Control of the 4D chromatin landscape underlying gene activity during development

摘要 现代生物学的重大挑战之一是了解基因活动在太空中是如何控制的，以及 时间，在天然染色体和个体活细胞的背景下。这项提议的目标是解决 正是这一挑战：我们将开发新的方法来测量和操纵远程染色体 并量化它们对基因表达的影响，实时和在活的细胞和组织中。通过 定量绘制转录因子组装之间的关系(例如，生物分子的形成 凝聚体)、染色体组织和转录动力学，我们的研究将定义基因表达如何 以前所未有的分辨率控制。 转录调控是生物体发育过程中细胞分化的基础，其 缺陷是各种疾病状态的基础，从发育障碍到癌症。然而，目前的方法是 局限性：传统的活体成像缺乏空间分辨率，无法准确定义 单个基因的规模，而使用固定材料的批量分析不适合研究时间动力学。在……里面 此外，通过液-液分离形成的无膜核冷凝被认为是 在转录调控中扮演关键但尚未确定的角色。 为了应对这些挑战，我们将开发新的成像方法来测量染色体距离 并建立光遗传工具来组装/拆解染色体环和核凝聚体。 我们将部署这些工具来检查基因组水平上的调控相互作用，这些相互作用具有增强子- 在果蝇和哺乳动物中的启动子相互作用(从几十到几百个碱基)，并研究它们在 果蝇胚胎发育过程中细胞命运指定的背景。由此产生的技术将是 应用于小鼠胚胎干细胞和从这些细胞衍生的器官中类似的转录位点。 总之，拟议的研究将有助于揭示细胞类型规范的健壮机制是如何从 随机过程，如转录突发、生物分子大小和稳定性的波动 凝聚体和染色质结构的动态不稳定性。这个项目的总体目标是建立一个 染色质结构和转录活性之间的定量联系，这最终将使我们能够 通过重新设计导致发育和疾病的转录程序来控制基因活性 流程。

Summary One of the grand challenges of modern biology is to understand how gene activity is controlled in space and time, in the context of native chromosomes and in individual living cells. The goal of this proposal is to tackle exactly this challenge: we will develop new approaches to measure and manipulate long-range chromosomal interactions and quantify their effects on gene expression, in real-time and in living cells and tissues. By quantitatively mapping the relationship between transcription factor assembly (e.g. formation of biomolecular condensates), chromosome organization and transcription kinetics, our study will define how gene expression is controlled at unprecedented resolution. Transcriptional regulation forms the basis of cellular differentiation during organismal development, and its defects underlie a variety of disease states, from developmental disorders to cancer. Yet current methods are limited: traditional live-imaging lacks the spatial resolution to accurately define chromosome organization at the scale of individual genes, while bulk assays using fixed material are ill-suited for studying temporal dynamics. In addition, membrane-less nuclear condensates, which form through liquid-liquid phase separation, are thought to play key but as-yet-undefined roles in regulating transcription. To address these challenges, we will develop new imaging methods to measure chromosomal distances in living cells and build optogenetic tools to assemble/disassemble chromosome loops and nuclear condensates. We will deploy these tools to examine regulatory interactions at genomic scales characteristic of enhancer– promoter interactions in flies and mammals (from tens to hundreds of kilobases), and study their implications in the context of cell fate specification in the developing Drosophila embryo. The resulting technologies will be applied to analogous transcriptional loci in mouse embryonic stem cells and organoids derived from these cells. Together, the proposed studies will help reveal how robust mechanisms of cell type specification emerge from stochastic processes such as transcriptional bursts, fluctuations in the size and stability of biomolecular condensates, and dynamic instability of chromatin architecture. The overall goal of this project is to establish a quantitative link between chromatin architecture and transcriptional activity, which will ultimately allow us to take control of gene activity by re-engineering the transcriptional programs underlying developmental and disease processes.