核小体重塑在果蝇母源向合子转化中的表观调控作用

After fertilization, Drosophila zygotic genome is inactive and gene transcription is driven by maternal components. Zygotic transcription is only initiated during maternal-zygotic transition (MZT). Chromatin appears largely decondensed and unstructured during first divistions and forms into domains along the process of MZT, for example, heterochromatin foci. We speculate that nucleosome remodeling changes chromatin configuration,and is likely one of the determinants of zygotic transcription initiation. In addition to this, histone modifications may invovle the establishment of chromatin signaure during MZT. Little is known about this critical question of embryonic development so far. We generated the first map of genome-wide nucleosome positions, and revealed that nucleosomes engaged in RNA Pol II pausing during transcrition. In this proposal, on the basis of our previous study, we plan to collect large amount of 1-2 h and 3-4 h fly embryos (representating before and after MZT, respectively), digest them with MNase to get mononucleosomes. ChIP assays are used to collect H3, H3K4me3, H3K27me3 nucleosomes, and RNA Pol II-bound DNA. Their DNA were sequenced with Solexa Hi-seq, respectively. Bioinformatics approaches are used to predict nucleosomes and RNA Pol II binding sites. The high-resolution maps of H3 wild-type nucleosomes, H3K4me3, H3K27me3 locations, and RNA Pol II binding sites in the whole genome are generated. The distribution patterns of nucleosomes, H3K4me3, H3K27me3, and RNA Pol II binding sites can be summarized. The difference of their positions before and after MZT can be obtained. Transcriptomes of embryos at these two stages are also prepared by RNA-seq. Correlation analysis of between nucleosome remodeling, histone modification changes, RNA Pol II binding, and gene expression will reveal epigenetic mechanisms by which nucleosome remodeling and histone modificatons regulate gene expression, and their functions in zygotic transcription initiation. In addition, indentification of the fragible nucleosomes will reveal the change of the open and close configuration of chromatin before and after MZT, and the potential role of histone modifications in this configuration change. This study will improve our understanding of functional roles of nucleosome remodeling in embryonic development, and pave the way to explore the defects in mammalian embryonic development caused by abnormal nucleosome remodeling, histone modifications.

果蝇胚胎发育从母源向合子转化(MZT)过程中，由母源成分驱动基因表达过渡到合子基因启动表达，染色质由解聚松散到凝聚形成异染色质。我们据此假设核小体重塑改变染色质构象，是合子基因启动转录的决定因素之一，以及组蛋白修饰参与其中染色质标记的建立。我们绘制了首张全基因组果蝇核小体图谱，并揭示核小体定位在基因转录调控中的作用。在此基础上，本项目以果蝇1-2与3-4小时胚胎(分别代表MZT前后)为实验材料，通过测序与生物信息分析，分别绘制这两时期核小体、组蛋白修饰、RNA Pol II结合图谱，和转录组。分析三者的分布规律、基因表达的变化、三者之间的关联性及调控基因激活与抑制的模式。这些分析将揭示果蝇胚胎发育MZT前后核小体重塑产生的染色质开放与屏蔽构象变化，组蛋白修饰在MZT中的作用，及协同激活合子基因转录的机制。本项目将为研究哺乳动物中由染色质重塑异常造成的发育缺陷的分子机制提供理论依据与方法。

母源-合子过渡（Maternal-Zygotic Transition，MZT）是胚胎发育中的重要事件。经过MZT，卵子带来的成分降解殆尽，合子基因组开始启动，基因转录翻译成蛋白，维持后续的胚胎发育。这一过程染色质发生巨大变化，由解聚松散到高度凝聚。然而，在MZT过程中，染色质结构如何变化，相应如何调控基因活性确保合子基因组正常启动并不清楚。本课题以受精后1-2与3-4小时果蝇胚胎（前两个时期为MZT前，3-4小时为MZT后）为实验材料，通过高通量测序与生物信息技术，分别绘制这两个时期全基因组高分辨率核小体定位、关键组蛋白修饰占有、RNA Pol II结合图谱、与转录组。结果发现，与其它物种MZT过程中染色质重塑不同，果蝇在MZT之前，TSS附近就已形成经典的核小体定位模式，而且核心组蛋白修饰也已经形成。MZT过程中核小体占有发生显著的全局性变化，MZT后基因组层面上核小体占有升高、更稳定，染色质更致密。但是TSS（转录起始位点）上及紧靠其上游的NDR（核小体缺失区）的核小体占有在MZT后却下降，暗示启动子区染色质的开放对MZT过程有重要作用。此外，MZT后，TSS附近的H3K9ac占有显著上升，或许参与调控MZT过程。纯粹合子基因启动子在母源期很少有H3K4me3修饰，而母源型合子基因启动子在母源期很多就有H3K4me3修饰确保这些基因在母源期就开始表达。有意思的是，有一组母源型合子基因启动子在MZT中虽然H3K9ac占有上升，但基因总体表达量却下降。这主要是合子期增加的表达量小于卵子带来的转录本的降解。本课题结果揭示果蝇早期胚胎发育MZT过程中染色质重塑与其它物种不同，完善我们对MZT分子机制的理解；也为治疗由染色质重塑异常造成的发育缺陷提供思路。