Structural and functional studies of histone acetyltransferase complexes

就像食谱一样，人类基因组包含了构建多细胞人类的详细说明。这本食谱是以基因组DNA的形式写成的，被组织成约20,000个被称为基因的“章节”，编码人体内的蛋白质。尽管人体中数以万亿计的活细胞中的每一个都含有这种基因组DNA的相同副本，但在任何给定的时间，每个细胞都只使用这些基因的一个子集来制造蛋白质，这就是区分肝细胞和肌肉细胞的原因。生命科学研究中的一项艰巨挑战是确定细胞如何在分子水平上准确地选择哪些基因在给定的时间和背景下表达。我们细胞中的基因组DNA被组织成一种被称为染色质的致密结构，它涉及到在细胞核中将DNA包裹在由称为组蛋白的蛋白质组成的支架上。细胞调节(开启或关闭)基因表达的一种方式是用不同的化学基团标记这些组蛋白蛋白，以标记在该细胞类型中哪些基因将是活跃的或保持不活跃的。这些标记或“染色质修饰”被称为染色质修饰复合体的一组专门的“蛋白质纳米机器”“写入”和“擦除”。我的研究计划的长期目标是了解染色质修饰复合体如何选择性地修饰组蛋白和调节基因表达的分子机制。我的第一笔NSERC发现补助金使我和我的学员能够建立一个综合的实验平台，用于表征染色质修饰复合体的生化特性。这一平台使我们能够完成三项试验性研究，对三个具有代表性的成员的分子结构产生了新的理解，这些成员来自一组专门的染色质修饰复合体，称为组蛋白乙酰转移酶(HAT)复合体。在此基础上，我们提议应用一种名为冷冻电子显微镜(Cryo-EM)的尖端分子成像方法，在原子水平上详细显示这些复合体中不同的成分是如何排列的，以及它们如何相互配合以添加化学标记来修饰组蛋白。从这项研究中获得的知识将有助于填补我们在理解细胞分化和多细胞生物体发育过程中如何建立和改变基因表达模式的关键知识空白。参与这项研究计划的HQP将获得独特的机会，发展先进的技术技能，如冷冻-EM、项目管理、沟通和科学写作技能，使他们能够在以研究为导向的就业市场上具有很强的竞争力，并为加拿大知识驱动型经济的增长做出贡献。

Much like a recipe book, the human genome contains detailed instructions for building a multi-cellular human being. This recipe book, written in the form of genomic DNA, is organized into ~20,000 individual “chapters” called genes, encoding proteins in the human body. Although every one of the trillions of living cells in the human body contains an identical copy of this genomic DNA, each cell only uses a subset of these genes to make proteins at any given time, and this is what differentiates, for example, a liver cell from a muscle cell. A formidable challenge in life science research is to determine how, at the molecular level, a cell precisely selects which genes to express at a given time and context. Genomic DNA in our cells is organized into a dense structure known as chromatin, which involves wrapping of DNA around a scaffold made by proteins called histones, in the nucleus of a cell. One way that a cell regulates (turns on or off) gene expression is by “labeling” these histone proteins with different chemical groups to mark which genes will be active or remain inactive in that cell type. These labels or “chromatin modifications” are “written” and “erased” by ensemble group of specialized “protein nanomachines” known as chromatin-modifying complexes. The long-term objective of my research program is to understand the molecular mechanisms of how chromatin-modifying complexes selectively modify histones and regulate gene expression. My first NSERC Discovery Grant has enabled my trainees and me to establish an integrated experimental platform for characterizing the biochemical properties of chromatin-modifying complexes. This platform allowed us to complete three pilot studies that generated novel understanding of the molecular structures of three representative members from a specialized group of chromatin-modifying complexes known as histone acetyltransferase (HAT) complexes. Building on this foundation, we are proposing to apply a cutting edge molecular imaging approach known as cryo-electron microscopy (cryo-EM) to visualize, at atomic level detail, how the different components are arranged within these complexes and how they work in concert with one another to add chemical labels to modify histones. Knowledge gained from this research will help fill a key knowledge gap in our understanding of how gene expression pattern is established and altered during cell differentiation and the development of a multicellular organism. The HQP involved in this research program will gain unique opportunities to develop advanced technical skills such as cryo-EM, project management, communications, and scientific writing skills that would allow them to be highly competitive in the research-oriented job market and contribute to the growth of Canada's knowledge-driven economy.