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

Abstract

T he zinc finger transcription factor Wilms tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia. 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, although there are conflicting data regarding the binding affinity and motifs of the different isoforms. Increased expression of the WT1 -KTS isoform at the expense of the WT1 +KTS isoform is associated with poor prognosis in acute myeloid leukemia. We determined the genome-wide binding pattern of WT1 -KTS and WT1 +KTS in leukemic K562 cells by chromatin immunoprecipitation and deep sequencing. We discovered that the WT1 -KTS isoform predominantly binds close to transcription start sites and to enhancers, in a similar fashion to other transcription factors, whereas WT1 +KTS binding is enriched within gene bodies. We observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. Additional analyses showed that both WT1 -KTS and WT1 +KTS target genes are more likely to be transcribed than non-targets, and are involved in cell proliferation, cell death, and development. Our study provides evidence that WT1 -KTS and WT1 +KTS share target genes yet still bind distinct locations, indicating isoform-specific regulation in transcription of genes related to cell proliferation and differentiation, consistent with the involvement of WT1 in acute myeloid leukemia.

Structure of WT1 zinc fingers bound to its cognate DNA: Implications of the KTS insert

“…While there are reports of the +KTS isoform functioning as a transcription factor [49] in certain cell models, comparative investigation of the two isoforms requires a more holistic cell-based approach. A recent deep sequencing study has revealed distinct global binding patterns for the +KTS and -KTS isoforms [50]. It is shown that even though both isoforms have common gene targets, binding of −KTS is specific to transcription start sites and to enhancers, whereas binding of +KTS is more abundant and is specific to regions within gene bodies.…”

DNA and RNA binding by the Wilms' tumour gene 1 (WT1) protein +KTS and −KTS isoforms—From initial observations to recent global genomic analyses

“…The aminoterminal of WT1 contains defined transcriptional activation, as well as repression, domains. 3,4 The −KTS and the +KTS isoforms in our investigation 102 were more highly expressed than non-bound genes, a correlation which was more pronounced for genes that had peaks both around the TSS and in other regulatory areas. Motamedi et al 99 also found that genes, whose expression was significantly different in metanephric rudiments in WT1 knockout mice embryos as compared to WT1-expressing embryos, predominantly reduced their expression in knockout cells, indicating WT1 as more often an activator in this system also.…”

“…The DNA binding motifs for the 2 WT1 isoforms in our investigation 102 are also not enough alike to cause the isoforms to bind the same locations in the genome. A possible explanation for the almost complete separation of the isoforms found in our analysis is a difference in binding partners which could influence choice of binding sites.…”

“…We recently performed the first isoform-specific ChIP-Seq investigation of the WT1 protein, using streptavidin-coated magnetic beads to affinity capture biotinylated exogenous WT1+17AA/−KTS and +17AA/+KTS proteins from leukaemic K562 cell lysate. 102 Our first finding was that binding peaks of both isoforms contained motifs that matched 2 WT1 position weight matrices available in the TRANSFAC database. 103 However, motifs matching the matrices were much more prevalent in the −KTS peaks (78% and 44% for the shorter and the longer matrix, respectively) than in the +KTS peaks (21% and 17%, respectively).…”

“…Also, the H3K4me3 histone mark, a well-established marker for actively transcribed genes, is more frequently present around the transcription start site of the target genes than it is around the TSS of non-target genes. However, the −KTS peaks have a greater enrichment of the H3K4me3 mark than the +KTS peaks, as well as a more distinct overrepresentation of highly expressed genes among the target genes, suggesting that the −KTS isoform may more often mediate transcriptional activation, as compared to the +KTS isoform 102. Dong et al100 found that in the overlap between their ChIP-Seq dataset, and a microarray expression assay after knockdown of WT1, WT1 seemed predominantly but not exclusively an activator, while Kann et al101 found that in RNA-seq data, the target genes of WT1…”