The role of cis-regulatory elements in the inheritance of transcriptional memory through mitosis.

顺式调节元件在通过有丝分裂遗传转录记忆中的作用。

• 批准号: 10751881
• 负责人: STEPHANIE NICOLE OPRESCU
• 金额: $ 6.87万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751881
• 关键词: Area; Behavior; Binding; Binding Proteins; Binding Sites; Biological Assay; CCAAT-Enhancer-Binding Proteins; Candidate Disease Gene; Cell division; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; DNA Sequence; DNase I hypersensitive sites sequencing; Data; Deposition; Development; EP300 gene; Elements; Endoderm; Enhancers; Ensure; Exclusion; GATA1 gene; GATA4 gene; Gene Expression; Genes; Genetic Transcription; Genome; Goals; HNF4A gene; Histone H2B; Human Cell Line; Inherited; Interphase; Knowledge; Liver; Maintenance; Measurement; Measures; Mediating; Memory; Methods; Mitosis; Mitotic; Mitotic Activity; Molecular; Mutagens; Physical condensation; Process; Proteins; Regulatory Element; Research; Residual state; Resolution; Role; Specific qualifier value; TATA-Box Binding Protein; Therapeutic; Trans-Activators; arm; cell fate specification; cell type; daughter cell; genome-wide; histone modification; human embryonic stem cell; improved; insight; preservation; programs; promoter; recruit; small molecule; stem cells; transcription factor; transmission process

PROJECT SUMMARY The goal of this proposal is to determine how cell identity, as defined by a transcriptional program, is inherited through mitosis by systematically evaluating the role of specific cis-regulatory elements and trans- acting factors. The central hypothesis is that cis-regulatory elements at gene promoters maintain transcriptional competency through mitosis while trans interactions with enhancers mediate the timely and appropriate level of gene expression during mitotic exit. This proposal will leverage how the underlying DNA sequence determines the multifaceted interactions at promoters and cell-type specific enhancers that ensure cells faithfully re- establish proper transcriptional programs. This will be evaluated in cells with a stable identity and expanded to understand how this is mediated in the context of early development, when cells undergo constant fate decisions. While it was previously thought that chromatin condensation during mitosis excluded most proteins and thereby transcription-related processes, recent studies originally emanating from the Zaret lab now indicate that transcription is active at a low level and promoters, but not most enhancers, are accessible and maintain active histone modifications [1-7]; and mitotic chromatin arms retain areas of dynamic chromatin [8]. Although genome-wide measurements of histone modifications, chromatin accessibility and chromatin organization suggest mitotic changes at various cis-regulatory elements, they are unable to garner high- resolution mechanistic insight into how mitotic memory is retained at promoters and acted upon, during mitotic exit, by enhancers. While components involved in transcription such as the TATA-binding protein TBP, the elongating form of RNAP2, and transcription factors (TFs) such as GATA1, FOXA1, and ESRRB are detected on mitotic chromatin; many of these TFs do not remain bound to their interphase enhancer targets, thus how these factors function at specific loci to transmit transcription memory through mitosis remains elusive [5-7, 9- 13]. Therefore, this proposal aims to determine how cell fate is preserved through cell division by first evaluating the functional requirement for promoters in mitotic transcription, and then the mechanisms employed by liver-specific transcription factors to mediate enhancer activation upon mitotic exit. This will be assessed in the liver-derived HUH7 human cell line, which is highly amenable to mitotic synchronization methods, and expanded to determine the role of identified factors in pluripotent and endoderm-differentiated human embryonic stem cells. This proposal will expand our understanding of the molecular mechanisms that maintain cell identity and cell fate specification, thereby improving our ability to target and modulate stem cells for therapeutic value.

项目概要 该提案的目标是确定转录程序定义的细胞身份如何 通过系统评估特定顺式调控元件和反式调控元件的作用，通过有丝分裂遗传 作用因素。中心假设是基因启动子处的顺式调控元件维持转录 通过有丝分裂来提高能力，而反式与增强子的相互作用则介导及时和适当的水平 有丝分裂退出期间的基因表达。该提案将利用基础 DNA 序列如何决定 启动子和细胞类型特异性增强子的多方面相互作用，确保细胞忠实地重新 建立适当的转录程序。这将在具有稳定身份的细胞中进行评估并扩展到 了解当细胞经历恒定的命运时，这是如何在早期发育的背景下介导的 决定。虽然以前认为有丝分裂期间染色质凝结排除了大多数蛋白质 以及与转录相关的过程，最近的研究最初来自 Zaret 实验室，现在 表明转录在低水平下活跃，并且启动子（但不是大多数增强子）是可接近的并且 维持活性组蛋白修饰 [1-7]；有丝分裂染色质臂保留动态染色质区域[8]。 尽管组蛋白修饰、染色质可及性和染色质的全基因组测量 组织表明各种顺式调控元件发生有丝分裂变化，它们无法获得高 解析有丝分裂记忆如何在启动子处保留并在有丝分裂过程中发挥作用的机制洞察 退出，通过增强器。虽然 TATA 结合蛋白 TBP 等参与转录的成分 检测到 RNAP2 的延长形式，以及 GATA1、FOXA1 和 ESRRB 等转录因子 (TF) 有丝分裂染色质；许多这些转录因子不再与它们的间期增强子靶标结合，因此如何 这些因子在特定位点发挥作用，通过有丝分裂传递转录记忆，但仍然难以捉摸 [5-7, 9- 13]。因此，该提案旨在首先确定如何通过细胞分裂来保存细胞命运 评估有丝分裂转录中启动子的功能需求，然后评估其机制 肝脏特异性转录因子利用它来介导有丝分裂退出时的增强子激活。这将是 在肝源性 HUH7 人类细胞系中进行评估，该细胞系非常适合有丝分裂同步 方法，并扩展以确定已识别的因子在多能和内胚层分化中的作用 人类胚胎干细胞。该提议将扩大我们对分子机制的理解 维持细胞身份和细胞命运规范，从而提高我们靶向和调节干细胞的能力 以获得治疗价值。