A chemical genetic method for monitoring genome-wide dynamics of O-GlcNAc turnover on chromatin-associated proteins.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA513484 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8417890.fastq.gz | Fastqsanger.gz | |||
| SRR8417900.fastq.gz | Fastqsanger.gz | |||
| SRR8417910.fastq.gz | Fastqsanger.gz | |||
| SRR8417920.fastq.gz | Fastqsanger.gz | |||
| SRR8417891.fastq.gz | Fastqsanger.gz |
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Project description:O-linked N-acetylglucosamine (O-GlcNAc) is a necessary protein modification installed onto hundreds of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). Recently, we developed an antibody-free metabolic feeding approach that enables unbiased mapping of O-GlcNAcylated proteins in a genome-wide manner, providing insight into the regulation of genes by O-GlcNAcylated proteins within Drosophila. Here we apply this O-GlcNAc chemical mapping strategy to a time course (TC) feeding experiment within Drosophila larvae, generated the first ever TC ChIP-seq experiment performed on both a protein modification and within a living organism. TC metabolic labeling experiments were performed in wild-type and O-GlcNAc hydrolase (OGA) deficient Drosophila. Analysis of the resulting sequencing data revealed that a loss of OGA causes a global increase in the retention of O-GlcNAc modification on proteins bound to the genome, suggesting most nuclear proteins are sensitive to effects of O-GlcNAc cycling. Interestingly, some loci are more sensitive to the impacts of a loss of OGA compared to others. This study will present an improved understanding of the regulation of gene expression by O-GlcNAc while providing the broader community with experimental methods for time-resolved analysis of genome-wide binding by proteins.
2020-01-07 | GSE124785 | GEO
Project description:We applied the chemical reporter-based metabolic labeling method to acquire O-GlcNAc modified proteins chromatin loci. Human breast cancer cell line MCF-7, as well as the genotoxic stress (Adriamycin) adapted cells MCF-7/ADR, were fed with 1 mM GalNAz. Metabolic labeled O-GlcNAz chromatin were crosslinked, sonicated and enriched by bioorthogonal chemistry. Then, the genomic DNA fragments bounded by O-GlcNAc mark were de-crosslinked, and constructed into libraries following by next-generation sequencing (Chemoselective O-GlcNAc chromatin sequencing, COGC-seq). To verify the robustness of this chemical reporter-based metabolic labeling method, we compared the results in MCF-7 and MCF-7/ADR cells with classical lectin succinylated wheat germ agglutinin (sWGA) ChIP-seq strategy. We also analyzed gene expression MCF-7 and MCF-7/ADR cells by RNA-seq.
2020-06-01 | GSE141698 | GEO
Project description:Advances in DNA sequencing are enabling new experimental modalities for studying chromatin. One emerging area is to use high-throughput DNA sequencing to monitor dynamic changes occurring to chromatin. O-Linked N-acetylglucosamine (O-GlcNAc) is a reversible protein modification found on many chromatin-associated proteins. The mechanisms by which O-GlcNAc regulates gene transcription are of high interest. Here we use DNA precipitation methods to enable monitoring time-dependent turnover of O-GlcNAc modified proteins associated with chromatin. Using an antibody-free chemical reporter strategy to map O-GlcNAc to the genome, we performed time course metabolic feeding experiments with wild-type Drosophila larvae alongside larvae lacking O-GlcNAc hydrolase (OGA), which are accordingly unable to remove O-GlcNAc. Analysis of resulting next-generation DNA sequencing data revealed that O-GlcNAc on chromatin-associated proteins at most genomic loci is processed with a half-life in hours. Notably, loss of OGA only increases this half-life by ∼3-fold. Interestingly, a small set of genomic loci are particularly sensitive to loss of OGA. In addition to these observations and new strategies to permit monitoring turnover of O-GlcNAc on chromatin, we also detail methods for coded blinding of samples alongside new normalization strategies to enable time-resolved, genome-wide analyses using chemical genetic methods. We envision these general methods will be applicable to diverse protein and nucleic acid modifications.
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Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.
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Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray
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Project description:Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure, with high confidence, turnover rates are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared to selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data is acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focussed studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways.
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Project description:Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O- glcnAcylation) via overexpression of the O-glcnAc–regulating enzymes O- glcnAc transferase (OGT) or O- glcnAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O- glcnAcylation either by pharmacological or genetic manipulation also alters metabolic function. Sustained O-glcnAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-glcnAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-Seq in SH-SY5Y cells indicated transcriptome reprogramming and down regulation of the NRF2-mediated antioxidant response. Sustained O-glcnAcylation in mice brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-glcnAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-glcnAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.
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