Identification And Analysis Of Human PRR11 And FAM33A Bidirectional Promoter

PRR11 (Proline-rich protein 11, PRR11) is a newly discovered tumor-related genes, which plays an important role in cell proliferation, cell cycle and other biological processes. Bioinformatics analysis indicated that the gene's upstream adjacent gene FAM33A (family with sequence similarity 33 A) also is a tumor-associated genes which involved in cell proliferation and cell cycle, and the transcription of these two gene is opposite directions, so we suggesting PRR11 and FAM33A genes may share a bidirectional promoter .In our preliminary work, we identified PRR11 and FAM33A transcription start site by 5' RACE (rapid amplification of cDNA ends) and bioinformatic analysis, then several overlapping genomic fragments from the 5'- flanking region of PRR11 and FAM33A gene were coloned into pGL3-basic vector to construct PRR11 and FAM33A promoter reporters. Promoter activity analysis showed that, PRR11 gene may have a promoter, FAM33A genes may have two promoters, one promoter and PRR11 gene are overlap, mainly located between the PRR11-P1496 and the PRR11-P594 (a region of -563 bp to +341 bp around the major transcriptional start site), suggesting PRR11 and FAM33A genes share a bidirectional promoter, which as a complete transcription unit is regulated. Transcription factor binding site prediction analysis software indicated that, PRR11 and FAM33A gene promoter region contains a typical GC box, and E2F, Sp1, Myb, and GATA-1 and other potential transcription factor binding sites, suggesting that E2F and Sp1 transcription factors may be involved in P PRR11 and FAM33A gene transcription regulation.To further investigate PRR11 and FAM33A genes' transcriptional regulatory mechanism and biological function, and find out the core bidirectional promoter region, based on the previous work we further identificate and analysis PRR11 and FAM33A genes' promoter region. At first construct a luciferase reporter named pGL3b-FAM33A-P688 of FAM33A gene promoter R1269/F1959 region by PCR method; then use pGL3b-PRR11- P1496 as template, which was constructed in previous work, construct a luciferase reporter by site-directed mutagenesis method, named pGL3b-PRR11-P788. Then based on pGL3b-FAM33A-P688 and PGL3b-PRR11-P788, we construct four luciferase reporter, which are pGL3b-FAM33A-P474, pGL3b-FAM33A-P436, pGL3b-PRR11-P574 and pGL3b-PRR11-P536, by site-directed mutagenesis method. Furthermore, use the four recombinant as templates, the same method construct pGL3b-FAM33A-P211, pGL3b-FAM33A-P80, pGL3b-PRR11-P211, and pGL3b-PRR11-P80. Luciferase reporter assay indicate that all of the promoter reporters have strong activities, further evident that in the PRR11 and FAM33A gene promoter overlap exist bidirectional promoter sequence can also control the transcription of the two genes, and common sequence in PRR11-P80 (1521-1600) and FAM33A-P80 (1521-1600) is the core bidirectional promoter region.