PRR11-FAM33A Bidirectional Promoter Is Negatively Regulated By P53through NF-Y

PRR11(proline-rich protein11) and it’s upstream adjacent gene,FAM33A (Family with sequence similarity,33member A), are twocancer-related genes, which were identified recently by our research groupand Nigg research group, respectively. They participate in cell proliferationand cell cycle. More importantly, they closely arranged in the chr17q23with a unique head-to-head way, that is a unique typical bidirectionalpromoter. Previously we have identified and analyzed this bidirectionalpromoter, and found that the core promoter may located in a region of80bp. Based on these study, we further explore the important cis-actingelements in the core PRR11-FAM33A bidirectional promoter region,especially p53and NF-Y.(1) Analysis of the important cis-acting elements in the corePRR11-FAM33A promoter regionBy analysis of transcription factor binding site and nucleotidesequence homology, we found that the core bidirectional promoter regioncontains a typical GC box, two CCAAT boxes, and two potentialtranscription factor binding sites of Myb, which were highly concerned in human, rat and mouse. The results strongly suggest that these transcriptionfactors play an important role in the transcriptional regulation of thePRR11-FAM33A bidirectional promoter.(2) Analysis of site-directed mutagenesis in the corePRR11-FAM33A bidirectional promoter regionIn order to confirm the bioinformatics analysis, site-directedmutagenesis was used to analyze the bidirectional promoter. Firstly, basedon the luciferase reporter gene construction PRR11-P80and FAM33A-P80,a series of mutants were constructed which have mutated binding sites forNF-Y, Sp1or Myb. Then, the Dual-Luciferase reporter assay was used tomeasure the promoter activity of the mutants. The results demonstrated thatpromoter activity of all the mutants were significantly reduced compared tothe wild-type construction, which significantly indicated that NF-Y, Sp1and Myb play an important role in the transcriptional regulation of thePRR11-FAM33A bidirectional promoter.(3) The regulation of PRR11-FAM33A bidirectional promoterdirectly by NF-YReporter gene assays with over-expression or knockdown of NF-Yexpression indicated that NF-Y may directly regulate PRR11-FAM33Abidirectional promoter. Firstly, co-transfection experiments in H1299cellswere carried out with a series of deletions and mutants of PRR11-FAM33Apromoter reporter gene constructions and pcDNA3.1-NF-Y. The results showed that over-expression of NF-Y could significantly increased thewild-type bidirectional promoter activity, but not to the mutants of NF-Ybinding site, suggesting that NF-Y could directly regulate the activities ofPRR11-FAM33A bidirectional promoter through interacting with theCCAAT box. Secondly, knockdown of NF-YB by siRNA experiment couldinhibit the expression of PRR11and FAM33A in mRNA level byReal-Time PCR.Then, ChIP assays and EMSA also confirmed that NF-Ycould directly bind to the core PRR11-FAM33A bidirectional promoter invivo and in vitro. So, these results indicated that NF-Y could directlyregulate PRR11-FAM33A bidirectional promoter, and it could beconsidered that PRR11and FAM33A were two newly NF-Y targeted gene.(4) p53can inhibit the expression of PRR11and FAM33A throughinterfering with NF-YFirst, PRR11-FAM33A bidirectional promoter constructions wereco-transfected with p53wildtype, p53mutant, and NF-Y wildtype.Reporter gene assays demonstrated that over-expression of wild-type p53could obviously repress the bidirectional promoter activity, however,mutated-type p53could not. Over-expression of wildtype p53could inhibitthe promoter activity when NF-Y was also over-expressed, and thisinhibition was significantly reduced or even disappeared when NF-Ybinding site in the bidirectional promoter region was mutated. The resultssuggest that p53could inhibit the activities of PRR11-FAM33A bidirectional promoter through NF-Y-dependent. Second, the endogenousexpression of PRR11and FAM33A were detected when NF-YB siRNAand/or wild-type p53plasmid were co-transfected into H1299cells.Quantitative RT-PCR and analysis illustrated that wild-type p53candecrease the endogenous expression of PRR11and FAM33A both inmRNA and protein levels, however, these effects were obviously depletedwhen NF-Y was inhibited. Third, p53could interact with NF-Y byco-immunoprecipitation experiments. Fourth, ChIP assays demonstratedthat more p53and less NF-Y was recruited to the bidirectional promoterwhen p53was over-expressed. In conclusion, our data suggest that p53canrepress the expression of PRR11and FAM33A via interfering with NF-Y.In summary, we found that some important transcription factorbinding sites such as NF-Y were located in the core PRR11-FAM33Abidirectional promoter region, confirmed that NF-Y can directly regulatethe activities of PRR11-FAM33A bidirectional promoter, and verified thatwildtype p53may indirectly inhibit the activities through interfering withNF-Y. This study not only provide theoretical basis for further exploringthe role of PRR11and FAM33A in cell cycle and carcinogenesis, but alsocontribute greatly to the theory of molecular oncology and transcriptionalregulation especially regarding bidirectional promoter...