Genome Architecture and Gene Control in Response to Stress

应激反应中的基因组结构与基因调控 基因组的3D拓扑在健康、疾病和疾病的转录调控中起着关键作用 发展。基因组采用跨细胞维持的离散的结构和调控结构域 种类，甚至生物体。然而，尽管这一标准拓扑中的变化正在变得越来越明显 在发育和疾病中都发挥着重要作用，但人们对这种动态变化的机制知之甚少 控制重组过程。此外，目前尚不清楚基因组的拓扑结构是否(或如何) 有助于协调对细胞应激反应至关重要的基因的表达。 我们已经建立了一个系统，在这个系统中，我们可以 引发戏剧性的架构重新安排 基因和基因之间的协调一致 在一组细胞中同步。这个 系统，萌芽酵母的热休克反应 酿酒酵母，使我们能够利用强大的 这种有机体的遗传可控性来定义 影响因素并揭示驱动因素的机制 基因组结构和核重组。 此外，热休克蛋白(HSP)基因和 控制其表达的转录调控因子， 热休克因子1(HSF1)在进化过程中 保守的，对健康和疾病至关重要的。使用 高度敏感和定量的版本 我们的染色体构象捕捉(3C) 实验室开发的，称为Taq I-3C，我们获得了证据表明，在对急性热应激的反应中， HSP基因经历了强烈的基因内相互作用，包括UA和启动子元件之间的循环， 启动子和终止子区域以及调控和编码区。更令人震惊的是，他们参与了 频繁的染色体内和染色体间的相互作用，聚集成离散的核内病灶。这些基因是 热休克-由另一种转录因子(TF)激活，MSN2，同样也是环，但似乎还没有 结合，或者与自身或与HSF1靶基因结合。同样，强劲的转录，构成 表达的基因经历了基因内循环，但这些基因似乎也没有合并。除了他们的 HSF1靶基因的基因内和基因间重组/重组是独特的结合 显著的动态：可在60秒内检测到，2.5分钟内达到峰值，30分钟内衰减。这些 观察提出了一些重要的问题。为了解决这些问题，我们提出了三个目标： 目的1：阐明酵母基因组在热休克和其他应激过程中的三维拓扑结构，以及 HSF1和POLII在协调这些变化中发挥的作用。我们将利用尖端的深度 基于测序的方法，主要是Taq I-Hi-C，以揭示热休克和因子依赖 整个基因组的染色体拓扑动态。我们将定义HSF1和POL II的作用，以及其他 在AIMS 2和AIMS 3中确定的因素，驱动暴露在热应激下的细胞的3D基因组结构。我们会 使用Taq I-3C确认关键发现。 目的2：阐明Pol II和HSF1在推动HSP基因融合中的决定因素和测试概念 HSP冷凝物通过液-液相分离进行聚集。我们将确定功能 HSF1和POL II中的结构域，负责将HSP基因驱动到暴露细胞的结合灶中 并探讨动态热休克蛋白冷凝物的生物物理性质。 目的3：揭示转录辅活化子、染色质重构体和建筑蛋白的作用 在驱动HSF1靶基因内部和之间的特定和动态相互作用方面。 我们将检验这一假设，即HSP基因合并代表多个辅助因子的协同作用- 由HSF1招募并与POL II协调行动--并调查选定因素所作的贡献， 重点关注那些在融合能力细胞中优先招募到热休克蛋白基因的基因。我们会 将Taq I-3C检测与一系列强大的酵母遗传技术相结合-有条件的 核耗竭、条件性蛋白质降解、基因组编辑--来询问这些因素的作用。 总之，提出的实验将揭示机械论的洞察力和广泛的、全基因组的 酵母热休克过程中依赖于HSF1的动态3D基因组重塑的研究进展 回应。他们将对热休克蛋白基因融合的生物学意义进行未来的探索， 由这里提出的实验结果提供信息。

Genome Architecture and Gene Control in Response to Stress The 3D topology of the genome plays a critical role in transcriptional regulation in health, disease and development. The genome adopts discrete structural and regulatory domains that are maintained across cell types and even organisms. However, while it is becoming clear that alterations in this standard topology play important roles in both development and disease, very little is known about the mechanisms that dynamically control the restructuring process. Moreover, it is unknown whether (or how) the topology of the genome contributes to the coordination of expression of genes critical to the cell’s response to stress. We have established a system in which we can induce dramatic architectural rearrangements concerted within and between genes and synchronized across a population of cells. The system, the heat shock response in the budding yeast S. cerevisiae, allows us to leverage the powerful genetic tractability of this organism to define the factors and uncover the mechanisms that drive genome architecture and nuclear reorganization. Moreover, Heat Shock Protein (HSP) genes and the transcriptional regulator that controls their expression, Heat Shock Factor 1 (Hsf1), are evolutionarily conserved and critical for health and disease. Using a highly sensitive and quantitative version of chromosome conformation capture (3C) that our laboratory developed, termed Taq I - 3C, we have obtained evidence that in response to acute thermal stress, HSP genes undergo intense intragenic interactions that include looping between UAS and promoter elements, promoter and terminator regions and regulatory and coding regions. Even more striking, they engage in frequent intra- and interchromosomal interactions, coalescing into discrete intranuclear foci. Genes that are heat shock-activated by an alternative transcription factor (TF), Msn2, likewise loop yet do not appear to coalesce, either with themselves or with Hsf1-target genes. Likewise, robustly transcribed, constitutively expressed genes undergo intragenic looping yet these genes too do not appear to coalesce. In addition to their distinctive coalescence, the intragenic and intergenic restructuring/reorganization of Hsf1-target genes is remarkably dynamic: detectable within 60 sec, peaking within 2.5 min and attenuating within 30 min. These observations raise important questions. To address these, we propose three aims: Aim 1: Elucidate the 3D topology of the yeast genome during heat shock and other stresses, and the role played by Hsf1 and Pol II in orchestrating these changes. We will utilize cutting-edge deep sequencing-based approaches, principally Taq I – Hi-C, to reveal heat shock- and factor-dependent chromosomal topology dynamics across the genome. We will define the role of Hsf1 and the Pol II, and other factors identified in Aims 2 and 3, in driving 3D genome architecture in cells exposed to thermal stress. We will confirm key findings using Taq I - 3C. Aim 2: Elucidate determinants of Pol II and Hsf1 in driving HSP gene coalescence and test notion that HSP condensates assemble through liquid-liquid phase separation. We will identify the functional domains within Hsf1 and Pol II that are responsible for driving HSP genes into coalesced foci in cells exposed to acute HS, and explore the biophysical nature of the dynamic HSP condensates. Aim 3: Unveil the roles of transcriptional coactivators, chromatin remodelers and architectural proteins in driving the specific and dynamic interactions within and between Hsf1-target genes. We will test that hypothesis that HSP gene coalescence represents the concerted action of multiple cofactors – recruited by Hsf1 and acting in concert with Pol II – and investigate the contribution made by select factors, focusing on those that are preferentially recruited to HSP genes in coalescence-competent cells. We will exploit the Taq I-3C assay in combination with an array of powerful yeast genetic techniques – conditional nuclear depletion, conditional protein degradation, genome-editing – to interrogate the role of these factors. Together, the experiments proposed will reveal both mechanistic insight and a broad, genome-wide perspective on the dynamic, Hsf1-dependent 3D genome remodeling that occurs during the yeast heat shock response. They will set up a future exploration of the biological significance of HSP gene coalescence, informed by results of experiments proposed here.