Quantitatively probing intra-nucleosomal chromatin variation and function

 描述(申请人提供)：许多表观遗传信息被认为编码在对包装基因组的组蛋白的潜在可遗传化学修饰的标识和定位中，修饰条件结合伙伴与其结合，从而转导下游功能结果。染色质的基本重复单位是核小体核心颗粒，它是组蛋白的两重对称八聚体，被两个超螺旋DNA所覆盖。这种体系结构将每个核心组蛋白的两个副本放置在指定的位置，每个副本都从结构的核心向受到密集翻译后修饰的非结构化尾巴伸出。对于给定的修改，为 每个染色质的基本重复单位有两个不同的可修改位点，从而编码了有意义的信息吗？有迹象表明，在这个水平上的变异是高度调控的，但由于缺乏可以测量这些特性的工具，人们对染色质修饰的这种规模知之甚少。我们开发了一种突破性的校准芯片技术，使我们能够第一次查询这种水平的核小体亚结构细节。在目标1中，我们将用我们校准的芯片方法直接量化核小体中组蛋白修饰的对称性，然后探索这种新的可测量的染色质属性的功能。鉴于结合伙伴的识别单位是整个核小体和侧翼DNA，而不仅仅是尾巴，这一水平上的确切差异在空间上是如何表现的，很可能是区分的一个重要因素。为此，我们最近发展了生化证据，证明核小体结合伙伴作为标记对称性的函数而区别对待。我们试图了解这种前所未有的认知水平的机制特性，无论是在其生物物理细节上，还是在其对细胞和生物体的功能后果方面。在目标2中，我们将使用校准的顺序芯片实验来定义二价结构域的分子性质-似乎是规范地激活和抑制组蛋白修饰修饰多能细胞中的关键发育基因-使用一套详尽的内部标准进行校准。然后，我们将研究它们的生物发生和预测能力作为分化的障碍。我们期望这项研究的结果将阐明亚核小体标记识别和功能的一般原理，形成一个令人信服的论点，即这种相对未被探索的染色质修饰水平对于基因组管理是重要的。

 DESCRIPTION (provided by applicant): Much epigenetic information is thought to be encoded in the identity and localization of potentially heritable chemical modifications to histone protein that package the genome, to which modification-contingent binding partners bind, thereby transducing downstream functional consequences. The fundamental repeating unit of chromatin, the nucleosome core particle, is a two-fold symmetric octamer of histone proteins enshrouded by two superhelical turns of DNA. This architecture places the two copies of each core histone in defined positions each projecting unstructured "tails" from the core of the structure to that are subject to dense posttranslational modification. For a given modification, is there meaningful information encoded by having two distinct modifiable sites per fundamental repeating unit of chromatin? There are hints that variation at this level is highly regulated, yet little is known about this scale of chromatin modifications owing to lack of tools that can measure these properties. We have developed a breakthrough calibrated ChIP technology that permits us to query this level of nucleosome sub-structure detail for the first time. In Aim 1 we will directly quantify the symmetry of histone modifications within nucleosomes with our calibrated ChIP method, then probe the function of this newly measurable chromatin property. Given that the unit of recognition for binding partners entire nucleosome and flanking DNA, as opposed to merely the tails, precisely how variation at this level spatially manifests is likely tobe an important element of discrimination. To this end, we have recently developed biochemical evidence that nucleosomal binding partners discriminate as a function of mark-symmetry. We seek to understand the mechanistic properties of this unprecedented level of recognition, both in its biophysical details and its functional consequences for cells and organisms. In Aim 2 we will define the molecular nature of bivalent domains-the seeming apposition of canonically activating and repressive histone modifications decorating critical developmental genes in pluripotent cells-- using calibrated sequential ChIP experiments calibrated with an exhaustive set of internal standards. We will then examine their biogenesis and predictive power as barriers to differentiation. We expect that the results of this study will illuminate the general principlesof sub-nucleosomal mark recognition and function, forming a compelling argument that this relatively un-explored level of chromatin modification is important for genome management.