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CUT&RUN to map WT1 binding in M15 cell lines that are genome edited for the KTS isoforms.

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE189871 | GEO | 2024/11/23

REPOSITORIES: GEO

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Project description:The t(11;22)(p13;q12) translocation is pathognomonic for the highly aggressive desmoplastic small round cell tumour (DSRCT). The translocation fuses exon 7 of the EWS gene to exon 8 of the WT1 gene (EWS/WT1). Two splice variants of EWS/WT1 exist, arising from the presence (+KTS) or absence (-KTS) of three amino acids: lysine, threonine and serine between zinc fingers 3 and 4. To investigate the oncogenic properties of both isoforms of EWS/WT1, we over-expressed EWS/WT1 in untransformed murine embryonic fibroblasts (MEFs). We demonstrate that neither isoform of EWS/WT1 is sufficient to transform wild type MEFs, however the oncogenic potential of both isoforms is unmasked by the loss of p53. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 resulted in enhanced cell proliferation, clonogenic survival and anchorage-independent growth. In addition EWS/WT1 expression in wild type MEFs attenuated several p53-dependent responses, including cell cycle arrest after irradiation and daunorubicin induced apoptosis. We show that DSRCT commonly have copy number amplification of MDM2 and MDMX, suggesting loss of p53 function in the tumours. Expression of either isoform of EWS/WT1 in MEFs induced characteristic mRNA expression profiles, including up-regulation of canonical Wnt pathway signaling. This was validated in cell lines and in a series of DSCRT, which confirmed canonical Wnt pathway activation in the tumours. We show for the first time that both isoforms of EWS/WT1 have oncogenic potential and that in addition to co-operating with loss of p53 function can also further attenuate p53-mediated responses. In addition we provide the first link between EWS/WT1 and Wnt pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterizes DSCRT. Four independently generated pools of wild type MEFs were infected with tetracycline repressible EWS/WT1+KTS, EWS/WT1-KTS or GFP, selected with hygromycin and protein expression confirmed. Four GFP, four KTS+ and four KTS- samples were hybridized to the Ilumina BeadChip.

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CUT&RUN to map WT1 binding in M15 cell lines that are genome edited for the KTS isoforms.

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