Harnessing the chromatin conformational code for epigenetic regulation

项目摘要 真核DNA包裹在核小体周围，核小体形成染色质链，染色质链进一步折叠成 三维装配。这些装配体的结构调节许多核功能，包括 基因组3D折叠和转录，并最终决定细胞的身份。核小体是一个众所周知的枢纽 染色质调控的作用，其中大部分被认为是通过各种翻译后修饰发生的， 组蛋白的突出的柔性尾部。基于这样的假设，即大多数染色质结构的调节和 与其他因子的相互作用发生在这些组蛋白尾部，核小体的球状核心已经被 被认为是严格的和最低限度的监管。令人兴奋的是，我最近的工作揭示了一个新的见解： 核小体核心是可塑性的，这种可塑性调节染色质折叠和基因阻遏。因此我 我认为核小体的球状延展性核心也是遗传和表观遗传调控的中心 作为潜在的新治疗靶点。为了验证这个挑战教科书范式的挑衅性假设 染色质调控，新的工具，能够探测在体外和体内原子尺度的动态大 必须开发大分子组装体如染色质。我的实验室将通过开发 构象特异性纳米抗体（NanoNucs），其充当不同核小体构象的传感器。 NanoNucs将从含有>2 X IO 9个不同纳米抗体的合成文库中发现。我们会委聘 NanoNucs获得核小体构象动力学的结构和生物物理学见解，并探测 并扰乱细胞中的核小体构象密码。具体而言，我们将：（i）获得原子理解 通过结合NMR，HDX-MS和cryo-EM，核小体交替状态;（ii）鉴定染色质因子， 感测和利用核小体可塑性;（iii）寻找生物学或生物学上的核小体构象， 病理生物标志物;和（iv）开发一种新的策略来操纵核小体形状和染色质 细胞中的状态通过实施这个雄心勃勃的，综合的，多学科的研究计划，我的实验室 将揭示核小体构象密码的分子机制和治疗潜力。我 我预计这些高风险、高回报的研究将揭示基因组的新的基本原理 改变长期存在的刚性组蛋白单位范式的监管，将广泛影响生物医学 科学在短期和长期。探索核小体的结构灵活性代表了一种 有机会确定新的治疗生物标志物和药物的疾病与表观遗传学缺陷， 癌症最终，在NIH主任的新创新者计划的关键支持下，我们的研究将丰富 我们对染色质的功能和生理学的了解，以及对染色质的原子尺度生物物理学见解， chromatin染色质architecture建筑itself本身.

Project summary Eukaryotic DNA is wrapped around nucleosomes, which form chains of chromatin that are further folded into three-dimensional assemblies. The architecture of these assemblies regulates many nuclear functions, including genome 3D folding and transcription, and ultimately dictates cellular identity. Nucleosomes are a well-known hub of chromatin regulation, most of which is thought to occur via a variety of post-translational modifications on the protruding flexible tails of histones. Based on the assumption that most regulation of chromatin's structure and interactions with other factors occurs at these histone tails, the globular core of nucleosomes has been considered rigid and minimally regulatory. Excitingly, my recent work has revealed a new insight: that the nucleosome core is malleable and that this plasticity regulates chromatin folding and gene repression. I therefore propose that the globular, malleable core of nucleosomes is a hub for genetic and epigenetic regulation as well as a potential novel therapeutic target. To test this provocative hypothesis that challenges the textbook paradigm of chromatin regulation, novel tools capable of probing both in vitro and in vivo atomic-scale dynamics of large macromolecular assemblies such as chromatin must be developed. My lab will close this gap by developing conformation-specific nanobodies (NanoNucs) that act as sensors of distinct nucleosome conformations. NanoNucs will be discovered from a synthetic library containing >2 x 109 distinct nanobodies. We will employ NanoNucs to gain structural and biophysical insights into nucleosome conformational dynamics and to probe and perturb the nucleosome conformational code in cells. Specifically, we will: (i) obtain atomic understanding of nucleosome alternative states by combining NMR, HDX-MS, and cryo-EM; (ii) identify chromatin factors that sense and leverage nucleosome plasticity; (iii) search for nucleosome conformations that are biological or pathological biomarkers; and (iv) develop a novel strategy to manipulate nucleosome shapes and chromatin states in cells. By carrying out this highly ambitious, integrated, and multidisciplinary research program, my lab will unveil the molecular mechanisms and therapeutical potential of the nucleosome conformational code. I anticipate that these high-risk, high-reward investigations will reveal new fundamental principles of genome regulation that shift the long-standing paradigm of rigid histone units and that will broadly impact biomedical science over the short and long terms. Exploring the structural flexibility of nucleosomes represents an opportunity to identify novel therapeutic biomarkers and drugs for diseases linked to epigenetics defects, such as cancer. Ultimately, with critical support from the NIH Director's New Innovator Program, our studies will enrich our knowledge of the function and physiology of chromatin with atomic-scale biophysical insights into the chromatin architecture itself.