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. ​​ ​ ​​ ​ ​

项目摘要 从沉积在鸡蛋中的母体转录本的依赖到新转录的合子的过渡 仔细调节成绩单，以确保适当的早期胚胎发展。为了洞悉 合子基因表达时间的调节，许多最近的研究都集中在母体转录上 因素激活果蝇早期胚胎中的合子基因组。另一方面，行动机制 尽管最近的研究表明它们也影响了普遍表达的复制品 空间定位基因的表达。这里提出的实验将研究无处不在的作用 控制增强子动作的阻遏物。具体而言，我们提出了序列特异性的，广泛表达的 阻遏物充当早期胚胎中的二元基因组激活的监护人，控制特定的作用 增强子来调节基因表达的时机，从而有效地管理母体 - 杂种过渡。 与激活剂相比，理解了转录的阻遏物，但我们提出了这两种类型的 转录调节器同样重要。我们假设是否决定是否 支持基因表达的顺式调节元件功能不仅取决于激活量，还取决于 还必须克服多少反思。提供有关广泛表达的复制品的见解 动作机制，我们的实验方法具有两个具体的目的。目标1。调查是否 染色质的可及性由广泛表达的代表调节。我们将使用测定 转座酶可访问的染色质，然后进行测序（ATAC-SEQ），以研究是否改变 阻遏物水平会影响染色质的可及性。此方法允许测定已经 仔细上演以提供早期胚胎中染色质可及性的时间。我们的目标 实验是通过测定突变体或引入靶向的，以提高或降低反射器水平 突变成增强子序列，以研究这些因素在确定染色质开放中的作用。目的 2。检验以下观点：宽大表达复制品调节生物类依赖性形态的输出。生物剂是 母体沉积的转录因子以浓度依赖性方式调节基因表达。 而不是将转录激活直接链接到绝对生物浓度，而是广泛表达 阻遏物可以起作用用于调节形态学有效浓度：例如，较高水平的双子 与未结合的增强剂相比，可能需要激活被复制品绑定的增强剂。我们将分析 激活因子与反射器之间的遗传相互作用，还使用染色质免疫沉淀（CHIP） 测试反射器是否影响双子体内的Bicoid DNA结合。关于在这里获得的见解 广泛表达的表示可能适用于包括脊椎动物在内的更高生物，例如 顺式调节机制通常在后生动物中保守。 になったんです。英语：您可以做的第一件事就是找到最好的方法。 になったんです。英语：您可以做的第一件事就是找到最好的方法。 になったんです。英语：您可以做的第一件事就是找到最好的方法。 になったんです。英语：您可以做的第一件事就是找到最好的方法。 になったんです。英语：您可以做的第一件事就是找到最好的方法。