Mechanisms of gene expression control in the p53 network

p53网络中基因表达控制的机制

• 批准号: 9732737
• 负责人: Joaquin M. Espinosa
• 金额: $ 16.5万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9732737
• 关键词: Adopted; Affect; Aging; Aneuploidy; Apoptosis; Apoptotic; Area; Autophagocytosis; Award; Binding; Binding Sites; Bioinformatics; Biological Assay; CCCTC-binding factor; Cancer cell line; Cell Cycle Arrest; Cell Death; Cell Separation; Cells; ChIP-seq; Chromatin; Cohort Studies; Comorbidity; Complex; Congenital chromosomal disease; DNA Binding; DNA-Binding Proteins; Deposition; Development; Disease; Dominant-Negative Mutation; Down Syndrome; ETS2 gene; Enhancers; Environment; Epigenetic Process; Fibroblasts; Funding; Future; Gene Cluster; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Histone Deacetylation; Histone H3; Histones; Human; Hyperactive behavior; Hypersensitivity; ISG15 gene; Immune system; Individual; Inflammatory; Interferon Activation; Interferon Receptor; Interferon Type I; Interferons; Investigation; Knowledge; Libraries; Life Expectancy; Ligands; Link; Longevity; Lysine; MDM2 gene; Malignant Epithelial Cell; Malignant Neoplasms; Measurement; Mediating; Mediator of activation protein; Medical; Mission; Molecular; Mutation; Nerve Degeneration; Nuclear; Oncogenes; PRC1 Protein; Parents; Pathway interactions; Phenotype; Phosphotransferases; Plasma; Polycomb; Population; Protein p53; Proteins; Proteomics; Publishing; Puma; RNA chemical synthesis; Receptor Gene; Regulation; Repression; Research; Research Project Grants; Role; STAT1 gene; Signal Transduction; Skin; Solid; Squamous cell carcinoma; Stimulus; Stress; Structure; Syndrome; TP53 gene; Techniques; Testing; Therapeutic; Transactivation; Transcript; Transcriptional Regulation; Tumor Suppression; Tumor Suppressor Proteins; United States National Institutes of Health; Untranslated RNA; Validation; Variant; addiction; cancer cell; cell type; cofactor; cytokine; deep sequencing; demethylation; design; epidemiology study; exhaustion; experimental study; flexibility; gene repression; genome-wide; global run on sequencing; in vivo; induced pluripotent stem cell; knock-down; leukemia; leukemogenesis; mouse model; multiple omics; novel; novel therapeutics; nutlin 3; overexpression; programs; promoter; response; senescence; small hairpin RNA; small molecule inhibitor; targeted treatment; therapy development; transcription factor; transcriptome sequencing; tumor; tumorigenesis

Trisomy 21 (T21) is the most common human chromosomal disorder, leading to the condition known as Down syndrome (DS) (1, 2). A remarkable observation enabled by the significant increase in the life expectancy of people with DS is that T21 protects these individuals from some medical conditions, while strongly predisposing them to others, providing a strong rationale for the NIH INCLUDE project (INvestigation of Co-occurring conditions across the Lifespan to Understand Down syndromE). In particular, and related to the mission of the NCI, epidemiological studies have revealed that people with DS display significantly lower rates of solid malignancies (3, 4), while being highly predisposed to many types of leukemias (5-8). Despite the obvious potential of the population with DS to advance our understanding of tumorigenesis and leukemogenesis, little is known about the molecular and cellular mechanisms by which T21 causes this differential ‘malignancy spectrum’. Clearly, research in this area will benefit not only people with DS, but also the typical population at large. Consistent with a key area of emphasis of the INCLUDE project (immune system dysregulation), we recently discovered that T21 causes constitutive activation of the Interferon (IFN) response across diverse cell types (9), which is likely due to the fact that four of the six IFN receptors (IFNRs) are encoded on chr21 (10). Accordingly, T21 cells are hypersensitive to IFN ligands, display hyperactivation of JAK/STAT signaling, and overexpression of IFN-Stimulated Genes (ISGs) (9-12). Furthermore, in a large plasma proteomics cohort study, we identified dozens of inflammatory cytokines with mechanistic links to IFN signaling that are dysregulated in people with DS (13). Strikingly, the IFN response was recently identified as a core pathway dysregulated in multiple mouse models of DS carrying triplication of the IFNR gene cluster (14). Additionally, several lines of evidence indicate that T21 may increase activity of the tumor suppressor p53 in both IFN-dependent and -independent ways. IFN-independent mechanisms could include aneuploidy itself, which is a known p53-activating stimulus (15), and the overexpression of chr21-encoded proteins, such as ETS2, a transcription factor, and DYRK1A, a kinase, both of which stimulate p53 activity (16, 17). IFN-dependent mechanisms include direct induction of the p53 promoter by Type I IFNs (18), interaction of the IFN-activated transcription factor STAT1 with p53 to enhance p53 activity (19), and three ISGs induced by T21 -ISG15, IFI16 and PYHIN1- that enhance p53 activity by diverse mechanisms (20-23). Altogether, these observations support the hypothesis that T21 can enhance the activity of the p53 network in people with DS, with potentially beneficial and deleterious effects (e.g. enhanced tumor suppression versus accelerated aging, senescence, and neurodegeneration). Therefore, consistent with Component 1 of the INCLUDE project, we hypothesize that hyperactive IFN signaling contributes to the different disease spectrum in people with DS. More specifically, within the scope of the parent NCI R01 award, we propose to investigate the interplay between T21, IFN signaling, and the p53 network. Our Specific Aims for this Supplement are: 1. To define the impact of trisomy 21 and IFN hyperactivity on the p53 transcriptional program. With NIH funding, our team was the first to employ genome-wide measurements of nascent RNA synthesis (i.e. GRO-seq) to identify the direct p53 transcriptional program using a small molecule inhibitor of the p53-MDM2 interaction called Nutlin (24, 25). Related to Aim 1 of the parent R01, we will use our published multi-omics pipeline combining p53 ChIP-seq, GRO-seq, and RNA-seq, to test the hypothesis that T21 enhances the p53 transcriptional program. We will use an available panel of iPSCs with and without T21 as a paradigm to identify quantitative (i.e. global) and/or qualitative (i.e. gene-specific) impacts of T21 on p53 signaling, and to define to what degree these changes are driven by enhanced JAK/STAT signaling. 2. To define the impact of trisomy 21 and IFN hyperactivity on the cellular response to non-genotoxic p53 activation in diverse cell types. With funding provided by the parent award, we completed an exhaustive characterization of the p53 signaling cascade elicited by Nutlin in diverse cancer cell lines (24-29). Now, in response to emphasis in the INCLUDE project on iPSCs and a pan-omics cohort study of people with DS, we will characterize the cellular response to Nutlin treatment in three available panels of matched cell types with and without T21: iPSCs, skin fibroblasts, and PBMCs derived from an ongoing cohort study of people with DS. We will use established cell phenotyping assays to test the hypothesis that T21 alters p53-mediated cellular responses, such as cell cycle arrest and apoptosis, and define to what degree these effects are dependent upon elevated JAK/STAT signaling. Altogether, completion of these experiments will enable future studies to define the interplay between IFN signaling and the p53 network in the differential development of co-morbidities in people with DS. QVR supplement abstract

三体21（T21）是最常见的人类染色体疾病，导致称为唐氏综合征（DS）的条件（1，2）。DS患者预期寿命的显著增加所带来的一个显著观察结果是，T21可以保护这些人免受某些医疗条件的影响，同时强烈地使他们倾向于其他疾病，为NIH INCLUDE项目提供了强有力的理由（调查整个生命周期中的共现条件以了解唐氏综合征）。特别是，与NCI的使命相关，流行病学研究表明，DS患者的实体恶性肿瘤发生率显著降低（3，4），同时高度倾向于多种类型的白血病（5-8）。尽管明显的潜力，人口与DS推进我们的理解肿瘤和白血病，鲜为人知的是T21导致这种差异的“恶性光谱”的分子和细胞机制。显然，这一领域的研究不仅有利于DS患者，也有利于一般人群。与INCLUDE项目（免疫系统失调）的重点领域一致，我们最近发现T21导致不同细胞类型中干扰素（IFN）反应的组成性激活（9），这可能是由于六种IFN受体（IFNRs）中有四种在chr 21上编码（10）。因此，T21细胞对IFN配体高度敏感，表现出JAK/STAT信号传导的过度活化和IFN刺激基因（ISG）的过表达（9-12）。此外，在一项大型血浆蛋白质组学队列研究中，我们鉴定了数十种与DS患者中失调的IFN信号传导机制相关的炎性细胞因子（13）。引人注目的是，最近在携带IFNR基因簇的三重化的DS的多个小鼠模型中，IFN应答被鉴定为失调的核心途径（14）。此外，一些证据表明，T21可以增加肿瘤抑制因子p53的活性，在IFN依赖性和非依赖性的方式。非IFN依赖性机制可能包括非整倍性本身，这是一种已知的p53激活刺激（15），以及chr 21编码蛋白的过表达，如ETS 2（一种转录因子）和DYRK 1A（一种激酶），两者都刺激p53活性（16，17）。IFN依赖性机制包括I型IFN对p53启动子的直接诱导（18），IFN激活的转录因子STAT 1与p53的相互作用以增强p53活性（19），以及由T21诱导的三种ISG-ISG 15、IFI 16和PYHIN 1-通过不同机制增强p53活性（20-23）。总而言之，这些观察结果支持了T21可以增强DS患者中p53网络的活性的假设，具有潜在的有益和有害作用（例如增强肿瘤抑制与加速老化，衰老和神经退行性变）。因此，与INCLUDE项目的第1部分一致，我们假设过度活跃的IFN信号传导导致DS患者的不同疾病谱。更具体地说，在父NCI R 01奖的范围内，我们建议调查T21，IFN信号和p53网络之间的相互作用。我们的具体目标是这个补充：1。明确21三体综合征和干扰素活性亢进对p53转录程序的影响。在NIH的资助下，我们的团队是第一个采用全基因组测量新生RNA合成（即GRO-seq）的人，使用称为Nutlin的p53-MDM 2相互作用的小分子抑制剂来识别直接p53转录程序（24，25）。与亲本R 01的目标1相关，我们将使用我们发表的组合p53 ChIP-seq、GR 0-seq和RNA-seq的多组学管道来测试T21增强p53转录程序的假设。我们将使用一组有和没有T21的iPSC作为范例，以确定T21对p53信号传导的定量（即全局）和/或定性（即基因特异性）影响，并定义这些变化在多大程度上是由增强的JAK/STAT信号传导驱动的。2.确定21三体和IFN活性亢进对不同细胞类型中非遗传毒性p53激活的细胞应答的影响。在母基金的资助下，我们完成了Nutlin在不同癌细胞系中引发的p53信号级联的详尽表征（24-29）。现在，为了响应INCLUDE项目对iPSCs的强调和DS患者的泛组学队列研究，我们将在三个可用的匹配细胞类型（有和没有T21）中表征Nutlin治疗的细胞反应：iPSCs，皮肤成纤维细胞和来自DS患者正在进行的队列研究的PBMC。我们将使用已建立的细胞表型分析来检验T21改变p53介导的细胞反应（如细胞周期阻滞和细胞凋亡）的假设，并确定这些效应在多大程度上依赖于JAK/STAT信号的升高。总之，这些实验的完成将使未来的研究，以确定IFN信号和p53网络之间的相互作用，在不同的发展与DS的人的共病。QVR补充摘要