Genome Architecture and Gene Control in Response to Stress

应对压力的基因组结构和基因控制

• 批准号: 10806024
• 负责人: David Samuel Gross
• 金额: $ 3.42万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10806024
• 关键词: 3-Dimensional; Acute; Address; Adopted; Affect; Architecture; Automobile Driving; Binding Sites; Biological; Biological Assay; Biophysics; Catalytic Domain; Cell Nucleus; Cells; Chemicals; Chromatin; Chromatin Remodeling Factor; Code; DNA Polymerase II; Development; Diffusion; Disease; Dissociation; Doctor of Philosophy; Elements; Exposure to; Fostering; Future; Gene Cluster; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Techniques; Genetic Transcription; Genome; Genomics; Health; Heat shock proteins; Heat-Shock Response; Hi-C; Laboratories; Light; Liquid substance; Mediator; Methodology; Methods; Molecular Conformation; Movement; Mutation; N-terminal; Nature; Nuclear; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Organism; Phase; Phosphorylation; Physical condensation; Play; Population; Positioning Attribute; Process; Promoter Regions; Proteins; RNA; Regulon; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Stress; Structural Genes; System; Tail; Techniques; Terminator Regions; Testing; Transcript; Transcription Coactivator; Transcriptional Regulation; Yeasts; cell type; chromosome conformation capture; cofactor; cohesin; cohort; condensin; deep sequencing; experimental study; genome editing; genome-wide; genome-wide analysis; genomic locus; heat-shock factor 1; insight; member; mutant; next generation sequencing; promoter; protein degradation; recruit; response; thermal stress; transcription factor; transcriptome sequencing; yeast genetics; yeast genome

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.

基因组结构与胁迫响应的基因调控 基因组的3D拓扑结构在健康、疾病和癌症的转录调控中起着关键作用。 发展基因组采用离散的结构和调控域， 类型，甚至是生物体。然而，虽然越来越清楚的是，在这个标准拓扑结构的变化发挥作用 在发育和疾病中起重要作用，但对动态地 控制重组过程。此外，还不知道基因组的拓扑结构是否（或如何） 有助于协调细胞对压力反应的关键基因的表达。 我们建立了一个系统， 引发戏剧性的建筑重组 在基因内部和基因之间协调一致， 在细胞群中同步。的 系统，芽殖酵母中的热激反应 S.酿酒厂，使我们能够利用强大的 这种生物体的遗传易处理性来定义 因素，并揭示驱动 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节因子， 热休克因子1（Hsf 1），在进化上 对健康和疾病至关重要。使用 高灵敏度和定量版本的 我们的染色体构象捕获（3C） 实验室开发的，称为Taq I - 3C，我们已经获得的证据表明，在响应急性热应力， HSP基因经历强烈的基因内相互作用，包括UAS和启动子元件之间的成环， 启动子和终止子区以及调节和编码区。更令人吃惊的是， 频繁的染色体内和染色体间相互作用，合并成离散的核内病灶。的基因 由另一种转录因子（TF）Msn 2激活热休克，同样地， 与自身或与Hsf 1靶基因结合。同样地，强转录，组成型 表达的基因经历基因内成环，然而这些基因似乎也不合并。除了它们 Hsf 1靶基因的基因内和基因间重组/重组是一种独特的结合， 非常动态：60秒内可检测到，2.5分钟内达到峰值，30分钟内衰减。 观察结果提出了一些重要问题。为了解决这些问题，我们提出三个目标： 目的1：阐明热休克和其他应激过程中酵母基因组的3D拓扑结构， Hsf 1和Pol II在协调这些变化中发挥的作用。我们将利用尖端的 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性 整个基因组的染色体拓扑动态。我们将定义Hsf 1和Pol II的作用， 目的2和3中鉴定的因子，在暴露于热应激的细胞中驱动3D基因组结构。我们将 使用Taq I - 3C确认关键发现。 目的2：阐明Pol II和Hsf 1在驱动HSP基因聚结中的决定因素，并验证 HSP冷凝物通过液-液相分离而聚集。我们将确定功能 Hsf 1和Pol II内的结构域，负责驱动HSP基因进入暴露于 急性HS，并探讨动态HSP冷凝物的生物物理性质。 目的3：揭示转录辅激活因子、染色质重塑因子和结构蛋白的作用 在驱动特定的和动态的相互作用和Hsf 1靶基因之间。 我们将检验这一假设，即HSP基因的结合代表了多种辅助因子的协同作用- 由Hsf 1招募并与Pol II协同行动-并调查选定因素的贡献， 集中在那些优先募集到HSP基因在凝聚能力的细胞。我们将 利用Taq I-3C检测结合一系列强大的酵母遗传技术-条件 核耗竭，条件性蛋白质降解，基因组编辑-以询问这些因素的作用。 总之，提出的实验将揭示机械的洞察力和广泛的，全基因组的 对酵母热休克过程中发生的动态、Hsf 1依赖的3D基因组重构的看法 反应他们将建立一个未来的探索HSP基因聚结的生物学意义， 根据这里提出的实验结果。

项目成果

Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.

相分离拮抗剂有效抑制转录并大致增加核小体密度。

• DOI: 10.1016/j.jbc.2022.102365
• 发表时间: 2022-10
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Meduri, Rajyalakshmi;Rubio, Linda S.;Mohajan, Suman;Gross, David S.
• 通讯作者: Gross, David S.

Shugoshin 2-a new guardian for heat shock transcription.

Shugoshin 2-热休克转录的新守护者。

• DOI: 10.15252/embj.2019104077
• 发表时间: 2020
• 期刊: The EMBO journal
• 作者: Kainth,AmoldeepS;Meduri,Rajyalakshmi;Pandit,Vickky;Rubio,LindaS;Gross,DavidS
• 通讯作者: Gross,DavidS

Primordial super-enhancers: heat shock-induced chromatin organization in yeast.

• DOI: 10.1016/j.tcb.2021.04.004
• 发表时间: 2021-10
• 期刊: Trends in cell biology
• 影响因子: 19
• 作者: Kainth AS;Chowdhary S;Pincus D;Gross DS
• 通讯作者: Gross DS

Inducible transcriptional condensates drive 3D genome reorganization in the heat shock response.

• DOI: 10.1016/j.molcel.2022.10.013
• 发表时间: 2022-11-17
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Chowdhary, Surabhi;Kainth, Amoldeep S.;Paracha, Sarah;Gross, David S.;Pincus, David
• 通讯作者: Pincus, David