Functions of the KDM5C and KDM5D tumour suppressor genes in clear cell renal cell carcinoma

染色质功能的表观遗传控制支配着基因转录模式，这些模式被维持并传递给子代细胞，以保护人类健康。调节染色质的基因突变和组蛋白的翻译后修饰证明了这种控制在癌症中的崩溃。这个项目研究了VHL肿瘤抑制基因和性染色体连锁的KDM5C和KDM5D肿瘤抑制基因的联合突变在肾透明细胞癌(CcRCC)的背景下的功能后果。基因组分析显示ccRCC具有独特的遗传组成，几乎所有的肿瘤都表现出VHL突变，以及一系列调控不同表观遗传过程的基因的一个或多个突变，包括X染色体基因KDM5C和KDM5D，其中X染色体基因KDM5C在大约20%的转移性ccRCC病例中发生突变，KDM5D在男性ccRCC病例中由于Y染色体的体细胞丢失而经常丢失。VHL蛋白通过HIF-α依赖的机制间接调节组蛋白H3赖氨酸27的甲基化，而高度同源的KDM5C/D蛋白编码组蛋白H3赖氨酸4二/三甲基去甲基酶。然而，目前还不清楚这些肿瘤抑制基因的协同突变是否以及如何影响转录输出或细胞和组织的表型。我们将采用一种简化的实验方法来简化ccRCC肿瘤的复杂性，使用干净的遗传学来解决这些问题。这将包括建立几种新的小鼠模型来单独或与VHL一起删除肾上皮细胞中的Kdm5c和/或Kdm5d，以分析对肾上皮细胞动态平衡和肿瘤形成的影响。为了补充这些基于小鼠的实验，我们将使用CRISPR-Cas9突变将KDM5C突变引入一系列女性人ccRCC细胞系，并将KDM5C/KDM5D单突变和双突变引入一系列雄性ccRCC细胞系。我们各种小鼠系的原代细胞和肿瘤细胞系以及改造的ccRCC细胞的可用性，将提供相关材料，使用细胞培养和异种移植分析这些基因变化对癌症相关细胞表型的影响，并将能够进行涉及RNA-Seq、ChIP-Seq和Exome-Seq的详细分子分析，以表征这些突变对组蛋白修饰以及对转录和突变谱的影响。利用可公开获得的来自人类ccRCC和ccRCC组织资源的基因表达和突变数据库，我们的目标是将我们实验系统的发现与人类ccRCC肿瘤相关联。这些研究有望为更好地理解ccRCC的分子亚集的发病机制奠定基础，并提供非常有用的细胞和小鼠模型，在未来的研究中将对识别和测试基因特异性治疗方法非常有用。

Epigenetic control of chromatin function governs gene transcription patterns that are maintained and transmitted to daughter cells to preserve human health. Breakdown of this control in cancer is evidenced by the predominance of mutations in genes regulating chromatin and the post-translational modification of histones. This project investigates the functional consequences of combined mutations in the VHL tumour suppressor gene and the sex chromosome-linked KDM5C and KDM5D tumour suppressor genes in the context of clear cell renal cell carcinoma (ccRCC), the most frequent form of kidney cancer. Genomic analyses have revealed that ccRCC has a unique genetic make-up, with almost all tumours exhibiting mutations in VHL, as well as one or more mutations in a series of genes that regulate different epigenetic processes, including the X-chromosome gene KDM5C, which is mutated in approximately 20% of cases of metastatic ccRCC, and KDM5D which is frequently lost in male ccRCC cases due to somatic loss of the Y-chromosome. The VHL protein indirectly regulates methylation of histone H3 lysine 27 via HIF-α-dependent mechanisms and the highly homologous KDM5C/D proteins encode histone H3 lysine 4 di-/tri-methyl demethylases. It is however currently not understood if and how cooperative mutations in these tumour suppressor genes affect transcriptional outputs or cellular and organismal phenotypes. We will undertake a reductive experimental approach to simplify the complexity of ccRCC tumours using clean genetics to address these issues. This will involve the generation of several new mouse models to delete Kdm5c and/or Kdm5d in renal epithelial cells alone or together with Vhl to analyse the effects on renal epithelial cell homeostasis and tumour formation. To complement these mouse-based experiments we will use CRISPR-Cas9 mutagenesis to introduce KDM5C mutation into a series of female human ccRCC cell lines and KDM5C/KDM5D single and double mutations into a series of male ccRCC cell lines. The availability of primary cells and tumour cell lines from our various mouse lines, as well as engineered ccRCC cells, will provide relevant material to analyse the effects of these genetic alterations on cancer-relevant cellular phenotypes using cell culture and xenograft assays and will also allow detailed molecular analyses involving RNA-Seq, ChIP-Seq and Exome-Seq to characterise the effects of these mutations on histone modifications and on transcriptional and mutational profiles. Using publically available gene expression and mutation databases from human ccRCCs and archival ccRCC tissue resources, we aim correlate the findings from our experimental systems to human ccRCC tumours. These studies are expected to lay the foundation for a better understanding of the pathogenesis of a molecular subset of ccRCC and to provide cellular and mouse models that will be highly useful for the identification and testing of genotype-specific therapies in future studies.