Mapping the indirect p53 gene regulatory network

癌症是一组与年龄相关的疾病，在欧盟每年导致超过120万人死亡，而转录因子p53是癌症发生的中心抑制因子。它通过调节过多的靶基因来控制细胞的增殖和凋亡。尽管在过去的十年中，人们进行了大量的努力，但仍然不清楚p53是如何调控其许多靶基因的，哪些辅助因素是上调或下调所必需的，以及它们的调控对抑制癌症的发生有什么作用。我们对P53基因调控的分子基础的不完整图景仍然是我们全面理解肿瘤抑制的关键障碍。我们假设P53使用几种因素来调节基因调控和控制细胞活性以抑制肿瘤生长；并且这些在P53基因调控网络中的通路贡献了独立的、不同的和必要的基因调控活动。利用我们先前开发的荟萃分析方法，我们确定了新的P53-RFX7信号通路。我们发现，转录因子RFX7控制着一个肿瘤抑制基因网络，以响应P53和压力。在RFX7的案例中，关于其兄弟RFX5的公开可用数据使我们能够确定RFX7是一个关键的转录调节因子，功能在p53下游。然而，这一相当幸运的情况并不能掩盖在p53的S网络中发现新的转录调控因子是困难的，当单独使用公共数据时。为了克服我们以前在p53基因调控网络中识别新的候选因子的方法的局限性，我们建立了识别未知转录因子的方法，并生成了使知情筛选方法成为可能的数据。我们将使用CAGE-SEQ数据来精确定位介导p53依赖的基因调控的启动子区域，并将其与已建立的DNA亲和纯化工作流程结合起来，然后进行质谱分析，以确定与调控DNA片段特异结合的转录因子。从知情的、全面的筛选方法中确定的候选转录因子随后将得到验证和表征。P53下游的新型转录调控因子的功能特征将有助于深入了解它们在P53调控过程中的作用，以及它们对靶基因和生物学过程的影响，而与P53无关。总而言之，该项目的目标是确定参与P53依赖的转录调控的未知转录因子。因此，该项目有助于更好地了解p53基因调控网络，并有助于确定已识别的转录因子在肿瘤抑制中的作用。除了在特定的p53功能障碍病例中获得治疗干预的新候选对象外，该项目还为其他转录网络的全基因组阐明提供了蓝图。

Cancer is a group of age-related diseases that leads to more than 1.2 million deaths per year in the EU, and the transcription factor p53 serves as a central suppressor of carcinogenesis. It controls cell proliferation and apoptosis by regulating a plethora of target genes. Despite intensive efforts in the past decade, it is still unclear how p53 regulates many of its target genes, what co-factors are necessary for up or down-regulation, and what their regulation contributes to the suppression of carcinogenesis. Our incomplete picture of the molecular basis of gene regulation by p53 remains a critical barrier to our overall understanding of tumor suppression. We hypothesize that p53 employs several factors mediating gene regulation and control cell viability to suppress cancer growth; and that these pathways in the p53 gene regulatory network contribute independent, distinct, and essential gene regulatory activities. Using a meta-analysis approach that we developed earlier, we identified the novel p53-RFX7 signaling pathway. We found that the transcription factor RFX7 governs a tumor suppressor gene network in response to p53 and stress. In the case of RFX7, publicly available data on its sibling RFX5 enabled us to identify RFX7 as a critical transcriptional regulator that functions downstream of p53. However, this rather lucky circumstance cannot conceal that the discovery of new transcriptional regulators in p53’s network is difficult when working with public data alone. To overcome the limitation of our previous approach to identify novel candidate factors in the p53 gene regulatory network, we established methods to identify uncharted transcription factors and generated data that enables an informed screening approach. We will use CAGE-seq data to pinpoint promoter regions that mediate p53-dependent gene regulation and combine it with an established workflow of DNA-affinity purification followed by mass spectrometry to identify transcription factors that specifically bind the regulatory DNA fragments. Candidate transcription factors identified from the informed, comprehensive screening approach will subsequently be validated and characterized. The functional characterization of novel transcriptional regulators that operate downstream of p53 will provide insights into their contribution to p53-regulated processes as well as to their effect on target genes and biological processes irrespective of p53. Together, the goal of this project is to identify uncharted transcription factors contributing to p53-dependent transcriptional regulation. Thus, the project contributes to a better understanding of the p53 gene regulatory network and helps to determine the roles of the identified transcription factors in tumor suppression. In addition to acquiring new candidates for therapeutic intervention in the specific case of p53 malfunction, the project also serves as a blueprint for the genome-wide elucidation of other transcriptional networks.