WT1 gene, originally identified as a tumor suppressor involved in the formation of Wilms’ tumor of the kidney, was subsequently described to have an oncogenic role in a variety of tumors from different origins, including leukemias. 1 In comparison to normal progenitor cells, it is overexpressed in acute lymphoblastic and myeloblastic leukemia, and in the blast crisis phase of chronic myelogenous leukemia; 1, 2 high levels of the protein are associated with a poor response to therapy. 1, 3 WT1 knockdown by antisense oligonucleotides or RNA interference was shown to induce apoptosis; conversely, its overexpression in myeloid leukemia cells protected against cell death. Modulation of some members of the bcl-2 family has been associated with apoptosis inhibition by WT1. 1, 3 Among proteins that regulate apoptosis in leukemia cells, a role is assigned to BAG3, a member of the family of proteins that, through their BAG domain, interact with HSC70/HSP70 heat shock proteins. bag3 gene expression is constitutive in some tumor types, including leukemias; in these cells, BAG3 protein has been shown to sustain cell survival and downmodulate cell apoptotic response to drugs, by either HSP70-dependent or-independent mechanisms. 4–7 Here we report that WT1 induces bag3 gene expression. This finding identifies a novel target of WT1 protein involved in apoptosis regulation in leukemia cells. As using in silico analysis we had found two putative WT1 binding sites on the bag3 promoter, we decided to investigate whether WT1 is directly recruited onto the bag3 promoter using a chromatin immunoprecipitation assay (ChIP) in K562 cells, which express significant amounts of endogenous WT1 and BAG3. Chromatin was immunoprecipitated with anti-WT1 antibodies. Subsequent PCR analysis, performed using oligonucleotides covering the putative WT1 binding sites, revealed that WT1 bound to the bag promoter sequences (350-bp band), whereas rabbit IgG antibody controls did not (Figure 1a). WT1 protein has two major isoforms, designated WT1 (-KTS) and

WT1 (+ KTS), containing an extra three aminoacids (KTS) between the third and fourth zinc fingers; WT1 (ÀKTS) appears to exert its effect mainly as a transcriptional factor, whereas WT1 (þKTS) is involved in RNA processing. 1 To investigate the transcriptional modulation of bag3 promoter by WT1, we introduced a luciferase reporter plasmid containing the bag3 promoter into HEK293 cells and analyzed luciferase levels in the presence of increasing amounts of WT1 (ÀKTS) or WT1 (þKTS) expression plasmids. As shown in Figure 1b, the ‘transcriptional isoform’, WT1 (ÀKTS), enhanced bag3 promoter activity in a dose-dependent manner, whereas transfection of the ‘posttranscriptional isoform’, WT1 (þKTS), did not influence the transcriptional activity.