Mechanisms of repressive interactions between developmental gene regulatory networks

发育基因调控网络之间的抑制相互作用机制

• 批准号: 10752429
• 负责人: William B Douglas
• 金额: $ 4.77万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752429
• 关键词: Adopted; Bacterial Artificial Chromosomes; Binding; Binding Sites; Biological Assay; Biology; Cell Lineage; Cell Reprogramming; Cell Therapy; Cells; ChIP-seq; Color; Development; Developmental Gene; Ectopic Expression; Electrophoretic Mobility Shift Assay; Embryonic Development; Endowment; Ensure; Exclusion; Experimental Models; Gene Expression; Gene Expression Profiling; Gene Transfer Techniques; Genes; Genetic Transcription; In Situ Hybridization; Invertebrates; Mammals; Measures; Mesoderm; Mesoderm Cell; Methods; Microscopy; Molecular; Mutate; Organism; Pathway interactions; Play; Process; Property; Proteins; Recombinants; Regenerative Medicine; Regulatory Element; Reporter; Reporter Genes; Repression; Resistance; Resolution; Role; Sea Urchins; Signal Transduction; Site; Skeleton; Somatic Cell; Specific qualifier value; Testing; Time; Transcription Repressor; Transcriptional Regulation; Transgenic Organisms; cell fate specification; cell type; cellular engineering; embryo cell; experimental study; gene interaction; gene network; gene regulatory network; gene repression; in vivo; inhibitor; knock-down; mutant; nano-string; notch protein; programs; skeletogenesis; synergism; transcription factor

Cells acquire their unique identities during development through the progressive emergence of distinct transcriptional programs. These transcriptional programs can be viewed as dynamic networks of interacting genes known as gene regulatory networks (GRNs). GRNs have been well studied for their positive role in activating the expression of genes that endow cells with their specialized properties; however, it is now apparent that an equally important function of these networks is to exclude other, potentially alternative, transcriptional programs. Repressive interactions of this kind play a widespread, fundamental role in determining cellular identities in organisms as diverse as invertebrates and mammals. They are also important in the context of regenerative medicine. The direct reprogramming of somatic cells by lineage-specific transcription factors (TFs) is accompanied by the comprehensive silencing of pre-existing transcriptional programs. Despite the pivotal role that repressive interactions between transcriptional networks play in both embryonic cell fate specification and somatic cell reprogramming, the underlying mechanisms are poorly understood. We will address this important problem using the sea urchin, a prominent experimental model for the analysis of developmental mechanisms and for GRN biology. One of the best characterized sea urchin GRNs underlies the development of cells that form the skeleton. A key component of this network is Alx1, a lineage- specific TF that provides direct, positive inputs into many genes that support skeletogenesis. In parallel with its positive role, Alx1 represses potential, alternative transcriptional programs that are ordinarily restricted to surrounding non-skeletogenic mesoderm (NSM) cells. Perturbation of Alx1 function in skeletogenic cells results in the ectopic deployment of NSM GRNs in these cells and causes them to adopt NSM fates. The repression of NSM GRNs by Alx1 thus provides an outstanding opportunity to uncover mechanisms by which transcriptional networks interact with one another, thereby ensuring the emergence of unique cellular identities. To dissect the mechanisms underlying this GRN interaction, I will begin by defining key spatial and temporal aspects of NSM GRN repression by Alx1, using both quantitative methods and spatial gene expression analysis to characterize changes in gene expression that occur after perturbing Alx1 function (Aim 1). Next, I will explore the hypothesis that Alx1 directly represses NSM genes by using fluorescent reporter constructs and transgenesis to dissect cis-regulatory elements of these genes (Aim 2). Finally, I will test the hypothesis that Alx1 controls a cell-autonomous unresponsiveness to Notch/Delta signaling, a pathway that drives NSM specification during normal development (Aim 3). These studies will shed light on the mechanisms by which Alx1 represses NSM GRNs. More broadly, they will lead to a better understanding of interactions between transcriptional networks that regulate cell identity.

细胞在发育过程中通过逐渐出现不同的 转录程序。这些转录程序可以被视为相互作用的动态网络 基因被称为基因调控网络(GRN)。GRN已经被很好地研究，因为它们在 激活赋予细胞特殊特性的基因的表达；然而，现在很明显 这些网络的一个同样重要的功能是排除其他潜在的替代转录 程序。这种抑制性相互作用在决定细胞 像无脊椎动物和哺乳动物一样，生物的身份也是多种多样的。它们在以下背景下也很重要 再生医学。谱系特异性转录因子对体细胞的直接重编程 伴随着对先前存在的转录程序的全面沉默。尽管扮演着关键的角色 转录网络之间的抑制性相互作用在胚胎细胞命运指定和 体细胞重新编程，其潜在机制还知之甚少。 我们将使用海胆来解决这个重要的问题，海胆是 对发育机制和GRN生物学的分析。最具特性的海胆GRN之一 是构成骨架的细胞发育的基础。这个网络的一个关键组成部分是ALX1，一种谱系- 为许多支持骨骼形成的基因提供直接、积极的输入的特定转铁蛋白。与ITS并行 积极作用，ALX1抑制潜在的、可选的转录程序，这些程序通常仅限于 围绕非骨骼发育的中胚层(NSM)细胞。骨骼细胞中ALX1功能的扰动结果 在这些细胞中异位部署NSM GRN并导致它们采用NSM命运。对中国的压制 因此，ALX1的NSM GRN提供了一个绝佳的机会来揭示转录 网络之间相互作用，从而确保出现独特的蜂窝身份。 为了剖析这种GRN交互作用背后的机制，我将首先定义关键的空间和 用定量方法和空间基因表达研究ALX1对NSM GRN抑制的时间特征 对干扰ALX1功能后发生的基因表达变化进行分析(目标1)。接下来，我 将探索ALX1通过使用荧光报告结构和 转基因以剖析这些基因的顺式调控元件(目标2)。最后，我将检验这一假设 ALX1控制细胞自主对Notch/Delta信号的无反应，这是驱动NSM的一条途径 正常开发过程中的规范(目标3)。这些研究将阐明 ALX1抑制NSM GRN。更广泛地说，它们将有助于更好地理解 调节细胞身份的转录网络。