A novel role for E2F4 as a transcriptional activator and a cell cycle driver

E2F4 作为转录激活剂和细胞周期驱动程序的新作用

• 批准号: 9108698
• 负责人: Jenny Hsu
• 金额: $ 3.49万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9108698
• 关键词: Adult; Automobile Driving; Binding; Bioinformatics; Biological Assay; Biology; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Nucleus; Cell model; Cells; Cellular Assay; ChIP-seq; Complex; Data; Development; Down-Regulation; E2F transcription factors; Event; Family; Family member; Gene Activation; Gene Expression; Gene Targeting; Genes; Genomic approach; Goals; Growth; Human; Malignant Neoplasms; Metabolism; Molecular; Mus; Normal Cell; Nuclear; Nuclear Localization Signal; Oncogenes; Organ; Outcome; Pathway interactions; Pluripotent Stem Cells; Population; Proliferating; Property; Protein Family; Research; Retinoblastoma Protein; Role; Somatic Cell; System; Testing; Therapeutic; Time; Tissues; Transcription Coactivator; Tumor Suppressor Proteins; Work; base; cancer cell; cancer therapy; cancer type; cdc Genes; cell type; cofactor; design; embryonic stem cell; innovation; insight; loss of function; novel; novel strategies; outcome forecast; overexpression; pluripotency; prevent; programs; public health relevance; research study; self-renewal; transcription factor; transcriptome sequencing; tumor; tumorigenesis

 DESCRIPTION (provided by applicant): For cancers to grow and thrive, normal cells must undergo changes in cell cycle regulation that allow them to proliferate rapidly and indefinitely. An incomplete understanding of these cell cycle adaptations hinders the development of cancer therapies. The Rb family proteins (Rb, p107, and p130), which regulate cell cycle arrest in normal cells by forming repressive complexes with E2F transcription factors, are rendered inactive in most, if not all, human cancers. Our long-term goal is to understand the molecular events that result in tumorigenesis upon loss of Rb family function. Among the nine E2F transcription factors, E2F4 is the major repressor E2F in adult tissues and organs, where it represses cell cycle genes in association with Rb family members. Here we hypothesize that in contexts in which Rb family proteins are inactive, E2F4 can act as a transcriptional activator driving rapid cell cycle progression. This hypothesis is supported by emerging evidence that E2F4 is highly expressed in multiple cancer types, and is nuclear and bound to some target genes in rapidly proliferating cells. However, we lack a complete understanding of which genes and pathways are regulated by E2F4 as an activator, and it is equally puzzling how E2F4, which lacks a nuclear localization signal, can enter the nucleus and bind to its targets in the absence of functional Rb family members. To investigate non-canonical roles for E2F4, we propose to use mouse embryonic stem cells (ESCs) as a cellular model. ESCs provide a genetically stable and tractable system with remarkable similarities to cancer cells, including functional inactivatio of the Rb family and hyperactivation of genes involved in metabolism and cell cycle progression. In addition, our preliminary studies indicate that E2F4 binds to highly expressed genes in ESCs in an Rb family-independent manner. We also found that loss of E2F4 leads to the downregulation of cell cycle targets. Based on these observations, we first propose to identify all the targets directly activated by E2F4 in ESCs using unbiased genomic approaches (ChIP-Seq and RNA-Seq). Second, we will identify the co- factors that regulate the nuclear localization and transcriptional activity of E2F4 in the absence of the Rb family proteins, using a combination of bioinformatics approaches and molecular assays. Third, we will investigate the role of E2F4 in the long-term self-renewal of ESCs by performing various cellular assays in gain- and loss-of-function experiments. This study will characterize a novel role for a canonical cell cycle repressor and will identify for the first time a functional role for an E2F family member in ESCs. Equally important, we expect that the results of the proposed experiments will directly inform research into the biology of cancer cells and other contexts where E2F4 appears to serve as a transcriptional activator.

 描述（由申请人提供）：对于癌症的生长和茁壮成长，正常细胞必须经历细胞周期调节的变化，使它们能够快速和无限期地增殖。对这些细胞周期适应的不完全理解阻碍了癌症治疗的发展。Rb家族蛋白（Rb、p107和p130）通过与E2 F转录因子形成抑制性复合物来调节正常细胞中的细胞周期停滞，在大多数（如果不是全部）人类癌症中变得无活性。我们的长期目标是了解Rb家族功能丧失后导致肿瘤发生的分子事件。在九种E2 F转录因子中，E2 F4是成人组织和器官中的主要阻遏物E2 F，在成人组织和器官中，它抑制与Rb家族成员相关的细胞周期基因。在这里，我们假设在Rb家族蛋白不活跃的情况下，E2 F4可以作为一个转录激活因子，驱动快速的细胞周期进程。这一假设得到了新证据的支持，即E2 F4在多种癌症类型中高度表达，并且在快速增殖的细胞中是核的并与一些靶基因结合。然而，我们缺乏对E2 F4作为激活剂调节哪些基因和途径的完整理解，同样令人困惑的是，缺乏核定位信号的E2 F4如何在缺乏功能性Rb家族成员的情况下进入细胞核并与其靶点结合。为了研究E2 F4的非经典作用，我们建议使用小鼠胚胎干细胞（ESCs）作为细胞模型。ESC提供了一个遗传稳定和易处理的系统，与癌细胞具有显著的相似性，包括Rb家族的功能失活和参与代谢和细胞周期进程的基因的超活化。此外，我们的初步研究表明，E2 F4以Rb家族独立的方式与ESCs中高表达的基因结合。我们还发现E2 F4的缺失导致细胞周期靶点的下调。基于这些观察，我们首先建议识别所有 使用无偏基因组方法（ChIP-Seq和RNA-Seq）在ESC中直接由E2 F4激活的靶标。其次，我们将使用生物信息学方法和分子测定的组合来鉴定在不存在Rb家族蛋白的情况下调节E2 F4的核定位和转录活性的辅因子。第三，我们将通过在功能获得和丧失实验中进行各种细胞测定来研究E2 F4在ESCs长期自我更新中的作用。这项研究将表征一个典型的细胞周期阻遏物的新作用，并将首次确定E2 F家族成员在ESCs中的功能作用。同样重要的是，我们希望拟议实验的结果将直接为癌细胞生物学和E2 F4似乎作为转录激活因子的其他背景的研究提供信息。