Nanoscale tools for functional studies of cancer-relevant chromatin modifications

描述(由申请人提供)：人类基因组进入染色质的动态组织调节转录的起始和延伸。染色质修饰、组装、拆解和重塑的缺陷会导致癌基因的失调，这些癌基因与卵巢、膀胱癌、前列腺癌和结直肠癌等多种癌症有关。先前的研究已经确定了参与染色质转录调控(CTR)的成分，包括组蛋白变体和翻译后修饰(PTM)、组蛋白修饰酶和组蛋白伴侣组装因子。值得注意的是，遗传、生化、结构、深度测序和单分子研究尚未完全揭示CTR的机制。因此，需要新的技术来探测目前无法在10-100 nm范围内获得的染色质组件的动力学和结构，其中包括调节DNA加工的关键分子事件。这项研究将通过开发纳米级工具来解决目前的技术差距，这些工具可以测量特定癌症相关修饰和加工部位的染色质的中尺度(10-100 nm)结构和动力学。具体地说，我们将开发1)带有多种抗体的DNA折纸纳米结构，这些抗体可以识别染色质标记(组蛋白修饰/变体和基因组DNA加工位点)的不同生理和癌症相关组合，以及2)DNA折纸位移传感器，以研究基因调控部位特定位置的中尺度动力学。这项工作的长期目标是开发工具和方法来探索体内与癌症相关的染色质修饰和癌基因调控部位的染色质功能和动态。在这项探索性研究的范围内，我们将重点放在染色质组件的设备开发、体外原理验证和特性方面。未来的工作将建立在这里建立的工具和实验框架的基础上，以实现DNA折纸设备来探索染色质组件的细胞内功能。

DESCRIPTION (provided by applicant): Dynamic organization of the human genome into chromatin regulates transcription initiation and elongation. Defects in chromatin modifications, assembly, disassembly and remodeling result in misregulation of oncogenes, which are associated with numerous cancers including ovarian, bladder, prostate, and colorectal tumors. Prior research has identified the components involved in chromatin transcriptional regulation (CTR), including histone variants and post-translational modifications (PTMs), histone modification enzymes, and histone chaperone assembly factors. Remarkably, genetic, biochemical, structural, deep sequencing and single molecule studies have not fully revealed the mechanisms of CTR. Therefore, new technologies are required to probe currently inaccessible dynamics and structure of chromatin assemblies at the 10-100 nm length scale, which encompasses critical molecular events the regulate DNA processing. This research will address current technological gaps through the development of nanoscale tools that measure mesoscale (10- 100nm) structure and dynamics of chromatin at specific cancer-relevant modification and processing sites. Specifically, we will develop 1) DNA origami nanostructures with multiple antibodies that recognize distinct physiological and cancer-relevant combinations of chromatin marks (histone modifications/variants and genomic DNA processing sites) and 2) DNA origami displacement sensors to study site-specific mesoscale dynamics at gene regulation sites. The long-term goal of this work is to develop tools and methods to probe chromatin function and dynamics at cancer-relevant chromatin modifications and oncogene regulation sites in vivo. Within the scope of this exploratory research, we will focus on the devic development, in vitro proof-of- principle, and characterizations of chromatin assemblies. Future work will build on the tools and experimental framework established here to implement DNA origami devices to probe intracellular function of chromatin assemblies.