NFATc1 regulates the transcription of DNA damage-induced apoptosis suppressor

DNA damage induced apoptosis suppressor (DDIAS), or human Noxin (hNoxin), is strongly expressed in lung cancers. DDIAS knockdown induced apoptosis in non-small cell lung carcinoma A549 cells in response to DNA damage, indicating DDIAS as a potential therapeutic target in lung cancer. To understand the transcriptional regulation of DDIAS, we determined the transcription start site, promoter region, and transcription factor. We found that DDIAS transcription begins at nucleotide 212 upstream of the DDIAS translation start site. We cloned the DDIAS promoter region and identified NFAT2 as a major transcription factor (Im et al., 2016 [1]). We demonstrated that NFATc1 regulates DDIAS expression in both pancreatic cancer Panc-1 cells and lung cancer cells.


Value of the data
These data provide information regarding the transcription site and promoter used to control DDIAS transcription.
NFATc1 plays a crucial role in DDIAS transcription in lung cancer cells. NFATc1 also controls DDIAS expression in the pancreatic cancer cell line PANC-1, as demonstrated by growth inhibition in a knockdown model.

Data
A 5 0 -rapid amplification of cDNA ends (RACE) assay revealed a transcription start site located 212 nucleotides upstream of the DDIAS translation start site or 32 base pairs (bp) downstream of the site reported in the National Center for Biotechnology Information (NCBI) database ( Fig. 1). This transcription start site was confirmed via RNA polymerase binding in a chromatin immunoprecipitation (ChIP) assay (Fig. 2). Putative transcription factor binding sites are located in the P3 region of the DDIAS promoter (Fig. 3A). In a P3-luciferase reporter assay, only NFATc1 knockdown significantly suppressed the promoter activity ( Fig. 3B and C). DDIAS mRNA expression was found to be regulated by NFAT signaling, as shown by treatment with cyclosporine A (CsA), a calcineurin inhibitor, phorbol 12-myristate 13-acetate (PMA), and the calcium ionophore A23187 (Fig. 4). Treatment with PMA andA23187 increased the cellular expression of DDIAS mRNA (Fig. 4A). In contrast, the cellular DDIAS mRNA level decreased after treatment with CsA (Fig. 4B). Previously, we showed that DDIAS knockdown inhibits the growth of lung cancer cells [2]. In the pancreatic cancer cell line PANC-1, NFATc1 knockdown clearly induces DDIAS depletion and results in growth inhibition, indicating the NFATc1mediated control of DDIAS expression (Fig. 5).

Experimental design, materials and methods
2.1. Determination of the transcription start site using 5 0 rapid amplification of cDNA ends (RACE) in HEK293 cells To identify the 5 0 UTR of human DDIAS, 5 0 RACE was performed using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) according to the manufacturer's protocol. Firststrand cDNA was synthesized using RNA from human embryonic kidney 293 (HEK293) cells. 5 0 -RACE PCR was performed using the Advantage 2 PCR kit (Clontech) and the following gene-specific primers: R1; 5 0 -TGTACCTGTGCAAACCAGTGGCAGT-3 0 or R2; 5 0 -AAAGCCTGCAATACCTGGGTCTGGT-3 0 . The PCR products were separated by electrophoresis on 1.5% agarose gels. After purification using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). PCR products were subcloned into the pGEM-T vector using the TA Cloning Kit (Promega, Madison, WI, USA) and sequenced. The first base pair after the adapter was identified as the transcription start site of the human DDIAS gene [1]. ChIP assay of RNA polymerase (ser5) in HEK293 cells. RNA polymerase II (phospho-ser5) binding to the DDIAS promoter in HEK293 cells. A ChIP assay was performed using an anti-RNA pol II antibody. An anti-IgG antibody was used as a negative control. PCR amplification was performed using the indicated DDIAS promoter-specific primers or control primers specific for GAPDH.

Luciferase reporter assays
Cells were co-transfected with the DDIAS promoter ( À1205) and pRL-TK, using 2 μl of Polyfect (Qiagen, USA) per well. Cells were harvested 36 h after transfection and lysed in 1 Â passive lysis buffer. Firefly and Renilla luciferase activities were assayed using a dual-luciferase reporter assay system (Promega, USA) and a luminometer (Victor X Light; Perkin Elmer, USA). Firefly luciferase activity was normalized to Renilla luciferase activity and expressed as the relative luciferase activity (RLA) to reflect promoter activity.

Cell viability
Cell viability was determined using the sulforhodamine B assay, as previously described [4]. The amount of sulforhodamine B dye bound to the cell matrix was quantified using a spectrophotometer at 530 nm.