Novel role of the polycomb repressive complex PRC2/EZH2 in glioblastoma

 描述(申请人提供)：组蛋白甲基转移酶EZH2在多形性胶质母细胞瘤(GBM)肿瘤和胶质瘤干细胞(GSCs)中过表达，并发挥癌基因的作用。已知它主要通过甲基化组蛋白H3K27作为基因表达的表观遗传抑制因子发挥作用。EZH2是多梳抑制复合体PRC2的催化成分，在癌症、多能性和其他基本细胞过程中具有重要作用。最近的工作表明，PRC2还与长的非编码RNA(LncRNAs)以及内含子和启动子相关的非编码RNA相关联。PRC2与非编码RNA结合的特异性以及这些相关RNA在调节PRC2功能中的作用正在积极研究中。 我们已经发现，在GBM癌细胞中，EZH2与来自蛋白质编码基因外显子的短RNA结合。EZH2的这种外显子RNA结合以前没有被注意到，而且这种结合对于调节EZH2功能的意义尚不清楚。然而，这些靶基因中有许多在GBM生物学中具有已知的功能。重要的是，我们还发现EZH2的缺失降低了其中几个靶RNA的表达，表明EZH2可以通过一种潜在的新机制发挥基因表达的激活作用。我们推测，在GBM中，EZH2/PRC2可能具有调控基因表达和/或剪接的作用，这一作用独立于其在组蛋白甲基化中的既定角色，而是通过其与外显子RNA的联系而介导。在这个项目中，PRC2的这一新作用将在GBM癌细胞和胶质瘤干细胞(GSCs)中进行测试。在目标1中，我们将使用RNA免疫沉淀和基于RNA交联的测序方法(RIP-seq和iCLIP-seq)来识别GBM癌细胞和GSCs中所有与EZH2相关的外显子RNA。我们将确定其他PRC2组分是否也与外显子RNA相关，这表明PRC2复合体作为一个整体以这种新的方式发挥作用。我们还将确定EZH2或PRC2是否被招募到与外显子RNA相关的区域的基因组位置，这将表明共转录机制。在目标2中，我们将确定EZH2的丢失是否影响基因表达水平和它与之结合的外显子靶标的选择性剪接，并测试这些RNA的异位表达是否可以将EZH2招募到特定的染色体位置。这些研究指出了GBM中EZH2或PRC2复合体的一种新的功能机制，并有望揭示其影响GBM生物学的新途径，从而为治疗干预提供新的途径。

 DESCRIPTION (provided by applicant): The histone methyltransferase EZH2 is overexpressed in glioblastoma multiforme (GBM) tumors as well as glioma stem cells (GSCs), and functions as an oncogene. It is known to function primarily as an epigenetic repressor of gene expression by methylating histone H3K27. EZH2 is the catalytic component of the polycomb repressive complex PRC2, which has important roles in cancer, pluripotency and other fundamental cellular processes. Recent work has shown that PRC2 also associates with long non-coding RNAs (lncRNAs), as well as intronic and promoter-associated non-coding RNAs. The specificity of PRC2 association with non-coding RNAs and the role of these associated RNAs in mediating the function of PRC2 is under active investigation. We have discovered that in GBM cancer cells, EZH2 binds to short RNAs that are derived from the exons of protein coding genes. This exonic RNA association of EZH2 has not been noted before, and the significance of this binding with regard to regulating EZH2 function is unknown. However, many of these target genes have known functions in GBM biology. Importantly, we have also found that loss of EZH2 reduces the expression of several of these target RNAs, indicating that EZH2 can function as an activator of gene expression by a potentially novel mechanism. We hypothesize that in GBM, EZH2/PRC2 could have a role in regulating gene expression and/or splicing that is independent of its established roles in histone methylation, and mediated instead by its association with exonic RNAs. This novel role for PRC2 will be tested in GBM cancer cells and glioma stem cells (GSCs) in this project. In Aim 1, we will use RNA immunoprecipitation and RNA crosslinking based sequencing approaches (RIP-seq and iCLIP-seq) to identify all EZH2-associated exonic RNAs in GBM cancer cells and GSCs . We will determine whether other PRC2 components also associate with exonic RNAs, indicative of the PRC2 complex as a whole functioning in this novel manner. We will also establish whether EZH2 or PRC2 is recruited to genomic loci at the regions where it associates with exonic RNAs, which would be indicative of a co-transcriptional mechanism. In Aim 2 we will establish whether loss of EZH2 affects gene expression levels and alternative splicing of the exonic targets that it binds to, and test if ectopic expression of these RNAs can direct EZH2 recruitment to specific chromosomal sites. These studies point to a novel mechanism of function for EZH2 or the PRC2 complex in GBM, and stand to reveal new ways in which it can impact the biology of GBM and thus offer new avenues for therapeutic intervention.