Engineering and Imaging 3D genome structure-function dynamics across time scales

哺乳动物基因组折叠成数以万计的长程循环相互作用。一个 关键的未知数是染色质环是否以及如何控制基因表达，以及一个主要的未知因素。 未解决的问题是循环的时间进展如何与转录动力学相关。 回答这个问题的一个主要障碍是循环在一系列时间尺度上发生变化， 需要使用适合跟踪和工程循环的工具和模型系统 纵向和实时的短时间和长时间。在此，我们建议开发和 应用新的工程和成像工具来精确测量、诱导和扰动环路 三种不同生物系统的时间控制跨越几分钟、几小时和几周。在 在最短的时间尺度（分钟，目标 1）中，我们将检查人类诱发的循环动力学 多能干细胞衍生的神经元对电刺激做出反应，揭示了如何 相互作用频率在功能上与立即早期和早期的转录爆发相关。 次级反应基因。在小时的时间尺度上（目标 3），我们将阐明如何 结构蛋白 YY1 连接增强子-启动子环，并在退出时重新组装 由红细胞有丝分裂产生。在周的时间尺度上（目标 2），我们将使用蜂窝“时间 机器”纵向追踪经历细胞重编程的稀有细胞，使我们能够 剖析单细胞和亚细胞环形成和溶解的功能 体细胞重编程为多能性和黑色素瘤转变过程中的解决 癌细胞产生耐药表型。我们的团队由高效且协作的团队组成 一组具有互补专业知识和重叠兴趣的初级和高级研究人员， 包括 Gerd Blobel 博士（表观遗传学、有丝分裂、环工程）、Eric Joyce 博士（Oligopaints） 成像），Bomyi Lim 博士（新生转录活细胞成像），Jennifer Phillips-Cremins 博士 （染色质结构、环工程、神经生物学），Stanley Qi 博士（CRISPR 基因组） 工程、活细胞成像）和 Arjun Raj 博士（单细胞基因组学、RNA 成像、 重新编程）。我们将开发并应用染色质活细胞和固定细胞成像技术 接触，并在同一细胞中对新生转录进行成像。我们将建设一支综合型干部队伍 以时间依赖性诱导方式设计循环的结构蛋白。成功 我们的工程和成像工具在生物系统中的应用将产生 全面、严格地评估循环之间的因果关系 以及跨时间尺度的独特生物表型。

The mammalian genome folds into tens of thousands of long-range looping interactions. A critical unknown is whether and how chromatin loops control gene expression, and a major unresolved question is how the temporal progression of loops relates to transcription dynamics. One major barrier to answering this question is that loops change on a range of timescales, necessitating the use of tools and model systems amenable to tracking and engineering loops longitudinally and in real time on both short and long timing. Here, we propose to develop and apply new engineering and imaging tools to measure, induce, and perturb loops with precise temporal control in three different biological systems spanning minutes, hours, and weeks. At the shortest timescale (minutes, Aim 1), we will examine loop dynamics in human induced pluripotent stem cell-derived neurons in response to electrical stimulation, revealing how interaction frequency is functionally connected to transcriptional bursting of immediate early and secondary response genes. On the timescale of hours (Aim 3), we will elucidate how the architectural protein YY1 connects enhancer-promoter loops that re-assemble upon the exit from mitosis by erythroid cells. On the timescale of weeks (Aim 2), we will use a cellular “Time Machine” to longitudinally track the rare cells that undergo cellular reprogramming, allowing us to dissect the functionality of loop formation and dissolution with single-cell and subcellular resolution during the reprogramming of somatic cells to pluripotency and transition of melanoma cancer cells to a resistant phenotype. Our team consists of a highly productive and collaborative set of junior and senior investigators with complementary expertise and overlapping interests, including Dr. Gerd Blobel (epigenetics, mitosis, loop engineering), Dr. Eric Joyce (Oligopaints imaging), Dr. Bomyi Lim (nascent transcript live cell imaging), Dr. Jennifer Phillips-Cremins (chromatin architecture, loop engineering, neurobiology), Dr. Stanley Qi (CRISPR genome engineering, live cell imaging), and Dr. Arjun Raj (single cell genomics, RNA imaging, reprogramming). We will develop and apply live and fixed cell imaging techniques for chromatin contacts, and in the same cells image nascent transcription. We will build a cadre of synthetic architectural proteins to engineer loops in a time-dependent inducible manner. Successful application of our engineering and imaging tools across biological systems will yield a comprehensive and rigorous assessment of the cause-and-effect relationship between loops and distinct biological phenotypes across timescales.