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

The 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.

Anti-apoptotic quinolinate phosphoribosyltransferase (QPRT) is a target gene of Wilms' tumor gene 1 (WT1) protein in leukemic cells.

Wilms' tumor gene 1 (WT1) is a zinc finger transcription factor that has been implicated as an oncogene in leukemia and several other malignancies. When investigating possible gene expression network partners of WT1 in a large acute myeloid leukemia (AML) patient cohort, one of the genes with the highest correlation to WT1 was quinolinate phosphoribosyltransferase (QPRT), a key enzyme in the de novo nicotinamide adenine dinucleotide (NAD+) synthesis pathway. To investigate the possible relationship between WT1 and QPRT, we overexpressed WT1 in hematopoietic progenitor cells and cell lines, resulting in an increase of QPRT expression. WT1 knock-down gave a corresponding decrease in QPRT gene and protein expression. Chromatin-immunoprecipitation revealed WT1 binding to a conserved site in the first intron of the QPRT gene. Upon overexpression in leukemic K562 cells, QPRT conferred partial resistance to the anti-leukemic drug imatinib, indicating possible anti-apoptotic functions, consistent with previous reports on glioma cells. Interestingly, the rescue effect of QPRT overexpression was not correlated to increased NAD + levels, suggesting NAD + independent mechanisms. We conclude that QPRT, encoding a protein with anti-apoptotic properties, is a novel and direct target gene of WT1 in leukemic cells.

The hematopoietic tumor suppressor interferon regulatory factor 8 (IRF8) is upregulated by the antimetabolite cytarabine in leukemic cells involving the zinc finger protein ZNF224, acting as a cofactor of the Wilms' tumor gene 1 (WT1) protein.

The transcription factor interferon regulatory factor-8 (IRF8) is highly expressed in myeloid progenitors, while most myeloid leukemias show low or absent expression. Loss of IRF8 in mice leads to a myeloproliferative disorder, indicating a tumor-suppressive role of IRF8. The Wilms tumor gene 1 (WT1) protein represses the IRF8-promoter. The zinc finger protein ZNF224 can act as a transcriptional co-factor of WT1 and potentiate the cytotoxic response to the cytostatic drug cytarabine. We hypothesized that cytarabine upregulates IRF8 and that transcriptional control of IRF8 involves WT1 and ZNF224. Treatment of leukemic K562 cells with cytarabine upregulated IRF8 protein and mRNA, which was correlated to increased expression of ZNF224. Knock down of ZNF224 with shRNA suppressed both basal and cytarabine-induced IRF8 expression. While ZNF224 alone did not affect IRF8 promoter activity, ZNF224 partially reversed the suppressive effect of WT1 on the IRF8 promoter, as judged by luciferase reporter experiments. Coprecipitation revealed nuclear binding of WT1 and ZNF224, and by chromatin immunoprecipitation (ChIP) experiments it was demonstrated that WT1 recruits ZNF224 to the IRF8 promoter. We conclude that cytarabine-induced upregulation of the IRF8 in leukemic cells involves increased levels of ZNF224, which can counteract the repressive activity of WT1 on the IRF8-promoter.