Project description:Pooled CRISPR screens are a powerful tool to identify regulators of a biological process, but have been limited to phenotypes that affect viability or can be monitored with fluorescent protein reporters. We evaluate two additional strategies for phenotypic enrichment based on quantification of RNA using a Flow-FISH assay or protein phosphorylation through intracellular phospho-staining. These tools will greatly expand the applicability of CRISPR screens since they increase the number of possible molecular phenotypes and assay designs.
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Peggy Farnham mailto:pfarnham@usc.edu for questions concerning data collection and usage and Philip Cayting mailto:pcayting@stanford.edu for data scoring and submission inquiries). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track, produced as part of the ENCODE Project, displays maps of histone modifications genome-wide using ChIP-seq in different cell lines. The ChIP-seq method involves first using formaldehyde to cross-link histones and other DNA-associated proteins to genomic DNA within cells. The cross-linked chromatin is subsequently extracted, sheared, and immunoprecipitated using specific antibodies. After reversal of cross-links, the immunoprecipitated DNA is sequenced and mapped to the human reference genome. The relative enrichment of each antibody-target (epitope) across the genome is inferred from the density of mapped fragments. Chemical modifications (e.g. methylation or acetylation) of the histone proteins present in chromatin influence gene expression by changing how accessible the chromatin is to transcription factors. Shown for each experiment (defined as a particular antibody and a particular cell type) is a track of enrichment for the specifically modified histone (Signal), along with sites that have the greatest enrichment (Peaks). Also included for each cell type is the input signal, which represents the control condition where no antibody targeting was performed. In general the following chemical modifications have associated genetic phenotypes: H3K4me3 and H3K9Ac are considered to be marks of active or potentially active promoter regions. H3K4me1 and H3K27Ac are considered to be marks of active or potentially active enhancer regions. H3K36me3 and H3K79me2 are considered to be marks of transcriptional elongation. H3K27me3 and H3K9me3 are considered to be marks of inactive regions. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
Project description:Polyamines are essential and evolutionarily conserved metabolites present at millimolar concentrations in mammalian cells. Cells tightly regulate polyamine homeostasis through complex feedback mechanisms, yet the precise role necessitating this regulation remains unclear. Here, we show that polyamines function as endogenous buffers of redox-active iron, providing a molecular link between polyamine metabolism and ferroptosis. Using genome-wide CRISPR screens, we identified a synthetic lethal dependency between polyamine depletion and the key ferroptosis suppressor, GPX4. Mechanistically, we show that polyamine deficiency triggers a redistribution of cellular iron, increasing the labile iron pool and upregulating ferritin. To directly visualize this iron buffering in living cells, we developed a genetically encoded fluorescent reporter for redox-active iron. Live-cell analysis revealed a striking inverse correlation between intracellular polyamine levels and redox-active iron at single-cell resolution. These findings reposition polyamines as key regulators of iron homeostasis, with implications for ferroptosis-linked disease states and cellular redox balance.
Project description:Actinorhodin is a blue-pigmented, redox-active pigmented secondary metabolite that is produced by the bacterium Streptomyces coelicolor. Although actinorhodin has been used as a model compound for studying secondary metabolism, its mechanism is not well understood. In this work, we have conducted a comprehensive chemical genetic investigation of actinorhodin’s antibacterial effect on target organisms.
Project description:Cancer development and progression is intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics emerge as a fundamental tool to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after the enrichment steps may occur. Considering the biological importance of thiol’s oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome limitations of the traditional method.
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Peggy Farnham mailto:pfarnham@usc.edu for questions concerning data collection and usage and Philip Cayting mailto:pcayting@stanford.edu for data scoring and submission inquiries). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track, produced as part of the ENCODE Project, displays maps of histone modifications genome-wide using ChIP-seq in different cell lines. The ChIP-seq method involves first using formaldehyde to cross-link histones and other DNA-associated proteins to genomic DNA within cells. The cross-linked chromatin is subsequently extracted, sheared, and immunoprecipitated using specific antibodies. After reversal of cross-links, the immunoprecipitated DNA is sequenced and mapped to the human reference genome. The relative enrichment of each antibody-target (epitope) across the genome is inferred from the density of mapped fragments. Chemical modifications (e.g. methylation or acetylation) of the histone proteins present in chromatin influence gene expression by changing how accessible the chromatin is to transcription factors. Shown for each experiment (defined as a particular antibody and a particular cell type) is a track of enrichment for the specifically modified histone (Signal), along with sites that have the greatest enrichment (Peaks). Also included for each cell type is the input signal, which represents the control condition where no antibody targeting was performed. In general the following chemical modifications have associated genetic phenotypes: H3K4me3 and H3K9Ac are considered to be marks of active or potentially active promoter regions. H3K4me1 and H3K27Ac are considered to be marks of active or potentially active enhancer regions. H3K36me3 and H3K79me2 are considered to be marks of transcriptional elongation. H3K27me3 and H3K9me3 are considered to be marks of inactive regions. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. Briefly, cells were crosslinked, chromatin was extracted and sonicated using a Bioruptor sonicator (Diagenode) to an average size of 300-500bp, and individual ChIP assays were performed using antibodies to modified histones. For the K562 and Ntera2 histone ChIP-seq samples, immunoprecipitates were collected using protein G-coupled magnetic beads; a detailed ChIP and library protocol can be found at http://www.roadmapepigenomics.org/protocols. For the U2OS histone ChIP-seq samples, immunoprecipitates were collected using StaphA cells; a detailed protocol can be found at http://expression.genomecenter.ucdavis.edu/chip.html. Library DNA was quantitated using either a Nanodrop or a BioAnalyzer and sequenced on an Illumina GA2. The sequencing reads were mapped to the genome using the Eland alignment program. ChIP-seq data was scored based on sequence reads (length ~30 bps) that align uniquely to the human genome. From the mapped tags, a signal map of ChIP DNA fragments (average fragment length ~ 200 bp) was constructed where the signal height is the number of overlapping fragments at each nucleotide position in the genome. For each 1 Mb segment of each chromosome, a peak height threshold was determined by requiring a false discovery rate <= 0.05 when comparing the number of peaks above threshold as compared to the number obtained from multiple simulations of a random null background with the same number of mapped reads (also accounting for the fraction of mapable bases for sequence tags in that 1 Mb segment). The number of mapped tags in a putative binding region is compared to the normalized (normalized by correlating tag counts in genomic 10 kb windows) number of mapped tags in the same region from an input DNA control. Using a binomial test, only regions that have a p-value <= 0.05 are considered to be significantly enriched compared to the input DNA control.
Project description:Hydroxylated polychlorinated biphenyls are the metabolites produced from polychlorinated biphenyls (PCBs) by drug-metabolizing enzyme cytochrome P450 1A1. These compounds are bound to transthyretin, a major plasma thyroid hormone-binding protein in amphibian tadpoles. The compounds-transthyretin complexes are transferred into the brain across the blood brain barrier in mammals. Thus these compounds are suspected to disrupt neural development in brain. We studied about the effects of hydroxylated PCBs on the thyroid system in brain using metamorphosing tadpoles of African clawed toad, Xenopus laevis. The metamorphosis assay revealed that these compounds had inhibitory effects on the thyroid hormone-induced metamorphosis. This in vivo assay was a powerful tool to detect thyroid-disrupting activities, because we were not able to detect the inhibitory effects of these compounds using thyroid hormone-responsive reporter gene assay in a cultured Xenopus cell line. A genome-wide gene expression analysis in brain following short-term exposure to these compounds demonstrated that the delay of metamorphosis and the morphological thyroid-disrupting changes could be caused partially by disruption of the thyroid hormone-induced gene expression by hydroxylated PCBs. Furthermore, we associated functional ontology terms with the transcripts whose expression were altered by thyroid hormone alone, or thyroid hormone and hydroxylated PCBs. We suggested that these approachs using a technique of bioinformatics revealed molecular mechanism of thyroid-disrupting activities in vivo.