| 摘要: | 基因的協同表現主導了多細胞生物胚胎發育的過程，包括了細胞增殖、分化、樣式形成以及細胞特化。此複雜與多變動的基因調控的網絡建立於轉錄因子 (transcription factor) 與增強子 (enhancer) 的交互作用。不同的增強子具有不同的 DNA 序列，有著與其專一的轉錄因子組合，如此的交互作用提供了基因表現精確的時間和空間的控制。雖然增強子在基因體的位置可以根據染色質的特徵、轉錄因子的交互作用、與DNA 序列，利用生物資訊學的工具加以預測，但此需要大量的實驗數據，而且對於增強子活性和增強子在發育過程中的活性的變化，無法加以預測。在果蠅早期胚胎發育的過程中， 發現了第一個胚胎初期發育的重大轉錄因子Zelda (Zld) ， 透過與 特定DNA序列模組結合，集體但有選擇性的活化子代基因體，並在 Maternal-to-Zygotic transition (MZT) 中，協同合子基因組的表現與作用網路 (Liang, et al., 2008; Nien, et al., 2011) 。並且發現 Zelda 不僅激活了合子基因體的級聯反應 (genetic cascade) ，同亦可以表觀遺傳因子的作用模式，改變染色質的結構，以促進其他轉錄因子與增強子 (enhancer) 的交互作用，達成基因表現的調控。在本研究中，我們將利用先端的生物科技，來分析 Zelda 蛋白質結構與功能，同時運用 STARR-Seq 的實驗方法來定位與量化果蠅早期胚胎發育中的增強子 (enhancer) 活性。最後我們以生物資訊學的分析，根據我們之前以高通量定序 RNA 聚合酶的染色質免疫沈澱所得的資料，並配合在資料庫中已發表的相關資料，以整合分析 (meta-analysis) 建構果蠅胚胎發育中 MZT 的“調控組” (regulatome) 。具體目標包含:(1) 運用 cryo-EM 興遺傳學方法分析 Zelda 蛋白質結構與功能(2) 運用 STARR-Seq 的實驗方法來定位與量化果蠅早期胚胎發育中的增強子 (enhancer) 活性(3) 以整合分析 (meta-analysis) 建構果蠅胚胎早期發育的調控組 ;The coordinated gene expression is responsible for many developmental processes during embryogenesis in multicellular organisms, including cell proliferation, pattern formation and cell fate specification. This complicated and dynamic gene regulatory network is established by interactions between transcription factors and enhancers. Different enhancers contain different sequences that interact with specific combinations of transcription factors, achieving the precise control of gene expression patterns, spatially and temporally. Although the genomic positions of enhancers could be predicted by some bioinformatics methods according to chromatin properties, transcription factor binding and sequence features, their activities and how they change in corresponding to development stages are poorly understood. In early Drosophila embryogenesis, it has been shown that a novel transcription factor, Zelda, coordinates zygotic gene-network during Maternal-to-Zygotic transition (MZT). Zelda not only activates a battery of genes initiated a genetic cascade for the zygotic genome activation (Liang, et al., 2008), but also functions as epigenetic factor, which could alter the chromatin structure and potentiate the binding of the other transcription factors. However, the underlying mechanism how Zelda reprograms the chromatin landscape and transcriptome remain elusive, In this study, we will utilize cutting-edge approaches to dissect the structure-function of Zelda, and then apply the STARR-Seq assay to globally identity and quantify the active enhancers in early Drosophila embryogenesis. Finally, we will apply bioinformatics method to incorporate our previous RNA-polymerase, ChIP-Seq and other available published datasets to reconstruct the “regulatome” corresponding to MZT in Drosophila development.Specific Aims include:(1) To analyze the protein structure-function of Zelda by cryo-EM followed by genetic assay.(2) To comprehensively map active early enhancers in Drosophila embryogenesis by STARR-seq.(3) To reconstruct gene-regulatory network in Drosophila embryo by meta-analysis |
