Abstract 2180: Identification of nuclear transcription factor Y (NF-Y) as a novel key transcriptional regulator of the tumor-suppressor gene DAPK1

Abstract

Abstract The death-associated protein kinase 1 (DAPK1) belongs to a family of multifunctional Ser/Thr-kinases that plays a crucial role in the mediation of cell death, autophagy and tumor suppression. Loss or reduced expression of DAPK1 occurs in the majority of patients with chronic lymphocytic leukemia (CLL) and many other non-hematological tumors commonly involving epigenetic silencing. To identify novel key DAPK1 transcriptional regulators, we conducted an siRNA screen in HEK293 cells using a custom library targeting over 60 individual genes with known or predicted DNA-binding function and general potential to modulate transcription. Candidate gene selection for siRNA-mediated knock-down was based on previous genome-wide siRNA screening experiments and in silico prediction of binding sites at the DAPK1 promoter region. As endogenous readout, DAPK1 mRNA expression was monitored by qRT-PCR.Using this strategy, we identified nuclear transcription factor Y (NF-Y) as the top positive regulator of DAPK1 that reduces its expression down to 55% of the level determined in untargeted control siRNA-treated cells. NF-Y is a well known transcriptional regulator of genes involved in cell cycle control, apoptosis and stress response. NF-Y consists of 3 subunits, NF-YA, -YB and -YC, forming a trimeric complex which is essential for specific binding of CCAAT boxes located proximal to the transcriptional start sites of genes. In addition to HEK293 cells, we were able to diminish DAPK1 mRNA expression upon siRNA-mediated NF-Y knockdown in a panel of 6 different cancer cell lines. Conversely, co-overexpression of NF-YA,-YB and -YC in HEK293 cells leads to increased expression levels of DAPK1. To investigate whether NF-Y contributes directly or indirectly to DAPK1 transcriptional regulation, DAPK1 luciferase reporter constructs harboring the predicted NF-Y binding site were generated. Point mutation of the reverse CCAAT-box in the DAPK1 promoter constructs significantly reduces the promoter activity down to 60% of the wild-type promoter. Furthermore, NF-Y knockdown significantly reduces the activity of the wild-type DAPK1 luciferase construct down to 55% in reporter assays. Strikingly, the CCAAT-box mutated constructs do not show altered activity after NF-Y depletion, suggesting a direct regulation of DAPK1 by NF-Y. Taken together, these results strongly indicate a direct role for NF-Y in the regulation of basal DAPK1 transcription levels, and also propose a novel mechanism for aberrant transcriptional silencing of DAPK1 in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2180. doi:1538-7445.AM2012-2180