Biological consequences of a lymphoma-associated mutation in Ezh2 in mice

描述(申请人提供)：EZH2是多梳抑制复合体2的酶组分，它催化组蛋白H3赖氨酸27(K27)的甲基化，组蛋白H3是与被抑制基因相关的组蛋白翻译后修饰。许多癌症类型的特点是基因失活。在各种肿瘤中，EZH2的mRNA水平升高，这与抑制关键基因的作用是一致的。然而，最近，在一种特定的癌症亚型：弥漫性大B细胞淋巴瘤的生发中心(GC)B细胞亚型(GCB-DLBCL)中发现了Ezh2(Y641)催化位点的一个特定突变，该突变使其H3K27三甲基转移酶活性过度激活，同时消除了其对H3K27单甲基化的能力。这种突变只发生在肿瘤细胞中，而不发生在生殖系DNA中，因此是一种体细胞获得性和正向选择的突变。如此独特的功能突变收获是前所未有的。EZH2在正常的GC B细胞中高表达，而在NA细胞中低表达。我们假设Ezh2在GC B细胞中的表达增加，以抑制特定的基因亚集，这些基因必须下调才能允许从原始B细胞分化为GC B细胞，和/或将GC B细胞分化为GC B细胞。我们进一步假设 Y461F突变将导致一组基因的过度抑制，这些基因通常被激活或重新激活，以便B细胞离开GC间隔室。如果这一假设是正确的，那么我们预测这种过度抑制可能会使GC B细胞在GC的突变环境中保持比平时更长的时间。这种Ezh2突变对B细胞的直接影响不能在患者的淋巴瘤样本中直接解决，因为每个人的基因组都是唯一的，而且淋巴瘤可能有易位或各种其他遗传或表观遗传变化。在这里，我们将直接在近交系小鼠身上测试这种高度活跃的Ezh2的效果，这样我们就可以明确地确定这种淋巴瘤相关突变的下游后果。我们将制作一只靶向rosa26基因的Ezh2 Y641F小鼠。靶向构建体在启动子和Ezh2 Y641F之间有一个花朵状的转录终止。当与C？1-Cre小鼠杂交时，突变的Ezh2将在GC B细胞中表达。我们将确定Ezh2 Y641F及其所有下游表观遗传、转录和生物学后果是否足以导致淋巴瘤。我们将确定它是否会导致 延迟退出GC，这可能会在GC的诱变环境中提供额外的时间。我们将对Ezh2 Y641F和WT小鼠的GC B细胞以及H3K27me3的CHIP-SEQ进行基因表达谱分析。我们将通过对PRC1组分和由PRC1催化的泛素化的H2AK119进行CHIP-SEQ来确定Ezh2 Y641的酶活性改变的下游后果。拟议的研究将深入了解该突变对基因表达、表观遗传学特征、抗原刺激后的B细胞分化和淋巴肿大的下游影响。如果Ezh2 Y641本身足以导致淋巴肿大，那么突变Ezh2的特定抑制剂可能被设计成一种治疗药物。)

DESCRIPTION (provided by applicant): Ezh2 is the enzymatic component of Polycomb Repressive Complex 2, and it catalyzes the methylation of lysine 27 (K27) of histone H3, a histone post-translational modification associated with repressed genes. Many cancer types are characterized by gene inactivation. Ezh2 mRNA levels are increased in a variety of tumors, consistent with a role in repressing critical genes. Recently, however, a specific mutation in the catalytic site of Ezh2 (Y641) that hyperactivates its H3K27 tri-methyltransferase activity while eliminating its ability to mono- methylate H3K27 been detected with high frequency in one specific subtype of cancer: the germinal center (GC) B cell subtype of diffuse large B cell lymphoma (GCB-DLBCL). This mutation occurs only in the tumor cell, not in the germline DNA, and therefore is a somatically acquired and positively selected mutation. Such a unique gain of function mutation is unprecedented. Ezh2 is highly expressed in normal GC B cells, but is low in na¿ve B cells. We hypothesize that Ezh2 expression is increased in GC B cells to repress a specific subset of genes that must be downregulated to allow the differentiation of a na¿ve B cell into a GC B cell, and/or of a GC B cell into a post-GC B cell. We further hypothesize that the Y461F mutation will result in over-repression of a subset of genes that normally are activated or re-activated in order for a B cell to exit the GC compartment. If this hypothesis is correct, then we predict that this over-repression might retain the GC B cells in the mutagenic environment of the GC longer than usual. The direct effect to B cells of this Ezh2 mutation cannot be directly addressed in patients' lymphoma samples, since the genome of each individual is unique, and because the lymphomas may have translocations or a variety of other genetic or epigenetic changes. Here we will directly test the effect of this hyperactive Ezh2 in an inbred strain of mouse, so that we can unambiguously determine the downstream consequences of this lymphoma-associated mutation. We will make a mouse with the Ezh2 Y641F targeted into the ROSA26 locus. The targeting construct has a floxed transcriptional stop between the promoter and Ezh2 Y641F. When crossed to C¿1-Cre mice, the mutant Ezh2 will be expressed in GC B cells. We will determine if Ezh2 Y641F, with all of its downstream epigenetic, transcriptional and biological consequences, will be sufficient to lead to lymphoma. We will determine if it results in delayed exit from the GC, which may provide additional time in the mutagenic environment of the GC. We will perform gene expression profiling of GC B cells from Ezh2 Y641F and WT mice, and also ChIP-seq for H3K27me3. We will determine the downstream consequences of the altered enzymatic activity of Ezh2 Y641 by ChIP-seq for PRC1 components and ubiquitinated H2AK119, which is catalyzed by PRC1. The proposed studies will provide insight into the downstream effects of this mutation on gene expression, epigenetic profile, B cell differentiation after antigen stimulation, and lymphomagenesis. If this Ezh2 Y641 is sufficient in itself to result in lymphomagenesis, then specific inhibitors of the mutant Ezh2 could potentially be designed as a therapeutic. )