Conformational Control of Heterochromatin Formation by the HP-1 Protein from Fission Yeast

项目摘要 真核生物基因组被组织成称为常染色质的活性和非活性结构域， 异染色质这种功能组织在染色体分离、端粒 维持和基因组稳定性。异染色质的一个关键组成部分是HP-1蛋白家族， 与组蛋白3赖氨酸-9甲基标记结合，并作为多种调节剂的平台。一种生化活动 认为对异染色质扩散很重要的是HP-1蛋白的粘附能力。最近 对裂殖酵母HP-1蛋白Swi 6的研究揭示了聚合受自抑制调节。一 一个关键的和知之甚少的问题是封闭的，无活性的， 以及驱动异染色质扩散的Swi 6的开放活性形式。这种知识上的差距源于这样一个事实， HP-1蛋白及其与核小体的复合物在溶液中是构象动态的， 结晶。在这里，我们将定义Swi 6-染色质复合物的结构动力学，并将结构动力学联系起来。 国家发挥作用。在目的1中，我们将使用整合的方法确定Swi 6的自抑制形式的结构。 采用溶液中的限制NMR光谱和小角X射线散射的建模方法 （SAXS）。结构模型的生物学意义将在基因沉默试验中进行测试， 裂殖酵母S.粟酒在目标2中，我们将确定以下化合物的构象重排程度： 当Swi 6与核小体接合以形成伸展能力状态时，染色质被激活。结构和 核小体-Swi 6复合物的动力学将通过生物物理学方法的组合进行研究， 例如甲基-TROSY NMR和HD-交换质谱（MS），其可以提供残基特异性 解决方案中的结构信息。将采用交联质谱法结合cryoEM 以获得模型的扩展能力的形式，如果Swi 6绑定到核小体。这种方法将使 深入了解裂殖酵母中Swi 6对异染色质形成的构象控制， 在人类细胞中异染色质是如何调节的概念基础。

Project Summary Eukaryotic genomes are organized into active and inactive domains referred to euchromatin and heterochromatin. This functional organization plays an important role in chromosome segregation, telomere maintenance and genome stability. A key component of heterochromatin are the HP-1 family of proteins, which bind to a histone 3 lysine-9 methyl mark and act as a platform for diverse regulators. A biochemical activity thought to be important for the spread of heterochromatin is the ability of HP-1 proteins to polymerize. Recent work on the fission yeast HP-1 protein, Swi6, reveals that polymerization is regulated by autoinhibition. A critical and poorly understood question is the extent of the conformational transition between closed, inactive and open, active forms of Swi6 that drive heterochromatin spread. This gap in knowledge derives from the fact that HP-1 proteins and their complexes with nucleosomes are conformationally dynamic in solution and difficult to crystallize. Here we will define the structural dynamics of the Swi6-chromatin complex and link the structural states to function. In Aim 1 we will determine the structure of the autoinhibited form of Swi6 using an integrated modeling approach that employs restraints NMR spectroscopy and small-angle x-ray scattering in solution (SAXS). The biological significance of the structural models will be tested in gene silencing assays in the fission yeast S. pombe. In Aim 2, we will determine the degree of conformational rearrangements of chromatin when Swi6 engages the nucleosome to form a spreading competent state. The structure and dynamics of the nucleosome-Swi6 complex will be interrogated by a combination of biophysical methods, such as Methyl-TROSY NMR and HD-exchange mass-spectrometry (MS), that can provide residue specific structural information in solution. Cross-linking mass-spectrometry in conjunction with cryoEM will be employed to obtain models of the spreading competent form if Swi6 bound to nucleosomes. This approach will shed insights into conformational control of heterochromatin formation by Swi6 in fission yeast and provide a conceptual foundation for how heterochromatin is regulated in human cells.