Integration of Cistromic and Transcriptomic Analyses Identifies Nphs2, Mafb, and Magi2 as Wilms’ Tumor 1 Target Genes in Podocyte Differentiation and Maintenance

Abstract

The Wilms' tumor suppressor gene 1 (WT1) encodes a zinc finger transcription factor. Mutation of WT1 in humans leads to Wilms' tumor, a pediatric kidney tumor, or other kidney diseases, such as Denys-Drash and Frasier syndromes. We showed previously that inactivation of WT1 in podocytes of adult mice results in proteinuria, foot process effacement, and glomerulosclerosis. However, the WT1-dependent transcriptional network regulating podocyte development and maintenance in vivo remains unknown. Here, we performed chromatin immunoprecipitation followed by high-throughput sequencing with glomeruli from wild-type mice. Additionally, we performed a cDNA microarray screen on an inducible podocyte-specific WT1 knockout mouse model. By integration of cistromic and transcriptomic analyses, we identified the WT1 targetome in mature podocytes. To further analyze the function and targets of WT1 in podocyte maturation, we used an Nphs2-Cre model, in which WT1 is deleted during podocyte differentiation. These mice display anuria and kidney hemorrhage and die within 24 hours after birth. To address the evolutionary conservation of WT1 targets, we performed functional assays using zebrafish as a model and identified Nphs2, Mafb, and Magi2 as novel WT1 target genes required for podocyte development. Our data also show that both Mafb and Magi2 are required for normal development of the embryonic zebrafish kidney. Collectively, our work provides insights into the transcriptional networks controlled by WT1 and identifies novel WT1 target genes that mediate the function of WT1 in podocyte differentiation and maintenance.

Distinct global binding patterns of the Wilms tumor gene 1 (WT1) −KTS and +KTS isoforms in leukemic cells

“…The motifs found to be most enriched for the WT1 -KTS isoform in our investigation are similar to those found in the four previous studies, closely resembling the established EGR-1 and WTE motifs. Our distribution of -KTS peaks, centering around the TSS, resembles the results of Dong et al 41 and Kann et al, 42 with comparable concentration in the promoter area (23.1% in our investigation as compared to 21% found by Kann et al and 16.9% by Dong et al) although due to differences in the analysis software exact comparisons are not possible. Motamedi et al 40 found a different distribution of peaks, with only 20% of peaks located within 5 kb of a TSS, whereas we found 46.5% of all -KTS peaks in this area.…”

“…Motamedi et al 40 found a different distribution of peaks, with only 20% of peaks located within 5 kb of a TSS, whereas we found 46.5% of all -KTS peaks in this area. Moreover, several of the GO groups enriched in our investigation were also found to be enriched in the earlier ChIP-Seqs and ChIP-chip investigations into WT1, such as cytoskeleton and adhesion, 39,41,42 cell cycle, 39,42 and development. 39 We found that the WT1 -KTS isoform binds as a traditional transcription factor around the TSS and to known T. Ullmark et al…”

“…This pattern suggests a mostly activating function for both isoforms, consistent with data from two of the earlier ChIP-Seq investigations 41,42 in which WT1 was found to be predominantly activating in mouse kidney glomeruli cells. When, instead, we analyzed the localization of all the WT1 peaks from our investigation, and compared the localizations with those of peaks from ChIP-Seq investigations present in the ENCODE database, we found that tracks for methylated DNA and the repressive histone mark H3K27me3 overlapped with our WT1 +KTS peaks to the extent of becoming top similarity hits.…”

“…One ChIPchip (chromatin immunoprecipitation coupled with DNA microarrays) and three ChIP-Seq investigations into the target genes of WT1, although not distinguishing WT1 -KTS and WT1 +KTS from each other, have been published. [39][40][41][42] All studies were performed on mouse kidney cells. The motifs found to be most enriched for the WT1 -KTS isoform in our investigation are similar to those found in the four previous studies, closely resembling the established EGR-1 and WTE motifs.…”

Transcription factor Wilms’ tumor 1 regulates developmental RNAs through 3′ UTR interaction

“…Accordingly, there is substantial evidence that WT1 binds genomic DNA and regulates transcription, acting as either an activator or a repressor (Essafi et al 2011;Toska and Roberts 2014). A growing number of physiological WT1 transcriptional targets have been identified in development, homeostasis, and disease (Motamedi et al 2014;Dong et al 2015;Kann et al 2015). However, the evidence suggests that transcriptional regulation is not the only WT1 function.…”