Histone arginine methylation and transcriptionally active chromosomal domains

组蛋白精氨酸甲基化和转录活性染色体结构域

• 批准号: RGPIN-2017-05927
• 负责人: Davie, James
• 金额: $ 2.04万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711890
• 关键词: Histone; arginine; methylation; transcriptionally; active

The eukaryotic genome is organized into domains, which contain one or more genes. Some domains contain expressed genes, while others have silenced genes. This research field called epigenetics is the study of the organization and expression of genes that is not dependent upon changes in the DNA sequence. For more than three decades, my research group has focused on the study of histones which play a key role in epigenetic regulation of gene expression. Histones bind to DNA forming a structure called the nucleosome. The nucleosomes are joined by a stretch of DNA to form chromatin. Histone modifications, which are different in expressed gene and silent gene chromatin, play a key role in the organization and expression of genes. My research uses red blood cells from egg-laying chickens to characterize the organization of expressed genes. My research group discovered a method to separate expressed gene chromatin from silent gene chromatin, and when combined with state-of-the-art DNA sequencing methods, we identified all the chromatin domains containing expressed genes in chicken red blood cells. Surprisingly the red blood cell expressed genes involved in the innate immune response, which defends the bird from infection by other organisms (viruses). My proposed discovery research will explore the role of two enzymes called protein arginine methyltransferases, PRMT1 and 5, in maintaining the histone modifications associated with the expressed gene chromatin domains. We believe that these enzymes are essential to maintaining the chromatin organization needed for expression of genes involved in oxygen transport and innate immunity. Three graduate and four summer students will be trained in state-of-the-art epigenetic methodology. Sanzida Jahan, a PhD trainee, and a Masters trainee, Tasnim Hussain, will determine the location of PRMT1/5 and the histone modifications produced by these enzymes (histone H3 dimethylated (symmetric) at R2 (H3R2me2s) and histone H4 dimethylated (asymmetric) at R3 (H4R3me2a)) using the chromatin immunoprecipitation (ChIP) assay and next generation DNA sequencing with chicken red blood cells and avian 6C2 cells, which are arrested at the colony forming unit erythrocyte stage. The second aim will find out how PRMT1 and 5 are directed to expressed genes. Sanzida and Tasnim will identify the proteins associated with these enzymes. In the third aim, the graduate students will use methods that reduce the expression of or inhibit PRMT1 and 5 and determine the consequences of the loss/inhibition of the enzyme's activity on the organization of expressed gene chromatin in chicken red blood cells. This discovery research will enhance our understanding of the mechanisms involved in epigenetic regulation of gene expression and highlight the central role of PRMTs in establishing a chromatin structure that is supportive of gene expression in a background of highly condensed chromatin.

真核基因组被组织成多个域，其中包含一个或多个基因。一些域包含表达的基因，而另一些域则包含沉默的基因。这个称为表观遗传学的研究领域是对不依赖于 DNA 序列变化的基因组织和表达的研究。三十多年来，我的研究小组一直专注于组蛋白的研究，组蛋白在基因表达的表观遗传调控中发挥着关键作用。组蛋白与 DNA 结合，形成称为核小体的结构。核小体由一段 DNA 连接形成染色质。组蛋白修饰在表达基因和沉默基因染色质中有所不同，在基因的组织和表达中发挥着关键作用。我的研究使用产蛋鸡的红细胞来表征表达基因的组织。我的研究小组发现了一种将表达基因染色质与沉默基因染色质分离的方法，并与最先进的 DNA 测序方法相结合，我们鉴定出了鸡红细胞中包含表达基因的所有染色质结构域。令人惊讶的是，红细胞表达了参与先天免疫反应的基因，这种反应可以保护鸟类免受其他生物体（病毒）的感染。我提出的发现研究将探索两种称为蛋白质精氨酸甲基转移酶（PRMT1 和 5）的酶在维持与表达基因染色质结构域相关的组蛋白修饰中的作用。我们相信这些酶对于维持参与氧运输和先天免疫的基因表达所需的染色质组织至关重要。三名研究生和四名暑期学生将接受最先进的表观遗传学方法的培训。博士生 Sanzida Jahan 和硕士生 Tasnim Hussain 将使用染色质确定 PRMT1/5 的位置以及这些酶产生的组蛋白修饰（R2 (H3R2me2s) 处的组蛋白 H3 二甲基化（对称）和 R3 (H4R3me2a) 处的组蛋白 H4 二甲基化（不对称）） 使用鸡红细胞和禽类 6C2 细胞进行免疫沉淀 (ChIP) 测定和下一代 DNA 测序，这些细胞被捕获在集落形成单位红细胞阶段。第二个目标是找出 PRMT1 和 5 如何针对表达基因。 Sanzida 和 Tasnim 将鉴定与这些酶相关的蛋白质。在第三个目标中，研究生将使用减少或抑制 PRMT1 和 5 表达的方法，并确定酶活性丧失/抑制对鸡红细胞中表达基因染色质组织的影响。这项发现研究将增强我们对基因表达表观遗传调控机制的理解，并强调 PRMT 在建立支持高度浓缩染色质背景下基因表达的染色质结构中的核心作用。