Epigenetic inactivation of the epithelial splicing factor ESRP2 in Wilms’ tumour


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Danny Legge1,Whei Moriarty1,Sebastian Oltean2,Karim Malik1,Keith Brown1
1University of Bristol,2University of Exeter



Wilms' tumour (WT) is a paediatric kidney cancer thought to originate from a block in differentiation of metanephric precursor cells. Around 40% of WT lack identifiable driver mutations, with epigenetic deregulation suggested to play a role in these cases.


We performed genome-wide analysis of DNA methylation in two WT cell lines using MCIP to identify hypermethylated genes in WT. 225 genes were identified, these were filtered by negative selection for genes that are PRC marked in ES cells and positively selected for genes upregulated in the renal vesicle during kidney development. Four candidate genes were identified. The methylation status of these was analysed by pyrosequencing in WT patient samples. Of these genes, ESRP2 (an RNA splicing factor) was found to be hypermethylated in WTs compared to normal/fetal kidney tissue. To investigate the function of ESRP2, we generated a WT cell line (WiT49 E200L) with doxycycline (Dox)-inducible expression of ESRP2, along with control cells transfected with empty vector (WiT49 V200). Colony formation and cell growth was analysed using functional assays and Incucyte analysis. Finally, to identify any ESRP2-induced alternative splicing events that may be important in WT development, we performed RNA-Seq analysis of E200L cells following ESRP2 induction.


ESRP2 induction following Dox addition was confirmed by PCR and western analysis. Alternative splicing of known ESRP2 targets was confirmed by PCR. Colony formation was significantly inhibited by around 50% upon induction of ESRP2. Cell growth was reduced by around 50% following ESRP2 induction in E200L cells. Initial GO analysis of RNA-Seq data revealed ESRP2-induced alternative splicing of genes involved in cell division and differentiation. Validation and further analysis of RNA-Seq data is currently ongoing.


Data generated from this analysis has the potential to highlight novel alternative splicing events that could potentially be targeted for more effective therapies for WT in the future.