Dual promoter regulation of death-associated protein kinase gene leads to differentially silenced transcripts by methylation in cancer. Carcinogenesis 2009

ow nloaded from Death associated protein kinase (DAPK), a mediator of apoptotic systems, is silenced by promoter hypermethylation in lung and breast tumors. This gene has a CpG island extending 2500 bp from the translational start site; however, studies characterizing its transcriptional regulation have not been conducted. Two transcripts for DAPK were identified that code for a single protein, while being regulated by two promoters. The previously identified DAPK transcript designated exon 1 transcript was expressed at levels 3-fold greater than the alternate exon 1b transcript. Deletion constructs of promoter 1 identified a 332 bp region containing a functional CP2 binding site important for expression of the exon 1 transcript. While moderate reporter activity was seen in promoter 2, the region comprising intron 1 and containing a HNF3B binding site sustained expression of the alternate transcript. Sequencing the DAPK CpG island in tumor cell lines revealed dense, but heterogeneous methylation of CpGs that blocked access of the CP2 and HNF3B proteins that in turn, was associated with loss of transcription that wa