitable for publication. CONCLUSIONS: BSXplorer facilitates efficient methylation data mining, contrasting and visualization, making it an easy-to-use package that is highly useful for epigenetic research.