Regulation of zygotic genome activation by Zelda

描述（由申请人提供）：有很多关于早期发展是不理解的。在受精后的某个时刻，合子基因组被激活，并且对即将到来的发育事件所必需的基因被转录。许多早期基因已经被描述并被放入基因网络中，但它们如何在适当的时间、以适当的顺序和以稳健的方式被激活还不清楚。在最后一个资助期，研究人员发现果蝇胚胎使用一种简单的策略来集体和选择性地激活基因-转录因子Zelda（Zld）识别并结合特定的序列基序（CAGGTA），随后将基因网络整合到连贯和不连贯的前馈回路系统中。在这样做的过程中，Zld驱动了发生在母体到合子过渡（MZT）期间的主要重编程事件，其中胚胎从母亲那里接管了发育的控制权。这是第一次在任何生物体中证明这样的调节器，因此，Zld为早期发育中基因网络的全球协调提供了一个范例。Zld类似于一类特殊的转录因子，称为先锋因子，它们首先与靶基因结合，当发育信号沿着出现时，为将来的激活提供能力。该提案的目标是揭示Zld用于调节合子基因组激活（ZGA）的潜在机制，并为关键发育调节因子的活动提供强大的时间控制。 Zld蛋白最初处于低水平，浓度在一小时内升高，与ZGA一致，然后在一小时内降低。 第三个小时。Zld蛋白动力学的调节以及Zld如何对早期基因的开关状态进行计时尚不清楚。将通过改变Zld浓度和监测靶基因活化来测试Zld阈值水平触发基因组活化的假设。将评估Zld本身是否被调节，重点是zld 3' UTR的潜在作用。Zld结合导致染色质景观的局部重塑，从而增加其他因子的可及性，导致靶基因的“表达性”增加的想法将被测试。在增强子和转录起始位点处的染色质状态的全局变化将在ZGA之前（当基因组失活时）、ZGA之后（当早期合子基因组被转录时）以及之后（当许多早期基因被关闭而其他基因被激活时）进行检查。重要的是，比较野生型和zld突变体中的染色质状态将揭示Zld是否在MZT期间介导表观遗传变化，以及某些染色质标记是否为未来的转录激活/抑制准备基因。遗传学、基因组学和生物化学方法将被用来检验这些假设。关键试剂包括zld突变体、改变Zld水平的zld拯救转基因和测定Zld靶基因对Zld结合位点变化的反应的转录报告转基因。实验方法将导致更好地了解合子基因组重编程从静止状态到一个强大的活跃状态在早期发展。

DESCRIPTION (provided by applicant): There is much about early development that is not understood. At some point after fertilization the zygotic genome is activated, and genes that are necessary for upcoming developmental events are transcribed. Many early genes have been characterized and placed into gene networks, but how they come to be activated at the proper time, in the proper order, and in a robust manner is unclear. In the last grant period, it was revealed that the Drosophila embryo uses a simple strategy to collectively and selectively activate genes - the transcription factor Zelda (Zld) recognizes and binds a specific sequence motif (CAGGTA), and subsequently integrates gene networks into a system of coherent and incoherent feed-forward loops. In doing so, Zld drives the major reprogramming event that occurs during the maternal-to-zygotic transition (MZT), where the embryo takes over control of development from the mother. This is the first demonstration of such a regulator in any organism, thus, Zld provides a paradigm for the global coordination of gene networks in early development. Zld resembles a special class of transcription factors called pioneer factors, which are the first to engage target genes, providing competence for future activation when developmental signals come along. The goal of this proposal is to reveal the underlying mechanisms by which Zld functions to regulate zygotic genome activation (ZGA) and to provide robust temporal control to the activities of the key developmental regulators. Zld protein is initially at low levels, rises in concentration by one hour, coincident with ZGA, then decreases in the third hour. The regulation of Zld protein dynamics and how Zld times the on-off state of early genes is unclear. The hypothesis that a threshold level of Zld triggers genome activation will be tested by altering Zld concentrations and monitoring target gene activation. Whether Zld itself is regulated will be assessed, with focus on a potential role of the zld 3' UTR. The idea that Zld binding leads to local remodeling of the chromatin landscape, thus increasing accessibility of other factors, leading to increased "expressivity" of target genes will be tested. Global changes in chromatin states at enhancers and transcription start sites before ZGA when the genome is inactive, after ZGA when the early set of zygotic genes are transcribed, and later when many early genes are turned off and others are activated will be examined. Importantly, comparing chromatin states in wild-type and zld mutants will reveal if Zld mediates epigenetic changes during the MZT, and if certain chromatin marks prepare genes for future transcriptional activation/repression. Genetic, genomic, and biochemical approaches will be used to test these hypotheses. Key reagents include zld mutants, zld rescue transgenes to alter Zld levels, and transcriptional reporter transgenes to assay Zld target- gene responses to changes in Zld binding sites. The experimental approaches will lead to a greater understanding of zygotic genome reprogramming from a quiescent state to a robustly active state in early development.