Reading the histone code:nanoscale morphology of Epigneomic Histone Modifications

描述（由申请人提供）：挑战领域：06使能技术。具体挑战题目：06-GM-101大分子配合物结构分析。染色质生物学和分子细胞学的一个主要挑战是如何在纳米分辨率下研究单细胞中特异性表观遗传染色质修饰的大分子结构。组蛋白的许多翻译后修饰在定义染色体的生物学功能中起着关键作用。有不同的修饰组与转录活性染色质、非活性染色质、复制染色质、染色体损伤位点以及关键的亚核室（如着丝粒和端粒）相关。这些表观遗传标记的缺陷，即“读”、“写”和“擦除”这些标记的酶和蛋白质的缺陷，已被发现出现在许多人类疾病中，包括癌症和神经退行性综合征。此外，这些表观遗传标记是干细胞生物学中的关键决定因素，在维持多能状态和驱动分化方面都很重要。目前，还没有技术可以在分辨率超过200-300 nm的单细胞中可视化表观遗传组蛋白修饰的大分子结构。光学显微镜的分辨率受限于衍射，而电子显微镜的分辨率受限于缺乏对比度。包括质谱、染色质免疫沉淀和染色体构象捕获在内的生化技术，在定义组蛋白标记的功能组合方面取得了长足的进步，但它们不能对细胞核内的结构进行成像，也不能在活细胞中跟踪它们的动态。这就是挑战所在。为了满足这一需求，我们设计了新的组蛋白修饰探针，通过将多价结合域融合到光激活的荧光团上，并在细胞中表达这些“解码器”结构。利用超分辨率显微镜的最新进展，这些报告者的位置可以以6-10纳米的定位精度确定，以低于30纳米染色质纤维宽度的分辨率重建修饰图像。该项目的目标是利用这一原理证明并开发技术，使研究人员能够在比目前可能的更精确的数量级上探索染色质的大分子结构。今后两年，我们将努力实现三个主要目标。(1)我们将构建一套高质量、多功能的解码器结构，代表所有已知的组蛋白修饰结合基序。(2)我们将通过比较这些解码器与传统抗体探针的共定位，在染色质免疫沉淀中对结合DNA进行全基因组测序，并在三维和活细胞中对报告者进行成像，来表征这些解码器的特性和结合特异性。(3)我们将通过合理设计嵌合、人工多价和合成结合基序组合来构建预计具有新的结合特异性的解码器。这些努力的结果将发展技术，使染色质表观遗传学的纳米视野的常规可视化。这将通过提供工具、试剂和协议来影响基础研究，这些工具、试剂和协议将引导染色质研究的范式转变。此外，由于染色质修饰对癌症、神经变性、胚胎发育、辅助生殖服务和未来的干细胞治疗具有实际意义，因此在单个活细胞中以纳米级分辨率快速成像表观遗传标记的能力有可能从根本上改善与广泛的人类健康问题相关的诊断、分类和治疗方式。染色体上蛋白质的复杂修饰在调节细胞生理和保持细胞正常和健康方面起着重要作用。研究这些变化如何起作用的一个严重限制是，我们无法看到它们，观察它们的结构组织，或者观察它们是如何来的和去的。克服这一限制是一项艰巨的挑战，将对基础和临床健康科学产生巨大影响，包括癌症、神经退行性综合征和干细胞治疗。该项目将利用荧光显微镜的突破，以及对修饰生物物理学的最新见解，开发技术，使研究人员能够首次在单个活细胞中以纳米级分辨率看到这些修饰的结构。

DESCRIPTION (provided by applicant): Challenge Area: 06 Enabling Technologies. Specific Challenge Topic: 06-GM-101 Structural Analysis of Macromolecular Complexes. A major challenge in chromatin biology and molecular cytology is how to study the macromolecular structures of specific epigenetic chromatin modifications in single cells at nanoscale resolution. The many post-translational modifications of histone proteins play critical roles in defining the biological functions of chromosomes. There are different sets of modifications associated with transcriptionally active chromatin, with inactive chromatin, with replicating chromatin, with sites of chromosome damage, and with key subnuclear compartments such as centromeres and telomeres. Defects in these epigenetic marks, in the enzymes and proteins that "read", "write", and "erase" them, have been found to occur in many human diseases, including cancer and neural degenerative syndromes. Furthermore, these epigenetic marks are key determinants in stem cell biology, and are important both in maintaining the pluripotent state and in driving differentiation. At present, there are no technologies that can visualize the macromolecular structures of epigenetic histone modifications in single cells at resolutions any better than approximately 200-300 nm. The resolution of light microscopy is limited by diffraction, and the resolution of electron microscopy is limited by lack of contrast. Biochemical techniques including mass spectroscopy, chromatin immunoprecipitation, and chromosome conformation capture, are making great strides in defining the functional combinations of histone marks, but they cannot image those structures within the nucleus or follow their dynamics in live cells. That is the challenge. To meet it, we have designed novel probes of histone modification by fusing multivalent binding domains to photoactivatable fluorophores and expressing these "decoder" constructs in cells. Using recent advances in super-resolution microscopy, the positions of these reporters can be determined with a localization precision of 6-10 nm, reconstructing the image of modifications at a resolution below the width of the 30 nm chromatin fiber. The goal of this project is to exploit this proof of principle and develop the technology to enable researchers to explore the macromolecular structures of chromatin at levels that are an order of magnitude more precise than is currently possible. Over the next two years we will address three major aims. (1) We will construct a high quality, versatile set of decoder constructs that represent all of the known histone modification binding motifs. (2) We will characterize the properties and binding specificities of these decoders by comparing their co-localization with traditional antibody probes, by conducting genome-wide sequencing of bound DNA in chromatin immunoprecipitations, and by imaging the reporters in three-dimensions and in live cells. (3) We will construct decoders that are predicted to have novel new binding specificities through the rational design of chimeric, artificial multivalent, and synthetic binding motif combinations. The results of these efforts will develop the technology to enable the routine visualization of the nanoscape of chromatin epigenetics. This will impact basic research by providing the tools, reagents, and protocols that will marshal a paradigm shift in how chromatin is studied. Moreover, since chromatin modifications have real practical importance for cancer, neural degeneracies, embryonic development, assisted reproductive services, and future stem cell therapies, the ability to image epigenetic marks rapidly, in single live cells, at nanoscale resolutions has the potential to radically improve the diagnosis, classification, and treatment modalities associated with a wide spectrum of human health issues. Complex modifications of proteins on the chromosomes have major roles in regulating cellular physiology and keeping cells normal and healthy. A severe limitation in studying how these modifications work is the fact that we cannot see them, observe their structural organizations, or watch how they come and go. Overcoming this limitation is a formidable challenge that will have enormous impact on both basic and clinical health science, including cancer, neurodegenerative syndromes, and stem cell therapies. This project will exploit breakthroughs in fluorescent light microscopy, and recent insights into the biophysics of the modifications, to develop technology that will enable researchers to see the structures of these modifications for the first time at nanoscale resolution in single live cells.