Regulation of zygotic genome activation by Zelda

项目摘要 受精后不久，转录组发生戏剧性的重编程， RNA被降解，合子RNA被合成，使胚胎能够快速而健壮地发育。 这在果蝇胚胎中尤其明显，在两小时内经历卵裂， 使胚盘细胞化，确定性别和X染色体剂量，设计身体计划， 准备好原肠胚形成。虽然这些过程背后的基因网络已经得到了很好的研究， 清楚它们是如何共同启动的，这一过程被称为合子基因组激活。上一 格兰特期间，我们证明了一个单一的因素，塞尔达（锌指早期果蝇激活剂），全球性的作用， 单独或与模式形成转录因子一起激活早期表达的基因。我们 发现塞尔达在基因组中结合了增强子，这些增强子具有固有的高核小体占有率 因为下面的序列有利于核小体的形成。我们进一步证明了塞尔达 降低了核小体屏障，从而促进了其他因子的结合， 下游靶基因。但塞尔达如何履行这一“先锋”角色？我们假设泽尔达首先 “侦察出”高核小体占有率的区域，然后在与CAGGTAG基序结合后， 核小体刚好足够其他因子进入基因组。这样塞尔达赋予“增强剂 能力”，一个新的概念，在如何发展计划的启动。我们还发现了新的 塞尔达的特点，使它有别于其他因素。首先，塞尔达扮演两个不同的分子角色， 靶基因的类型-作为直接转录激活因子或作为间接增强因子（需要其他因子）。 我们建议启动子特异性决定塞尔达扮演的角色。其次，塞尔达有两个DNA结合 结构域结合不同的基序，典型的CAGGTAG基序和一个新发现的G丰富的基序。是什么 这一新主题的作用？在这项资助中，我们的目标是确定塞尔达如何机械地运作， 多尺度水平-从不同的蛋白质结构域，到独特的转录因子作用，再到全局染色质 交互.我们对每个目标都有强有力的支持证据。在目标1中，我们将使用体外DNA结合 分析来评估塞尔达如何与染色质相互作用，以及创新的实时成像技术来研究如何与染色质相互作用。 塞尔达调节基因组激活过程中的转录动力学。目的2检验启动子- 使用一系列启动子交换分析，特异性是塞尔达将发挥的分子作用的基础。目标3 研究了新的DNA结合结构域的功能， 基因组测定（ChIP-seq和RNA-seq），以确定其在靶基因转录中的作用。我们还将使用 DNA结合试验，以揭示富G基序和CAGGTAG之间的相互作用。如果这些目标得以实现， 影响当前对转录程序如何在发育和人类健康中启动的思考。

PROJECT SUMMARY Shortly after fertilization, a dramatic reprogramming of the transcriptome occurs whereby maternally deposited RNAs are degraded and zygotic RNAs are synthesized, enabling the embryo to develop quickly and robustly. This is especially exemplified in the Drosophila embryo, which within a two-hour period undergoes cleavage, cellularizes the blastoderm, determines sex and X-chromosome dosage, patterns the body plan, and gets ready for gastrulation. While the gene networks underlying these processes have been well studied, it is not clear how they are collectively initiated, a process referred to as zygotic genome activation. In the previous grant period we demonstrated that a single factor, Zelda (zinc finger early drosophila activator), acts globally to activate early-expressed genes, either solely or together with the pattern-forming transcription factors. We found that Zelda binds enhancers across the genome, which have intrinsically-high nucleosome occupancy because the underlying sequences are favorable for nucleosome formation. We further showed that Zelda lowers this nucleosome barrier, thus facilitating the binding of other factors and thus increasing expressivity of downstream target genes. But how does Zelda fulfill this “pioneering” role? We hypothesize that Zelda first “scouts out” regions of high nucleosome occupancy, then upon binding to CAGGTAG motifs, displaces nucleosomes just enough for other factors to access the genome. In this way Zelda imparts “enhancer competence”, a new concept in how developmental programs are initiated. We also found additional novel features of Zelda that set it apart from other factors. First, Zelda plays two different molecular roles on two types of target genes – as a direct transcriptional activator or as an indirect potentiator (other factors required). We propose that promoter-specificity dictates which role Zelda plays. Second, Zelda has two DNA binding domains that bind different motifs, the canonical CAGGTAG motif and a newly identified G-rich motif. What is the function of this novel motif? In this grant, we aim to determine how Zelda functions mechanistically at multiscale levels - from different protein domains, to unique transcription factor roles, to global chromatin interactions. We have strong supporting evidence for each aim. In Aim 1, we will use in vitro DNA binding assays to assess how Zelda interacts with chromatin, and innovative live-imaging techniques to study how Zelda regulates transcriptional dynamics during genome activation. Aim 2 tests the hypothesis that promoter- specificity underlies which molecular role Zelda will play using a series of promoter-swap assays. Aim 3 investigates the function of the novel DNA binding domain using a deletion mutant without the domain in genomic assays (ChIP-seq and RNA-seq) to determine its role in target gene transcription. We will also use DNA binding assays to reveal interactions between the G-rich motif and CAGGTAG. If achieved these aims will impact current thinking of how transcriptional programs are initiated in development and human health.