ARID1A IDR Targets EWS-FLI1 Condensates and Finetunes Productive Chromatin Remodeling
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1096325 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28558400_1.fastq.gz | Fastqsanger.gz | |||
| SRR28558400_2.fastq.gz | Fastqsanger.gz | |||
| SRR28558401_1.fastq.gz | Fastqsanger.gz | |||
| SRR28558401_2.fastq.gz | Fastqsanger.gz | |||
| SRR28558402_1.fastq.gz | Fastqsanger.gz |
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Project description:Samples 1012398_F1-F8: Using recombinant purified mCherry-tagged FUS-IDR we reconstituted condensates in a soluble nuclear extract isolated from U2OS cells. The extract was fractionated by centrifugation into a supernatant and pellet. Labels 126, 127, 128 are supernatant replicates and labels 129, 130, 131 are pellet replicates.
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Project description:It remains largely unclear how material properties of biomolecular condensates impact physiological and pathological processes such as cancer. Here we show that AKAP95, a nuclear protein involved in gene regulation, plays an important role in tumorigenesis through directly regulating mRNA splicing. We show that AKAP95, through an intrinsic disordered region (IDR), forms liquid-like droplets in vitro and dynamic foci in nucleus. We have also identified key residues in IDR whose mutations to different residues perturb AKAP95 condensation in opposite directions. Importantly, the biological activity of AKAP95 in splice regulation is affected by either disruption of liquid condensation or enhancing the gelation of the assembly. We then show that the ability of AKAP95 in overcoming oncogene-induced senescence requires AKAP95 in an appropriate window of liquidity. These results thus link phase separation to tumorigenesis and uncover an important role of regulating the material property of protein condensates in disease including cancer.
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Project description:Cancer-associated genetic aberrations, such as the nucleoporin 98 (NUP98) gene rearrangement detected in human leukemias, often produce condensates, a type of membrane-less biomolecular assemblies. How exactly the cancer-related condensation contributes to oncogenesis remains far from clear. Here, we investigate NUP98-PHF23, a leukemia-causing chimeric protein that fuses NUP98’s sequence enriched in phenylalanine-and-glycine repeats (FG repeats, also known as intrinsically disordered region [IDR]) in-frame with PHF23’s PHD finger, a domain that specifically ‘reads’ and binds H3K4 trimethylation (H3K4me3). Our integrated analyses using protein module mutagenesis, cell imaging, genomic profiling and condensation reconstitution collectively demonstrate a multifaced role for NUP98’s FG repeats in driving fusion condensation while recruiting and co-mixing with a set of histone modifiers and chromatin remodelers, notably the MLL family of H3K4 methylation-‘writing’ enzymes. The H3K4me3-‘reading’ PHD finger and NUP98’s IDR are cooperative in mediating efficient targeting of NUP98-PHF23 and its coactivators onto leukemic genes, leading to active transcription. Together, we show that NUP98-PHF23 coordinates a set of homotypic and heterotypic interactions (IDR:IDR, IDR:coactivator and PHD:histone) to organize formation of the chromatin-bound multi-component condensates, wherein a feedforward loop involving the ‘reading’ and ‘writing’ of H3K4 methylation acts to enforce an open chromatin landscape at leukemogenic loci, thereby driving oncogenic transformation.
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Project description:Synthetic, innate defense regulators (IDR) peptides, designed based on natural host defenses peptides, have enhanced immunomodulatory activities and reduced toxicity leading to protection in infection and inflammation models that is dependent on macrophages/monocytes. Here we measured the effect of IDR-1018 on macrophage gene expression during differentiation. Differentiation in the presence of IDR-1018 induced a unique signature of immune responses suggesting that IDR-1018 drives macrophage differentiation towards an intermediate M1-M2 state, enhancing anti-inflammatory functions while maintaining certain pro-inflammatory activities important to the resolution of infection. RNA-seq was performed using the Illumina Genome Analyzer IIx platform. Monocytes were isolated from 3 healthy donors, and left unstimulated or stimulated for 4 hours with 20 μg/ml IDR-1018. For library preparation, 500 ng of total RNA was processed according to the Illumina TruSeq RNA sample preparation guide (Illumina catalogue number FC-122-1002). Briefly, mRNA was purified using poly-dT beads, followed by synthesis of the first and second cDNA strands, end repair addition of an poly-A overhang, and ligation of adapters and unique barcodes, as per the manufacturer’s instructions. DNA enrichment was carried out via a 15-cycle PCR. Following quantification, 8 pM of dsDNA was used for cluster generation on a CBOT instrument (Illumina, San Diego, CA). RNA sequencing was done on a GAIIx instrument (Illumina), performed as a single read run with 51 amplification cycles. Data processing was carried out in house, using CASAVA to convert raw data and demultiplex to FASTQ sequence files. Reads were aligned to the reference genome using TOPHAT, and then mapped to genes using the Bioconductor package GenomeRanges.
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Project description:Effect of peptide IDR-1 on CD14+ human monocytes. >br< >br< Effect of peptide IDR-1 on LPS-induced responses in CD14+ human monocytes.>br< >br< Human peripheral blood mononuclear cells (PBMC) were treated with innate defence regulator peptide IDR-1 in the presence and absence of LPS (purified from Pseudomonas aeruginosa) for 4hrs. The objective was to obtain a transcriptional profile of responses induced by IDR-1 and the effect of IDR-1 on LPS-induced transcription.
2007-04-10 | E-FPMI-8 | biostudies-arrayexpress
Project description:Ewing sarcoma (EwS) is a rare and predominantly pediatric malignancy of bone and soft tissue in children and adolescents. Although international collaborations have greatly improved the prognosis of most EwS, the occurrence of macrometastases or relapse remains challenging. The prototypic oncogene EWS-FLI1 acts as an aberrant transcription factor that drives the cellular transformation of EwS. In addition to its involvement in RNA splicing and the DNA damage response, this chimeric protein directly binds to GGAA repeats, thereby modifying the transcriptional profile of EwS. Direct pharmacological targeting of EWS-FLI1 is difficult because of its intrinsically disordered structure. However, targeting the EWS-FLI1 protein complex or downstream pathways provides additional therapeutic options. This review describes the EWS-FLI1 protein partners and downstream pathways, as well as the related target therapies for the treatment of EwS.
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Project description:The EWS-FLI1 chimeric protein uniquely expressed in Ewing's sarcoma has an obligate role in its aetiology. In our previous report we showed that ectopic expression of the DNA sequences form the junction region (a.a 251-280) can inhibit Ewing's sarcoma cell growth. In the present report, we introduced a peptide (TAT/NLS/EWS-PEP) comprising of thirty amino acids spanning the junction in conjunction with HIV-1-trans-activating (TAT) and nuclear localization signal sequence (NLS). Peptide uptake and localization studies revealed presence of peptide in ~99% of transduced cells and in the nucleus. Peptide transfection induced cytotoxicity relative to untreated and TAT-NLS peptide treated Ewing's sarcoma cells. The peptide inhibited clonogenicity, cell cycle, bromo-deoxy uridine (BrdU) uptake and invasion capacity of treated cells. The treatment also affected epithelial to mesenchymal transition (EMT) markers and EWS-FLI1 target gene expression levels. Co-immunoprecipitation experiments involving ectopically expressed full-length EWS-FLI1 protein and the peptide revealed an interaction. Additionally, we found that peptide interaction also occurs with the protein-GGAA microsatellite sequences complex known to contain EWS-FLI1. Further, in the pull-down assay, the peptide was found to interact with proteins known to potentially interact with EWS-FLI1. Based on these results we conclude that peptide could be applied in targeting EWS-FLI1 protein.
| S-EPMC5543137 | biostudies-literature