Regulation of zygotic genome activation by Zelda

项目总结 受精后不久，转录组发生了戏剧性的重新编程，由此母体储存了 RNA被降解，合子RNA被合成，使胚胎快速而健壮地发育。 这一点在果蝇胚胎中尤为明显，果蝇胚胎在两小时内经历卵裂， 胚层的细胞化，决定性别和X染色体的剂量，设计身体计划，并获得 准备好原肠形成了。虽然这些过程背后的基因网络已经得到了很好的研究，但还没有。 明确它们是如何集体启动的，这一过程被称为合子基因组激活。在上一次 在授权期内，我们证明了一个单一的因子，塞尔达(锌指早期果蝇激活剂)，在全球范围内作用于 激活早期表达的基因，单独或与形成模式的转录因子一起激活。我们 发现Zelda结合了整个基因组的增强子，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。我们进一步证明泽尔达 降低这种核小体屏障，从而促进其他因子的结合，从而增加 下游靶基因。但塞尔达如何履行这一“开拓性”角色呢？我们假设泽尔达首先 高核小体占有率的“侦察”区域，然后在与CAGGTAG基序结合时，移位 核小体刚好足以让其他因子访问基因组。通过这种方式，塞尔达传授“增强剂” 能力“，这是如何启动发展方案的一个新概念。我们还发现了另外一本小说 塞尔达的特点使其有别于其他因素。首先，泽尔达在两个分子上扮演着两个不同的角色 靶基因的类型--作为直接转录激活子或作为间接增强子(需要其他因素)。 我们认为启动子的特异性决定了塞尔达所扮演的角色。其次，塞尔达有两个DNA结合 结合不同基序的结构域，典型的CAGGTAG基序和新发现的富含G的基序。是什么 这个小说母题的作用是什么？在这项资助中，我们的目标是确定塞尔达如何在 多尺度水平-从不同的蛋白质结构域，到独特的转录因子角色，再到全球染色质 互动。对于每个目标，我们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 评估塞尔达如何与染色质相互作用的分析，以及研究如何与染色质相互作用的创新实时成像技术 Zelda在基因组激活过程中调节转录动态。AIM 2测试了启动子- 使用一系列启动子交换分析，塞尔达将扮演什么样的分子角色是其特异性的基础。目标3 利用缺失突变体研究了新的DNA结合域的功能。 基因组分析(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结合分析揭示富含G的基序与CAGGTAG之间的相互作用。如果实现了这些目标，将 影响目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.