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

工程与成像跨时间尺度的 3D 基因组结构-功能动态

基本信息

批准号：
10456233
负责人：
Gerd A Blobel
金额：
$ 110.84万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-16 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10456233
关键词：
3-Dimensional Adopted Amalgam Architecture Biological Biological Models Biological Process Cell Cycle Stage Cell Nucleus Cell Separation Cells ChIP-seq Characteristics Chromatin Chromatin Loop Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complex DNA DNA sequencing Data Development Electric Stimulation Engineering Enhancers Epigenetic Process Erythroid Cells Event Frequencies G1 Phase Gene Expression Genes Genetic Materials Genetic Transcription Genome Genome engineering Hi-C Hour Human Image Imaging Device Imaging Techniques Imaging technology Immediate-Early Genes Induced pluripotent stem cell derived neurons Light Link Maps Measures Mediating Metaphase Methodology Mitosis Mitotic Monitor Mus Neurobiology Neurons Peptide Sequence Determination Pharmaceutical Preparations Phase Transition Phenotype Plant Roots Population Proteins Qi RNA Reporter Research Personnel Resistance Resolution Role Somatic Cell Stimulus Structure Structure-Activity Relationship System Technology Testing Time Transcript YY1 Transcription Factor base biological systems cancer cell cancer therapy cell type cellular imaging cohesin design experimental study genome-wide genomic RNA induced pluripotent stem cell insight interest live cell imaging mammalian genome melanoma new technology pluripotency promoter response single molecule time use tool ultra high resolution

项目摘要

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.

哺乳动物的基因组折叠成成千上万的远程循环相互作用。一个关键未知是染色质是否以及如何控制基因表达和主要未解决的问题是与转录动力学相关的回路的临时进展。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，需要使用工具和模型系统，可用于跟踪和工程循环在短时间和长时间进行纵向和实时。在这里，我们建议开发和应用新的工程和成像工具以精确度量，诱导和散发循环在三种不同的生物系统中进行临时控制，跨越了几分钟，小时和几周。最短的时间尺度（分钟，目标1），我们将检查人类诱导的循环动态响应电刺激的多能干细胞衍生的神经元，揭示了如何相互作用频率在功能上连接到立即的转录爆发，次要反应基因。在小时的时间尺度上（AIM 3），我们将阐明如何建筑蛋白YY1连接重新组装出出口的增强剂启动器循环来自红细胞细胞有丝分裂。在几周的时间尺度上（AIM 2），我们将使用蜂窝机器”纵向跟踪经历细胞重编程的稀有单元，使我们允许我们剖析循环形成的功能，并用单细胞和亚细胞溶解在体细胞重新编程中分辨为多能性和黑色素瘤过渡癌细胞具有抗性表型。我们的团队由高产和协作组成具有完整专业知识和重叠兴趣的初级和高级调查员集，包括Gerd Blobel博士（表观遗传学，有丝分裂，Loop Engineering），Eric Joyce博士（寡头成像），Bomyi Lim博士（新生的转录本活细胞成像），Jennifer Phillips-Cremins博士（染色质体系结构，循环工程，神经生物学），Stanley Qi博士（CRISPR基因组工程学，活细胞成像）和Arjun Raj博士（单细胞基因组学，RNA成像，重新编程）。我们将开发和应用染色质的实时和固定细胞成像技术触点，在相同的细胞中图像新生的转录。我们将建立一个合成的干部建筑蛋白以时间依赖性的诱导方式进行工程循环。成功的我们的工程和成像工具在生物系统中的应用将产生循环之间的因果关系的全面和严格评估以及跨时标的不同的生物表型。