CUT&RUN to map WT1 binding in M15 cell lines that are genome edited for the KTS isoforms.
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ABSTRACT:
PROVIDER: PRJNA784936 | ENA |
REPOSITORIES: ENA
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Project description:throughput profiling of WT1 in M15 mouse mesonephric cells and M15 genome edited for the KTS isoforms. The genome-edited lines were derived from the M15 cells by transfection and puromycin selection of cells with the guide RNA expressing plasmids. Briefly, the guide RNAs were designed against the splice site that is essential for KTS isoform. Out of the designed guide RNAs, some of them were cloned into the pX601 vector with the Sa Cas9 variant, and clone confirmation was done using sequencing. Isoform-specific lines were established by providing oligos for repair which represented the region corresponding to the codons coding for KTS or not.
2024-11-23 | GSE189871 | GEO
Project description:EWS fusion oncoproteins underlie the pathogenesis of several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive mesenchymal tumor driven by fusions between the disordered domain of EWS and the developmental transcription factor WT1. Here we combined chromatin occupancy and long-range interaction profiles to identify EWS-WT1-dependent gene regulation networks and directly controlled target genes. We show that EWS-WT1 operates primarily as a powerful activator of distal regulatory elements and controls an oncogenic gene expression program that characterize primary DSRCTs. Moreover, EWS-WT1 has two isoforms that differ by three amino acids in their DNA binding domain (+/- KTS), as observed for wild type WT1, and we show that each fusion isoform has a specific DNA binding profile that is distinct from its wild type counterparts and requires a functional EWSR1 prion like domain. Remarkably, xenograft experiments using human mesothelial cells, candidate cells of origin of DSRCT, reveal that both isoforms are required to generate viable tumors that resemble DSRCT. Finally, we identify new candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex isoform-dependent oncogenic activity of EWS-WT1.
2024-10-01 | GSE248842 | GEO
Project description:EWS fusion oncoproteins underlie the pathogenesis of several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive mesenchymal tumor driven by fusions between the disordered domain of EWS and the developmental transcription factor WT1. Here we combined chromatin occupancy and long-range interaction profiles to identify EWS-WT1-dependent gene regulation networks and directly controlled target genes. We show that EWS-WT1 operates primarily as a powerful activator of distal regulatory elements and controls an oncogenic gene expression program that characterize primary DSRCTs. Moreover, EWS-WT1 has two isoforms that differ by three amino acids in their DNA binding domain (+/- KTS), as observed for wild type WT1, and we show that each fusion isoform has a specific DNA binding profile that is distinct from its wild type counterparts and requires a functional EWSR1 prion like domain. Remarkably, xenograft experiments using human mesothelial cells, candidate cells of origin of DSRCT, reveal that both isoforms are required to generate viable tumors that resemble DSRCT. Finally, we identify new candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex isoform-dependent oncogenic activity of EWS-WT1.
2023-10-04 | GSE212978 | GEO
Project description:EWS fusion oncoproteins underlie the pathogenesis of several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive mesenchymal tumor driven by fusions between the disordered domain of EWS and the developmental transcription factor WT1. Here we combined chromatin occupancy and long-range interaction profiles to identify EWS-WT1-dependent gene regulation networks and directly controlled target genes. We show that EWS-WT1 operates primarily as a powerful activator of distal regulatory elements and controls an oncogenic gene expression program that characterize primary DSRCTs. Moreover, EWS-WT1 has two isoforms that differ by three amino acids in their DNA binding domain (+/- KTS), as observed for wild type WT1, and we show that each fusion isoform has a specific DNA binding profile that is distinct from its wild type counterparts and requires a functional EWSR1 prion like domain. Remarkably, xenograft experiments using human mesothelial cells, candidate cells of origin of DSRCT, reveal that both isoforms are required to generate viable tumors that resemble DSRCT. Finally, we identify new candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex isoform-dependent oncogenic activity of EWS-WT1.
2023-10-04 | GSE212977 | GEO
Project description:EWS fusion oncoproteins underlie the pathogenesis of several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive mesenchymal tumor driven by fusions between the disordered domain of EWS and the developmental transcription factor WT1. Here we combined chromatin occupancy and long-range interaction profiles to identify EWS-WT1-dependent gene regulation networks and directly controlled target genes. We show that EWS-WT1 operates primarily as a powerful activator of distal regulatory elements and controls an oncogenic gene expression program that characterize primary DSRCTs. Moreover, EWS-WT1 has two isoforms that differ by three amino acids in their DNA binding domain (+/- KTS), as observed for wild type WT1, and we show that each fusion isoform has a specific DNA binding profile that is distinct from its wild type counterparts and requires a functional EWSR1 prion like domain. Remarkably, xenograft experiments using human mesothelial cells, candidate cells of origin of DSRCT, reveal that both isoforms are required to generate viable tumors that resemble DSRCT. Finally, we identify new candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex isoform-dependent oncogenic activity of EWS-WT1.
2023-10-04 | GSE212976 | GEO
Project description:EWS fusion oncoproteins underlie the pathogenesis of several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive mesenchymal tumor driven by fusions between the disordered domain of EWS and the developmental transcription factor WT1. Here we combined chromatin occupancy and long-range interaction profiles to identify EWS-WT1-dependent gene regulation networks and directly controlled target genes. We show that EWS-WT1 operates primarily as a powerful activator of distal regulatory elements and controls an oncogenic gene expression program that characterize primary DSRCTs. Moreover, EWS-WT1 has two isoforms that differ by three amino acids in their DNA binding domain (+/- KTS), as observed for wild type WT1, and we show that each fusion isoform has a specific DNA binding profile that is distinct from its wild type counterparts and requires a functional EWSR1 prion like domain. Remarkably, xenograft experiments using human mesothelial cells, candidate cells of origin of DSRCT, reveal that both isoforms are required to generate viable tumors that resemble DSRCT. Finally, we identify new candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex isoform-dependent oncogenic activity of EWS-WT1.
2023-10-04 | GSE242863 | GEO
Project description:Absence of WT1 during kidney organoid development from human induced pluripotent stem cells (iPSCs) induces hallmarks of Wilms tumorigenesis. To define underlying transcriptional alterations and similarities to human patients, we performed timecourse RNA-seq of kidney organoid development from control iPSCs (control, not edited) and in the absence of WT1. Two timepoints for knockout (KO) of WT1 were investigated: In iPSCs (KO in iPSCs), and between day 4 and day 7 of organoid formation (KO d4-7).
2021-08-01 | E-MTAB-9957 | biostudies-arrayexpress
Project description:This exon array analysis was performed on mice carrying splice specific mutations at the end of exon9 of the Wilms tumor supressor gene that interfere with the production of WT1(+KTS) isoforms (Hammes et al., 2001; Cell 106:309) 4 wildtype and 4 wt1(+KTS) mutant E18.5 mouse embryos were transcardially perfused with magnetic beads, glomeruli isolated and RNA extracted and analyzed for differential expression on Affymetrix Exon Arrays
2015-03-27 | E-GEOD-67313 | biostudies-arrayexpress
Project description:This exon array analysis was performed on mice carrying splice specific mutations at the end of exon9 of the Wilms tumor supressor gene that interfere with the production of WT1(+KTS) isoforms (Hammes et al., 2001; Cell 106:309)
2015-03-27 | GSE67313 | GEO
Project description:Background: Wilms' tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia (AML). A naturally occurring alternative splice event between zinc fingers three and four, removing or retaining three amino acids (KTS), is believed to change the DNA binding affinity of WT1. Altered balance between WT1 -KTS and WT1 +KTS expression associates with poor prognosis in AML. Methods: We characterized the DNA binding patterns of biotin-tagged WT1 -KTS and WT1 +KTS in K562 cells by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Results: We discovered that WT1 -KTS preferentially binds near transcription start sites (TSS) and in enhancers, whereas WT1 +KTS binds within gene bodies. Additionally, we observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif analysis showed enrichment of two TRANSFAC derived motif matrices within peaks for both isoforms, and enrichment of several previously published WT1 motifs which, however, differed between isoforms. Additional analyses showed that WT1 -KTS and WT1 +KTS target genes are transcribed to a higher extent than non-targets, and involved in cell proliferation, cell death, and development. Conclusions: Our results provide the first evidence that WT1 -KTS and WT1 +KTS bind principally different regions of the genome, yet share target genes. Our results indicate isoform-specific regulation of processes related to cell proliferation and differentiation, consistent with the involvement of WT1 in AML.
2016-12-24 | GSE81009 | GEO