Structural and functional studies of histone acetyltransferase complexes

就像一本食谱一样，人类基因组包含了构建多细胞人类的详细说明。这本食谱以基因组DNA的形式编写，被组织成约20，000个称为基因的独立“章节”，编码人体中的蛋白质。虽然人体内数万亿个活细胞中的每一个都含有相同的基因组DNA拷贝，但每个细胞在任何给定时间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个巨大挑战是确定细胞如何在分子水平上精确地选择在给定时间和环境下表达哪些基因。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，它涉及将DNA包裹在细胞核中由称为组蛋白的蛋白质制成的支架周围。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，以标记哪些基因在该细胞类型中将是活跃的或保持不活跃。这些标记或“染色质修饰”被称为染色质修饰复合物的专门的“蛋白质纳米机器”的集合组“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物如何选择性地修饰组蛋白和调节基因表达的分子机制。我的第一个NSERC发现补助金使我的学员和我建立了一个综合的实验平台，用于表征染色质修饰复合物的生化特性。该平台使我们能够完成三项试点研究，这些研究对三个代表性成员的分子结构产生了新的理解，这些成员来自一组被称为组蛋白乙酰转移酶（HAT）复合物的染色质修饰复合物。在此基础上，我们建议应用一种称为冷冻电子显微镜（cryo-EM）的尖端分子成像方法，以原子水平的细节来可视化这些复合物中不同组分的排列方式，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理解基因表达模式在细胞分化和多细胞生物体发育过程中如何建立和改变的关键知识空白。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cryo-EM，项目管理，沟通和科学写作技能，使他们在以研究为导向的就业市场中具有高度竞争力，并为加拿大知识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.