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

项目摘要 从依赖母体转录进入卵子到新转录的受精卵的转变 转录本受到严格的监管，以确保早期胚胎的正常发育。让我们深入了解 合子基因表达时间的调控，最近的许多研究都集中在母体转录如何 因子激活果蝇早期胚胎中的合子基因组。另一方面，它的作用机制是 虽然最近的研究表明，无处不在表达的抑制因子也会影响 空间定位基因的表达。这里提出的实验将调查无处不在的 控制增强子作用的抑制物。具体地说，我们建议序列特定的、广泛表达的 抑制子在早期胚胎中作为合子基因组激活的守护者，控制特定的 增强剂调节基因表达的时间，从而有效地管理母体-受精卵的转变。 与激活因子相比，转录抑制因子的研究还不够深入，然而我们提出了两种类型的转录抑制因子 转录调控同样重要。我们假设一项决定是否 顺式调控元件支持基因表达的功能不仅取决于激活量，而且还取决于 还有就是必须克服多大程度的压制。来洞察广泛表达的抑制者的 作用机制(S)，我们的实验方法有两个具体目标。目标1.调查是否 染色质的可及性受广泛表达的抑制物调控。我们将使用Assay来 转座酶可及染色质后测序(ATAC-seq)以研究 抑制物水平影响染色质的可及性。这种方法可以分析已经被 小心翼翼地在早期胚胎中提供染色质可及性的时间进程。我们的目标是 实验是通过分析突变体或引入靶向来增加或降低抑制物水平。 突变为增强子序列，以研究这些因子在调节染色质开放中的作用。目标 2.验证广泛表达的抑制物调节双标依赖的形态原输出的想法。双曲面是一种 母体存放的转录因子，以浓度依赖的方式调节基因表达。 而不是转录激活直接与绝对双核浓度有关，广泛表达 抑制物可能起到调节形态原有效浓度的作用：例如，较高水平的双核生物 可能需要激活抑制物结合的增强子，而不是未结合的增强子。我们将进行化验 激活物和抑制物之间的遗传相互作用，也使用染色质免疫沉淀(CHIP)来 在体内测试抑制物是否影响类生物素与DNA的结合。在这里获得的关于角色的见解 广泛表达的抑制子很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。 ​​ ​ ​​ ​ ​

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. ​​ ​ ​​ ​ ​