Deciphering p53-regulated alternative transcription and splicing

确定遗传变异产生影响肿瘤发展的表型差异的机制是癌症研究的主要目标。在这方面的努力，以前集中在mRNA水平的改变，通过表观遗传和转录调控。然而，最近的研究表明，改变异构体丰度也是癌症发展中的常见现象。大多数人类基因产生多个转录本，这些转录本可以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种主要机制，即。选择性剪接和通过辅助转录起始位点（TSS）的选择性转录受转录因子（TF）调节。例如，研究表明剪接可能取决于转录因子驱动的启动子活性，而启动子活性又与延伸率相关。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景观。通过与替代启动子或增强子结合，转录因子可以更直接地诱导替代转录。值得注意的是，DNA甲基化似乎影响替代启动子的使用。虽然已知转录因子可以影响给定细胞中的同种型景观，但详细的潜在分子机制在很大程度上尚未解决。p53信号通路是人类癌症中失调的最突出的例子之一，但是p53信号通路对mRNA同种型丰度的影响在很大程度上未被探索。本研究计划的总体目标是建立一系列的协议和方法来分析转录因子（TF）p53对表观基因组和转录组景观的影响。在这里，我们特别关注的假设，转录因子p53调节选择性剪接，并可能导致使用的直接或间接的靶基因的选择性转录起始位点。由此产生的改变可能有助于p53的肿瘤抑制功能。我们建议结合联合收割机的下一代测序实验和分析策略，以评估p53依赖的异构体丰度，p53依赖的转录起始，p53诱导的表观基因组变化，和p53依赖的选择性剪接。我们寻求获得TF介导的异构体及其潜在机制的更深入的了解，从而提供资源，以简化未来的研究工作。在更广泛的范围内，拟议的研究为全面调查TF对表观基因组和转录组水平的影响提供了蓝图。这里开发的生物信息学解决方案将转录组的定量和定性变化与染色质构象和DNA甲基化组的变化联系起来，这将促进未来类似的研究。

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.