Deciphering p53-regulated alternative transcription and splicing

确定遗传变异产生表型差异影响肿瘤发展的机制是癌症研究的主要目标。在这方面的努力以前主要集中在通过表观遗传和转录调控改变的mRNA水平。然而，最近的研究表明，改变异构体丰度也是癌症发生中的一种常见现象。大多数人类基因产生多个转录本，这些转录本可能编码具有不同甚至拮抗生物学功能的蛋白质。重要的是，产生这种异构体多样性的两个主要机制，即。辅助转录起始点(TSS)的选择性剪接和选择性转录受转录因子(Tf)调控。例如，已有研究表明，剪接可能取决于转录因子驱动的启动子活性，而转录因子驱动的启动子活性与延伸率有关。此外，转录因子能够通过控制剪接调节因子的表达来改变基因组剪接的格局。通过与交替启动子或增强子结合，转录因子甚至可以更直接地诱导交替转录。值得注意的是，DNA甲基化似乎影响了替代启动子的使用。虽然已知转录因子可以影响特定细胞中的异构体景观，但详细的潜在分子机制在很大程度上仍未解决。P53信号通路是人类癌症中最突出的调节异常的例子之一，但P53信号对mRNA异构体丰度的影响在很大程度上尚不清楚。这个研究计划的总体目标是建立一系列的方案和方法来分析转录因子(TF)P53对表观基因组和转录组的影响。在这里，我们特别关注的假设是，转录因子p53调节选择性剪接，并可能导致使用直接或间接靶基因的替代转录起始点。由此产生的改变可能与P53‘S抑癌基因的功能有关。我们建议结合一些下一代测序实验和分析策略来评估P53依赖的异构体丰度、P53依赖的转录启动、P53诱导的表观基因组变化和P53依赖的选择性剪接。我们试图更深入地了解转铁蛋白介导的异构体及其潜在的机制，从而为简化未来的研究工作提供资源。在更广泛的范围内，拟议的研究为全面研究转铁蛋白在表观基因组和转录水平上的影响提供了蓝图。这里开发的生物信息学解决方案将转录组的数量和质量变化与染色质构象和DNA甲基组的变化联系起来，将促进未来的类似研究。

The determination of mechanisms by which genetic variation produces phenotypic differences affecting tumor development is a primary goal of cancer research. Efforts in this respect have previously focused on mRNA levels altered through epigenetic and transcriptional regulation. Recent studies, however, show that altering isoform abundance is also a common phenomenon in cancer development. The majority of human genes give rise to multiple transcripts that may code for proteins with diverse or even antagonistic biological functions. Importantly, the two major mechanisms that produce this isoform diversity, ie. alternative splicing and alternative transcription via auxiliary transcription start sites (TSS) are regulated by transcription factors (TF). For instance, it has been shown that splicing may depend on transcription factor-driven promoter activity, which is coupled to the elongation rate. In addition, transcription factors are able to alter the genomic splicing landscape by controlling the expression of splicing regulators. Via binding to alternate promoters or enhancers, transcription factors may induce alternative transcription even more directly. Notably, DNA methylation appears to influence alternative promoter usage. Although it is known that transcription factors can influence the isoform landscape in a given cell, the detailed underlying molecular mechanisms are largely unresolved. The p53 signaling pathway is one of the most prominent examples of dysregulation in human cancer, but the consequences of p53 signaling on mRNA isoform abundance are largely unexplored. The overall objective of this research program is to establish a series of protocols and methods to analyze the influence of the transcription factor (TF) p53 on the epigenomic and transcriptomic landscape. Here, we specifically focus on the hypothesis that the transcription factor p53 regulates alternative splicing and may lead to the use of alternative transcription start sites of direct or indirect target genes. The resulting alterations may contribute to p53’s tumor suppressor function. We propose to combine a number of next-generation sequencing experiments and analysis strategies to assess p53-dependent isoform abundance, p53-dependent transcription initiation, p53-induced epigenomic changes, and p53-dependent alternative splicing. We seek to obtain a deeper understanding of TF-mediated isoforms and their underlying mechanisms and will thereby provide resources to simplify future research endeavors. On a broader scale, the proposed research provides a blueprint for comprehensively investigating the effect of TFs on the epigenomic and transcriptomic level. The bioinformatics solutions developed here to link quantitative and qualitative changes of the transcriptome with changes in thechromatin conformation and DNA methylome will facilitate similar research in the future.