儿童胶质母细胞瘤K27突变型核小体动态定位及其基因调节作用分析

Glioblastoma multiforme(GBM) is one of the most aggressive primary malignant brain tumors,2-year suvival rate is less than 20% .Recently, somatic mutation of gene H3F3A (encoding histone variant H3.3) that results in a Lys 27-to-methionine (K27M) change in H3.3 was frequently identified in the pediatric GBM. The mutation globally reduces H3K27 tri-methylation (H3K27me3) and locally increased the histone modification. Genes that gain or lose H3K27me3 are significantly enriched at cancer pathways. This made K27M-mutant-glioblastoma to be new subtype of such cancer.Does nucleosome dynamic position in transcription start site(TSS)has unique feature? Does it has relationship with hypermethylation of GBM candidate genes and histone code disruption? How does it affect GBM genesis,progression and outcome through gene silencing? In the project, through next generation sequencing technique, nucleosomes positions, genomic DNA sequence and gene expression will be determined in pediatric K27M mutant glioblastoma cells as well as tumor-surrounding cells(TSC) and pediatric non K27M mutant glioblastoma cells(NGBM). Nucleosome dynamics will be analyzed between GBM,TSCand NGBM, and the role of the dynamics in gene regulation will be resolved. Whether K27M mutation intimately associates nucleosome organization of H3F3A will be determined. New epigenetic mechanism-based targets will be identified for diagnosis, treatment and prognosis.Link between the abnormal nucleosome organization and glioma development will be thoroughly elucidated.

儿童脑胶质母细胞瘤是原发于中枢神经系统的高度恶性肿瘤，2年生存率小于20%。最近研究表明编码组蛋白变体H3.3的基因H3F3A在儿童和青少年胶质母细胞瘤中呈现特征性K27M突变，突变引起H3K27me3全基因组尺度降低但部分位点增加，且得失位点的基因在肿瘤通路中富集，这使得具有该特点的肿瘤成为一种新的肿瘤亚型。基因H3F3A转录区核小体位置（占位和位置改变）是否会呈特征性改变？这种改变与该基因的甲基化以及组蛋白代码中断是否相关？它是如何调节基因表达从而进一步影响肿瘤的发生、发展？本研究对比分析儿童K27M突变型脑胶质母细胞瘤、瘤旁组织以及K27M突变和非突变胶质母细胞瘤H3F3A基因启动子区核小体精确位置和占位的异同，分析核小体动态定位差异与基因沉默、甲基化之间的关系，进一步解析核小体定位的动态变化所引起的基因转录调节的变化与胶质母细胞瘤发生、发展之间的关系。

儿童脑胶质母细胞瘤是原发于中枢神经系统的高度恶性肿瘤，2年生存率小于20%。有研究表明编码组蛋白变体H3.3的基因H3F3A在儿童和青少年胶质母细胞瘤中呈现特征性K27M突变，突变引起H3K27me3全基因组尺度降低但部分位点增加，且得失位点的基因在肿瘤通路中富集，这使得具有该特点的肿瘤成为一种新的肿瘤亚型。项目拟研究儿童胶质瘤特征性H3.3K27M突变与核小体定位之间的关联；分析H3.3K27M突变胶质瘤中，核小体以及包含H3.3核小体的定位的变化(动态)，以及这种动态与基因转录调节的关联。最终阐明儿童胶质瘤H3.3K27M突变的染色质结构因素、揭示突变导致的核小体动态对基因转录调节的作用，为这类疾病药物研发、预防治疗提供依据。项目对30例儿童脑胶质母细胞瘤K27M突变型的患者,进行自身配对，收集其新鲜肿瘤和瘤旁组织进行分离、纯化细胞，对同一患者胶质母细胞瘤K27突变型和瘤旁组织细胞以及K27非突变型核小体的动态定位模式。以ChIP-Seq试验测定儿童胶质母细胞瘤K27突变型、瘤旁组织细胞、K27非突变型的核小体位置，RAN-Seq测定基因的表达量，测定的胶质瘤基因组。通过基于隐马尔科夫模型计算分析实验数据，识别了胶质瘤细胞的K27M突变与H3F3A基因(编码H3.3)的核小体特定定位有关，胶质瘤中核小体定位的基因调节作用基因H3F3A转录区核小体位置（占位和位置改变）有特征性改变，这种改变与该基因的甲基化以及组蛋白代码中断相关，阐明了核小体动态在H3.3K27M突变的胶质瘤细胞中的基因调节作用。核小体定位的改变导致的基因表达改变的作用、以及这些基因的功能、与胶质瘤表型之间的关系H3.3K27M突变是由于特定的核小体定位模式引起，那么分布在H3F3A基因上的核小体就是一个新的靶点，适当调整该基因的核小体分布，就可以抑制H3.3K27M突变。由于核小体定位在H3.3K27M突变的胶质瘤细胞中有特定作用，核小体定位改变而导致的基因转录水平显著上调和下调的基因，发现抑制 H3F3A 基因发生 K27M 突变的靶点，作为儿童胶质瘤预防、药物开发和治疗的靶点。