Fiorino Antonio, Manenti Giacomo, Gamba Beatrice, Bucci Gabriele, De Cecco Loris, Sardella Michele, Buscemi Giacomo, Ciceri Sara, Radice Maria T, Radice Paolo, Perotti Daniela
Department of Predictive & Preventive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Italy.
Department of Predictive & Preventive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Italy.
Int J Biochem Cell Biol. 2016 Sep;78:162-172. doi: 10.1016/j.biocel.2016.07.013. Epub 2016 Jul 15.
Retina-derived POU domain Factor 1 (RPF-1), a member of POU transcription factor family, is encoded by POU6F2 gene, addressed by interstitial deletions at chromosome 7p14 in Wilms tumor (WT). Its expression has been detected in developing kidney and nervous system, suggesting an early role for this gene in regulating development of these organs. To investigate into its functions and determine its role in transcriptional regulation, we generated an inducible stable transfectant from HEK293 cells. RPF-1 showed nuclear localization, elevated stability, and transactivation of promoters featuring POU consensus sites, and led to reduced cell proliferation and in vivo tumor growth. By addressing the whole transcriptome regulated by its induction, we could detect a gross alteration of gene expression that is consistent with promoter occupancy predicted by genome-wide Chip-chip analysis. Comparison of bound regulatory regions with differentially expressed genes allowed identification of 217 candidate targets. Enrichment of divergent octamers in predicted regulatory regions revealed promiscuous binding to bipartite POUS and POUH consensus half-sites with intervening spacers. Gel-shift competition assay confirmed the specificity of RPF-1 binding to consensus motifs, and demonstrated that the Ser-rich region upstream of the POU domain is indispensable to achieve DNA-binding. Promoter-reporter activity addressing a few target genes indicated a dependence by RPF-1 on transcriptional response. In agreement with its expression in developing kidney and nervous system, the induced transcriptome appears to indicate a function for this protein in early renal differentiation and neuronal cell fate, providing a resource for understanding its role in the processes thereby regulated.
视网膜源性POU结构域因子1（RPF-1）是POU转录因子家族的成员，由POU6F2基因编码，在肾母细胞瘤（WT）中7号染色体p14区域的间质缺失中被发现。已在发育中的肾脏和神经系统中检测到其表达，表明该基因在调节这些器官的发育中起早期作用。为了研究其功能并确定其在转录调控中的作用，我们从HEK293细胞中生成了一种可诱导的稳定转染子。RPF-1显示出核定位、稳定性增强以及对具有POU共有位点的启动子的反式激活，并导致细胞增殖减少和体内肿瘤生长受抑制。通过分析其诱导调控的整个转录组，我们检测到基因表达的总体变化，这与全基因组芯片分析预测的启动子占据情况一致。将结合的调控区域与差异表达基因进行比较，可鉴定出217个候选靶标。预测调控区域中不同八聚体的富集揭示了其与具有间隔序列的二分体POUS和POUH共有半位点的混杂结合。凝胶迁移竞争试验证实了RPF-1与共有基序结合的特异性，并表明POU结构域上游富含丝氨酸的区域对于实现DNA结合是必不可少的。针对一些靶基因的启动子-报告基因活性表明RPF-1对转录反应的依赖性。与其在发育中的肾脏和神经系统中的表达一致，诱导的转录组似乎表明该蛋白在早期肾脏分化和神经元细胞命运中具有功能，为理解其在由此调控的过程中的作用提供了资源。
