Abstract 3505: Regulation of Cyclin A1 promoter by the Wilms tumor gene, WT1

Cyclin A1(CCNA1) is a unique type of Cyclin A expressed predominantly in highly proliferative tissues such as the testis and bone marrow. In hematopoietic cells CCNA1 expression has been observed in CD34+ progenitor cells, leukemia stem cells and leukemia cell lines. Similarly, Wilms tumor 1 (WT1) gene expression has been observed in these highly proliferative, undifferentiated cells. Regulation of CCNA1 expression is poorly understood, but the CCNA1 gene promoter is a highly GC-rich TATA-less sequence, containing three potential WT1 binding sites. Chromatin immunoprecipitation analysis of two of the three sites revealed binding of the WT1 protein to chromatin in K562 leukemia cells. To assess the role of WT1 in CCNA1 regulation, activation of the CCNA1 promoter by WT1 was measured using luciferase reporter constructs in K562 cells. The distal construct (-1180/+145 ) contains two potential WT1 sites, and the activity of this reporter construct was up-regulated by WT1 co-transfection. In contrast, the proximal (-454/+145) promoter region contains no canonical WT1 binding sites. Suprisingly, the proximal promoter was also up-regulated by WT1 co-transfection. Transcription factor binding site analysis revealed 6 GC boxes -within the core promoter region of the proximal promoter construct that we predicted could serve as non-canonical WT1 binding sites. To determine the importance of these GC boxes for WT1 binding, we treated K562 cells with Mithramycin A, which blocks binding at GC rich sequences. We found that Mithramycin A treatment decreased the activity of both the distal and proximal promoter constructs. Finally we demonstrated that WT1 mediated activation of CCNA1 promoter required an intact DNA binding domain, as a mutant WT1 expression construct lacking the zinc finger domain failed to activate any of the CCNA1 promoter constructs. Overall these results show that WT1 binding can up-regulate the CCNA1 promoter. This observation supports our previous finding that overexpressing WT1 in K562 cells increased CCNA1 mRNA. These data suggest that elevated WT1 expression could mediate increased CCNA1 expression in leukemia cells and thereby enhance proliferation. Overall, our results are consistent with the hypothesis that both WT1 and CCNA1 behave as oncogenes in leukemia. Citation Format: Sony Pandey, Mustafa Moazam, Steven J. Kuerbitz, Gail C. Fraizer. Regulation of Cyclin A1 promoter by the Wilms tumor gene, WT1. [abstract]. In: Proceedings of the 105th An