Identification Of The Distinct Promoters For The Two Transcripts Of Apoptosis Related Protein 3 And Their Transcriptional Regulation By NFAT And NFκB

Gene expression is essential for cell differentiation and development. Regulation of individual gene is vital for its function under certain circumstances. Gene structure is the determinant of the regulation of expression, in which transcriptional regulation is dominant. To this end, elucidating the structure is important for the study of the function of a novel gene.Human APR3 (apoptosis related protein 3,GeneID: 51347) was first cloned in our laboratory from HL60 cells treated with ATRA to induce cell differentiation and apoptosis. APR3 is a novel gene highly conserved across species. According to the data in NCBI, there are two transcripts of this gene (NM₀16085.3 and NM₀80592.2), which are assumed to arise from alternative splicing. Analysis of the APR3 related mRNA sequence submitted to NCBI by different labs revealed that BI 194121.1 and CR 600041 had the longest 5'end corresponding to the two transcripts. Interestingly, an about 320 bp fragment at the 5' end of the gene is present in CR 600041 while absent in BI 194121.1, suggesting the possibility of different transcription start site of the two transcripts. In other words, an about 320 bp fragment at the 5' end of the gene is present in CR 600041 while absent in BI 194121.1, suggesting the possibility of different transcription start site of the two transcripts. To test this possibility, we used two sets of primers (FP3 plus RP3; FP4 plus RP3) to amplify APR3 from the cDNA of HL60. In view of these, we confirmed that the two transcripts start with different transcription start site by PCR data using different sets of primers and we have identified the distinct promoters responsible for the two transcripts by reporter assay. Results from series of depletion have localized the promoters for the two transcripts to -245/-173 and -113/+148 respectively.Though no literature about APR3 has been reported, analysis of the data about APR3 available at GEO profiles sheds light on its function. Currently, totally 307 records referred to APR3 were accessible at GEO profiles and about 10 records revealed consistent and significant changes of APR3 under the experimental treatments. Most of them were differentiation or inflammation associated. It was reported that sustained activation of NFAT was involved in the process of T cell differentiation. NFκB is also extensively involved in the development of T cells and B cells, and its primary function is to ensure lymphocyte survival at various developmental stages. Besides, NFκB are also activated under the irradiation and DNA damage circumstances. LPS can activate NFκB through TLR in immune cells. Thus, it is fascinating to explore the regulations of APR3 promoter under NFAT and NFκB. Consistent with the potential role of APR3 in developmental and inflammatory process, bioinformatics analysis found that the 5'flanking regulatory region contains putative binding sites for NFκB and NFAT. In this study, we have characterized the response of the two transcripts to transcription factor NFAT and NFκB. Our results suggest that NFAT and/or NFκB activation might be responsible for the altered expression of APR3 in these processes and this novel gene may be functionally important in these processes.In summary, here we have identified the two separate promoters of APR3 for the two transcripts and characterized their transcriptional regulations by NFAT and NFκB, which may explain some of the altered expressions reported at GEO profiles and propose the importance of APR3 under these conditions.