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

哺乳动物的基因组折叠成数万个长距离循环相互作用。一 关键的未知是染色质环是否以及如何控制基因表达， 尚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个时间尺度范围内变化， 这就需要使用适合于跟踪和工程循环的工具和模型系统 纵向地并且在真实的时间上在短定时和长定时两者上。在这里，我们建议发展和 应用新的工程和成像工具来测量，诱导和扰动回路， 时间控制在三个不同的生物系统跨越分钟，小时和星期。在 最短的时间尺度（分钟，目标1），我们将研究循环动力学在人类诱导 多能干细胞衍生的神经元对电刺激的反应，揭示了如何 相互作用频率在功能上与即时早期的转录爆发有关， 次级反应基因在小时的时间尺度上（目标3），我们将阐明 结构蛋白YY 1连接增强子-启动子环， 由红系细胞的有丝分裂而成。在以周为单位的时间尺度上（目标2），我们将使用一个细胞“时间 机器”纵向跟踪经历细胞重编程的稀有细胞， 用单细胞和亚细胞的方法来剖析环的形成和溶解的功能， 在体细胞重编程为多能性和黑色素瘤转变期间的分辨率 转化为耐药表型。我们的团队由一个高效和协作的 一组初级和高级调查员，他们具有互补的专门知识和重叠的兴趣， 包括Gerd Blobel博士（表观遗传学，有丝分裂，环工程），Eric Joyce博士（Oligopaints 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， 重编程）。我们将开发和应用染色质的活细胞和固定细胞成像技术 接触，并在相同的细胞图像新生转录。我们将建立一个综合的干部 结构蛋白以时间依赖性诱导方式工程化环。成功 我们的工程和成像工具在生物系统中的应用将产生一个 全面、严格地评估循环之间的因果关系 和不同的生物表型。

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.