Determination of the role of CHD5 tumor suppressor in the zebrafish model

描述（由申请人提供）：CHD5染色质重塑子在人类肿瘤抑制中起关键作用。CHD5基因座的缺失与多种肿瘤的形成有关，包括结直肠癌、白血病、肺癌和神经母细胞瘤。此外，在多种肿瘤中，CHD5表达的缺失与预后不良密切相关，而异位表达研究表明，肿瘤细胞中CHD5表达的增加可以抑制其生长。CHD5抑制癌细胞致瘤性的能力表明，增强CHD5功能的医疗策略将抑制与CHD5表达缺失或减少相关的肿瘤生长。制定这样的医疗策略需要深入了解CHD5的作用机制。因此，我们的长期目标是了解CHD5如何抑制肿瘤的形成和发展，并利用这一认识来开发新的抗肿瘤策略。系统发育分析显示，CHD5蛋白仅在脊椎动物中发现，这反过来表明CHD5重塑蛋白在该谱系中具有保守功能。本研究旨在研究斑马鱼CHD5基因CHD5在肿瘤抑制和斑马鱼基因表达中的作用，目的是建立这一强大的脊椎动物系统，作为确定CHD5染色质重塑剂抑制肿瘤机制的合适背景。初步数据显示，chd5在成人中以与哺乳动物相似的方式表达，并揭示了早期胚胎中chd5的一种新的表达模式以及chd5在胚胎发生中的作用。该提案的第一个目的是使用表达突变chd5的转基因细胞系来确定chd5是否像在哺乳动物中一样在斑马鱼中起肿瘤抑制作用。第二个目标是对Chd5蛋白的辅助因子进行生化筛选，这将有助于揭示Chd5如何起作用，并且也将成为有助于Chd5依赖性肿瘤抑制的因子的候选因子。第三个目标采用RNA-seq策略来鉴定表现出chd5依赖性表达的基因，以鉴定介导chd5抑制肿瘤形成能力的靶基因。我们的实验目标的完成将建立斑马鱼作为表征CHD5重塑物如何抑制肿瘤的模型，这将作为小鼠模型的重要补充。特别是，我们提出的研究将使我们能够在斑马鱼中进行全面的化学遗传筛选，以确定抑制chd5依赖性缺陷的化合物，从而确定用于开发治疗chd5依赖性肿瘤的药物的先导化合物。同样，完成所提出的目标将使我们能够使用斑马鱼中可用的强大的正向和反向遗传工具来识别有助于chd5介导的肿瘤抑制的其他表观遗传机制。因此，这种机制的鉴定将揭示chd5依赖性肿瘤抗肿瘤治疗的其他靶点。

DESCRIPTION (provided by applicant): The CHD5 chromatin remodeler plays a critical role in tumor suppression in humans. Loss of the CHD5 locus is associated with formation of a wide range of tumors, including colorectal cancer, leukemia, lung cancer, and neuroblastoma. Furthermore, loss of CHD5 expression is strongly correlated with poor prognosis in a variety of tumors whereas ectopic expression studies have shown that increased expression of CHD5 in tumor cells can suppress their growth. The ability of CHD5 to suppress the tumorigenicity of cancer cells suggests that medical strategies that augment CHD5 function will suppress the growth of tumors associated with loss or reduced expression of CHD5. Development of such medical strategies requires an in depth understanding of the mechanism(s) by which CHD5 acts. Our long-term goal is therefore to understand how CHD5 suppresses tumor formation and development and to use this understanding to develop novel anti-tumor strategies. Phylogenetic analysis reveals that CHD5 proteins are found only in vertebrates, which in turn suggests a conserved function for the CHD5 remodeler in this lineage. This proposal examines the role of the zebrafish CHD5 gene chd5 in tumor suppression and gene expression in zebrafish with the goal of establishing this powerful vertebrate system as a suitable context for determining the mechanism of tumor suppression by the CHD5 chromatin remodeler. Preliminary data show that chd5 is expressed in a similar fashion to mammalian CHD5 in adults and reveal a novel pattern of expression in early embryos as well as a role for chd5 in embryogenesis. The first aim of the proposal uses transgenic lines expressing mutated versions of chd5 to establish whether chd5 acts as a tumor suppressor in zebrafish as it does in mammals. The second aim is a biochemical screen for co-factors of Chd5 protein that will help reveal how Chd5 acts and that will also be candidates for factors that contribute to CHD5-dependent tumor suppression. The third aim employs an RNA-seq strategy to identify genes that exhibit chd5-dependent expression to identify target genes that mediate the ability of chd5 to suppress tumor formation. Completion of our experimental aims will establish zebrafish as a model for characterization of how CHD5 remodelers suppress tumors that will serve as a vital complement to the mouse model. In particular, our proposed studies will enable us undertake the type of comprehensive chemical genetic screens that are practical in zebrafish to identify compounds that suppress chd5-dependent defects and thereby identify lead compounds for development for pharmaceuticals to treat CHD5-dependent tumors. Similarly, completion of the proposed aims will enable us to use the formidable forward and reverse genetic tools available in zebrafish to identify additional epigenetic machinery that contributes to CHD5-mediated tumor suppression. Identification of such machinery will therefore reveal additional targets for anti-tumor therapies for CHD5-dependent tumors.