Mechanisms of gene expression control in the p53 network

21三体(T21)是人类最常见的染色体疾病，导致唐氏综合症(DS)(1，2)。唐氏综合症患者预期寿命的显著增加带来了一个显著的观察结果，即T21保护这些人免受某些疾病的影响，同时强烈地使他们容易患上其他疾病，这为NIH Include项目(了解唐氏综合症的整个生命周期的共生条件调查)提供了强有力的理由。特别是，与NCI的使命相关的流行病学研究表明，DS患者的实体恶性肿瘤发病率(3，4)显著较低，同时极易患多种类型的白血病(5-8)。尽管患有DS的人群有明显的潜力促进我们对肿瘤发生和白血病发生的理解，但人们对T21导致这种不同的“恶性谱系”的分子和细胞机制知之甚少。显然，这一领域的研究不仅将使DS患者受益，也将使普通人群受益。与INCLUDE项目的一个关键重点领域(免疫系统失调)一致，我们最近发现T21引起跨不同细胞类型的干扰素(干扰素)反应的结构性激活(9)，这可能是由于六个干扰素受体(IFNRs)中的四个是在chr21(10)上编码的。因此，T21细胞对干扰素配体高度敏感，表现出JAK/STAT信号的高度激活，以及干扰素刺激基因(ISGs)的过度表达(9-12)。此外，在一项大型血浆蛋白质组学队列研究中，我们确定了数十种与干扰素信号机制有关的炎性细胞因子，它们在DS患者中处于失调状态(13)。值得注意的是，干扰素反应最近被确定为在携带IFNR基因簇三倍体的多种DS小鼠模型中调节失调的核心途径(14)。此外，一些证据表明，T21可能以干扰素依赖和非依赖的方式增加肿瘤抑制因子P53的活性。干扰素不依赖的机制可能包括非整倍体本身，这是一种已知的p53激活刺激(15)，以及chr21编码的蛋白质的过度表达，如转录因子ETS2和DYRK1A，这两者都能刺激p53的活性(16，17)。干扰素依赖的机制包括I型IFN直接诱导P53启动子(18)，干扰素激活的转录因子STAT1与P53相互作用以增强P53的活性(19)，以及T21诱导的三种ISG-ISG15、IFI16和PYHIN1-通过不同的机制提高P53的活性(20-23)。总之，这些观察结果支持这样的假设，即T21可以增强DS患者的P53网络的活性，具有潜在的有益和有害的影响(例如，增强肿瘤抑制相对于加速衰老、衰老和神经退化)。因此，与INCLUDE项目的第一部分一致，我们假设过度活跃的干扰素信号与DS患者的不同疾病谱有关。更具体地说，在父母NCI R01奖的范围内，我们建议调查T21、干扰素信号和p53网络之间的相互作用。本附录的具体目的是：1.明确21三体和干扰素过度活动对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)的团队，以确定直接的p53转录程序，使用一种名为Nutlin(24，25)的p53-MDM2相互作用的小分子抑制剂。与亲本R01的目标1相关，我们将使用我们发表的结合p53芯片-seq、Gro-seq和RNA-seq的多组学流水线来验证T21增强p53转录程序的假设。我们将使用一个带有和不带有T21的IPSCs小组作为范例，以确定T21对p53信号的定量(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受到增强的JAK/STAT信号的驱动。2.明确21三体和干扰素过度激活对不同类型细胞对非遗传毒性P53激活的细胞反应的影响。利用父母奖提供的资金，我们完成了Nutlin在不同癌细胞系(24-29)中引发的P53信号级联的详尽表征。现在，为了回应INCLUDE项目对IPSCs和对DS患者的泛组学队列研究的强调，我们将在三个可用的具有和不具有T21的匹配细胞类型的小组中表征Nutlin治疗的细胞反应：来自于正在进行的DS患者队列研究的IPSCs、皮肤成纤维细胞和PBMC。我们将使用已建立的细胞表型分析来检验T21改变P53介导的细胞反应的假设，如细胞周期停滞和凋亡，并确定这些影响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能够确定干扰素信号和p53网络在DS患者合并疾病的不同发展中的相互作用。QVR增刊摘要

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