Mechanisms of Broadly-Expressed Repressors in Zygotic Gene Expression in an Animal Model

动物模型中合子基因表达中广泛表达的阻遏蛋白的机制

• 批准号: 9789684
• 负责人: Angelike Stathopoulos
• 金额: $ 8.38万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789684
• 关键词: Affect; Animal Model; Animals; Binding; Biological Assay; Chromatin; Chromosome Mapping; DNA Binding; DNA Sequence; Dependence; Deposition; Development; Drosophila genus; Embryo; Enhancers; Ensure; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Goals; Health; Hour; Human; Link; Methods; Mutation; Organism; Output; Pattern; Regulation; Regulatory Element; Repression; Role; System; Testing; Time; Transcript; Transcription Coactivator; Transcription Repressor/Corepressor; Transcriptional Activation; Transcriptional Regulation; Transposase; Vertebrates; activating transcription factor; chromatin immunoprecipitation; egg; experimental study; gastrulation; in vivo; insight; morphogens; mutant; transcription factor; transcriptome; virtual

Project Summary The transition from dependence on maternal transcripts deposited into the egg to newly transcribed zygotic transcripts is carefully regulated to ensure proper development of early embryos. To provide insight into the regulation of zygotic gene expression timing, many recent studies have focused on how maternal transcription factors activate the zygotic genome in Drosophila early embryos. On the other hand, the mechanism of action of ubiquitously expressed repressors is not well understood, though recent studies have shown they also impact expression of spatially-localized genes. The experiments proposed here will investigate a role for ubiquitous repressors in controlling enhancer action. Specifically, we propose that sequence-specific, broadly-expressed repressors act as guardians of zygotic genome activation in early embryos, controlling the action of particular enhancers to regulate timing of gene expression, and thus effectively manage the maternal-zygotic transition. Repressors of transcription are understudied compared to activators, yet we propose both types of transcriptional regulators are equally important. We hypothesize that the decision of whether or not a cis-regulatory element functions to support gene expression depends not only on the amount of activation but also on how much repression must be overcome. To provide insight into broadly-expressed repressors’ mechanism(s) of action, our experimental approach has two specific aims. Aim 1. Investigate whether chromatin accessibility is regulated by broadly-expressed repressors. We will use Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) to investigate whether changes in repressor levels affect chromatin accessibility. This method allows assay of single embryos that have been carefully staged to provide a time-course of chromatin accessibility in the early embryo. The goal of our experiments is to increase or decrease repressor levels, either by assaying mutants or introducing targeted mutations into enhancer sequences, to investigate a role for these factors in regulating chromatin opening. Aim 2. Test the idea that broadly-expressed repressors regulate Bicoid-dependent morphogen outputs. Bicoid is a maternally deposited transcription factor that regulates gene expression in a concentration-dependent manner. Rather than transcriptional activation being linked directly to absolute Bicoid concentration, broadly-expressed repressors may act to modulate the morphogen effective concentration: for example, higher levels of Bicoid may be required to activate enhancers bound by repressors compared to those not bound. We will assay for genetic interaction between activators and repressors and also use chromatin-immunoprecipitation (ChIP) to test whether repressors influence Bicoid’s DNA-binding in vivo. The insights gained here regarding the role of broadly-expressed repressors will likely be applicable to higher organisms, including vertebrates, as cis-regulatory mechanisms are generally conserved in metazoan animals. ​​ ​ ​​ ​ ​

项目概要 从依赖沉积在卵子中的母体转录物到新转录的合子的转变 转录本经过仔细调节，以确保早期胚胎的正常发育。 合子基因表达时间的调控，最近许多研究都集中在母体转录如何 另一方面，激活果蝇早期胚胎中合子基因组的作用机制。 尽管最近的研究表明，普遍表达的阻遏蛋白也会影响 这里提出的实验将研究无处不在的基因的表达。 具体来说，我们提出序列特异性的、广泛表达的阻遏物。 阻遏蛋白充当早期胚胎中合子基因组激活的守护者，控制特定的作用 增强子调节基因表达的时间，从而有效地管理母本-合子转变。 与激活子相比，转录抑制子的研究还不够充分，但我们提出这两种类型 我们认为转录调节因子是否是一个决定性的因素同样重要。 支持基因表达的顺式调控元件功能不仅取决于激活量，还取决于 还涉及必须克服多少压制，以深入了解广泛表达的压制者。 作用机制，我们的实验方法有两个具体目标 目标 1. 调查是否。 染色质可及性受广泛表达的阻遏蛋白调节。我们将使用 Assay 进行检测。 转座酶可及的染色质随后进行测序（ATAC-seq）以研究染色质是否发生变化 阻遏蛋白水平影响染色质可及性，该方法允许对已染色质的单个胚胎进行分析。 精心分阶段，以提供早期胚胎中染色质可及性的时间过程，这是我们的目标。 实验的目的是通过检测突变体或引入靶向基因来增加或减少阻遏物水平 增强子序列突变，以研究这些因素在调节染色质开放中的作用。 2. 检验广泛表达的阻遏蛋白调节 Bicoid 依赖性形态发生素输出的观点。 母体沉积的转录因子，以浓度依赖性方式调节基因表达。 转录激活并非与绝对 Bicoid 浓度直接相关，而是广泛表达 阻遏物可能会调节形态发生素的有效浓度：例如，较高水平的 Bicoid 与未结合的增强子相比，可能需要激活被阻遏物结合的增强子。 激活子和阻遏子之间的遗传相互作用，并使用染色质免疫沉淀 (ChIP) 测试阻遏物是否影响 Bicoid 的体内 DNA 结合。 广泛表达的阻遏蛋白可能适用于高等生物，包括脊椎动物，因为 顺式调节机制在后生动物中通常是保守的。 ​​ ​ ​​ ​ ​