Nanoscale tools for functional studies of cancer-relevant chromatin modifications

描述（由申请人提供）：人类基因组动态组织成染色质调节转录起始和延伸。染色质修饰、组装、拆卸和重塑的缺陷导致癌基因的失调，这与许多癌症相关，包括卵巢癌、膀胱癌、前列腺癌和结肠直肠癌。先前的研究已经确定了参与染色质转录调控（CTR）的组分，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达到的10-100 nm长度尺度的染色质组装体的动力学和结构，这包括调节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位点的中尺度（10- 100 nm）结构和染色质动态。具体来说，我们将开发1）具有多种抗体的DNA折纸纳米结构，这些抗体识别染色质标记（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，以研究基因调控位点的位点特异性中尺度动力学。这项工作的长期目标是开发工具和方法，以探测体内癌症相关染色质修饰和癌基因调控位点的染色质功能和动力学。在这项探索性研究的范围内，我们将专注于设备开发，体外原理验证和染色质组装的表征。未来的工作将建立在这里建立的工具和实验框架，以实现DNA折纸装置探测染色质组装的细胞内功能。

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.