Role of Polycomb-mediated epigenetic regulation in diffuse large B cell lymphoma

描述（由申请人提供）：越来越清楚的是，许多形式的癌症的发病机制具有重要的表观遗传成分。许多癌症类型的特征在于基因失活，并且基因可以通过表观遗传机制（例如DNA甲基化和/或获得抑制性组蛋白3修饰（例如赖氨酸27或9的甲基化））沉默。H3 K27的甲基化由Polycomb蛋白Ezh 2催化，Polycomb蛋白Ezh 2是Polycomb抑制复合物2（PRC 2）的催化组分。最近，在一种特定的癌症亚型中高频率地检测到Ezh 2的催化位点中的特异性突变，该突变过度激活其H3 K27 me 3甲基转移酶活性，同时消除其单甲基化H3 K27的能力：弥漫性大B细胞淋巴瘤（GCB-DLBCL）的生发中心（GC）B细胞亚型。这种过度活跃的Ezh 2突变不存在于所检查的任何其他类型的淋巴瘤中，这表明它可能与GCB-DLBCL淋巴瘤发生独特相关。Ezh 2转录在naove B细胞中被抑制，但在GC B细胞中被上调。我们假设Ezh 2表达在此阶段正常增加以抑制必须下调的特定基因亚组，以允许幼稚B细胞分化为GC B细胞，以及GC B细胞分化为GC后B细胞。一旦Ezh 2甲基化H3 K27，H3 K27 me 3可以被Polycomb抑制复合物1（PRC 1）识别。这种抑制性PRC 1复合物的催化组分是Ring 1b，其使赖氨酸119（uH 2A）上的H2 A单倍化，导致进一步的抑制。然而，H2 AK 119也可以被其他复合物泛素化。此外，与果蝇PRC 1不同，哺乳动物PRC 1中的基本4个组分具有许多替代的旁系同源物，从而产生对H3 K27 me 3具有不同亲和力的各种不同PRC 1复合物。尽管已知组蛋白结合亚基的4种哺乳动物旁系同源物对H3 K27 me 3具有不同的亲和力，但对具有不同组成的PRC 1复合物的生物学后果知之甚少。我们假设在GCB-DLBCL亚组中更多H3 K27 me 3的直接下游结果可能是将有更多的PCR 1募集和/或不同的PRC 1复合物的募集。这可能会导致更多的uH 2A和/或更多的DNA甲基化的一些基因。因此，这些基因可能表现出最多的失调。该项目将表征Polycomb介导的表观遗传变化，这些变化发生在正常人B细胞中，因为它们分化为GC B细胞，然后分化为GC后记忆B细胞。我们将进行ChIP-seq以鉴定与H3 K27 me 3结合的PRC 1组分，并将其与表达谱和DNA甲基化谱进行比较，以确定哪些基因通常以Polycomb介导的方式被抑制。我们将分析大量GC-DLBCL，确定携带或不携带Ezh 2突变的淋巴瘤的表观遗传特征，并将其与基因表达谱相关联。这些研究将提供对GCB-DLBCL中基因失调的了解，并且还将提供关于正常表观遗传机制的新信息，通过该机制，基因在B淋巴细胞分化的后期步骤期间以Polycomb依赖性方式被抑制。 公共卫生相关性：弥漫性大B细胞淋巴瘤（DLBCL）是一种侵袭性疾病，占所有非霍奇金斯淋巴瘤的40%，并且GCB亚群已被描述为在组蛋白甲基转移酶Ezh 2中具有独特的突变。发现由于Ezh 2突变而特异性失调的基因，并了解携带这种突变Ezh 2的下游后果，将是确定可能参与GCB-DLBCL发病机制的关键分子靶点的重要一步。我们将获得关于正常生发中心B细胞（经历恶性转化为GCB-DLBCL的细胞类型）中Polycomb介导的抑制的各种机制的新信息，这对于理解淋巴瘤发生中发生的误调节是必不可少的。

DESCRIPTION (provided by applicant): It has become increasingly clear that the pathogenesis of many forms of cancer have a significant epigenetic component. Many cancer types are characterized by gene inactivation, and genes can be silenced through epigenetic mechanisms such as DNA methylation and/or the acquisition of repressive histone 3 modifications such as the methylation of lysines 27 or 9. Methylation of H3K27 is catalyzed by the Polycomb protein Ezh2, the catalytic component of Polycomb Repressive Complex 2 (PRC2). Recently, a specific mutation in the catalytic site of Ezh2 that hyperactivates its H3K27me3 methyltransferase activity while eliminating its ability to monomethylate 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 hyperactive Ezh2 mutation is not present in any other types of lymphomas examined, suggesting that it may be uniquely relevant for GCB- DLBCL lymphomagenesis. Ezh2 transcription is repressed in naove B cells, but it is upregulated in GC B cells. We hypothesize that Ezh2 expression is normally increased at this stage to repress a specific subset of genes that must be downregulated to allow the differentiation of a naove B cell into a GC B cell, and of a GC B cell into a post-GC B cell. Once Ezh2 methylates H3K27, H3K27me3 can be recognized by the Polycomb Repressive Complex 1 (PRC1). The catalytic component of this repressive PRC1 complex is Ring1b, which monoubiquitinates H2A on lysine 119 (uH2A), leading to further repression. However, H2AK119 can be ubiquitinylated by other complexes also. Furthermore, unlike Drosophila PRC1, the basic 4 components in mammalian PRC1 have many alternative paralogs, resulting in a variety of different PRC1 complexes with different affinities for H3K27me3. Very little is known about the biological consequences of PRC1 complexes having different compositions, although it is known that the 4 mammalian paralogs of the histone binding subunit have varying affinities for H3K27me3. We hypothesize that a direct downstream consequence of more H3K27me3 in a subset of GCB-DLBCL may be that there will be more PCR1 recruitment and/or recruitment of different PRC1 complexes. This could result in more uH2A and/or more DNA methylation on some genes. Consequently, these genes are likely to display the most dysregulation. This project will characterize the Polycomb-mediated epigenetic changes that take place in normal human B cells as they differentiate into GC B cells, then into post-GC memory B cells. We will perform ChIP-seq to identify PRC1 components binding to H3K27me3, and compare this to the expression profile and DNA methylation profile to determine which genes are normally repressed in a Polycomb-mediated manner. We will analyze a large set of GC-DLBCL, will determine the epigenetic profile of lymphomas that bear or do not bear the Ezh2 mutation, and will relate this to their gene expression profile. These studies will provide insight into the dysregulation of genes in GCB- DLBCL, and will also provide novel information on the normal epigenetic mechanisms by which genes are repressed in a Polycomb-dependent manner during the late steps of B lymphocyte differentiation. PUBLIC HEALTH RELEVANCE: Diffuse large B cell lymphoma (DLBCL) is an aggressive disease that accounts for 40% of all non- Hodgkins lymphomas, and the GCB subset has been described to have a unique mutation in the histone methyltransferase, Ezh2. Finding genes that are specifically misregulated due to the Ezh2 mutation, and understanding the downstream consequences of bearing this mutant Ezh2, will be a major step forward in pinpointing key molecular targets that may be involved in the pathogenesis of GCB-DLBCL. The novel information we will gain regarding the various mechanisms of Polycomb- mediated repression in normal germinal center B cells, the cell type that undergoes the malignant transformation to GCB-DLBCL, will be essential to understand the misregulation that occurs in lymphomagenesis.