多細胞生物發育包含許多生物途徑，例如細胞增長，模式發育與分化。歷程中所有參與的基因表現必需精密的控制，如何整合這些複雜又動態的基因網絡，仰賴反式作用因子（轉錄因子）與順式作用因子（增強子）。增強子通常包含複合反應元件，可與不同組合的轉錄因子交互作用，以因應不同的發育階段，並促進正確的基因表現。雖然生物資訊的運算邏輯已能根據各種功能性基因體數據，預測增強子的位置，但要探討增強子的動態活性與如何因動應不同細胞型態及發育階段，仍然是很大的挑戰。轉錄因子Zelda在果蠅早期的胚胎發育中扮演啟動基因體表現的角色。利用簡單的策略與一組特定的基因序列交互作用，Zelda可以整體性與選擇性的激活所有早期胚胎發育所需要的基因，但Zelda的作用機制卻很複雜。Zelda的生化特性與結構仍尚未解開，因此在本實驗中，我們將利用Cryo-EM分析來研究Zelda的結構並了解相對應的功能。此外，先前Zelda ChIP-seq 的結果顯示Zelda會與上萬的序列片段結合，但並非每一個片段都是有活性的增強子。因此本研究將利用S2 細胞為基礎的STARR-seq系統，來鑑定並定量全基因體中的增強子活性。整合這些數據以及先前已建立的資料組作分析，我們將可以定義胚胎發育早期基因活化增強子的特徵。具體目標包含:1以cryo-EM分析Zelda蛋白質結構與利用遺傳學方法學習其功能2用 STARR-Seq的實驗方法來測定與量化果蠅早期胚胎發育基因體中增強子活性3利用生物大數據整合分析以建構Zelda調控的果蠅胚胎早期發育之順式調控組 ;The development processes in multi-cellular organism, including cell proliferation, pattern formation and differentiation all depend on the proper spatiotemporal controls of gene expression. Cells coordinate such a complicated and dynamic gene network through the interactions between trans-acting factors (transcriptional factors) and cis-regulatory elements (enhancers). Enhancers contains different sequences could interact with different combinations of transcription factors in response to different development stages and give arise proper activities for gene expression. Although bioinformatic algorithms with information provide by functional genomic datasets could predict the genomic position of enhancers, how their dynamic activities change corresponding to cell types and development stages are difficult to analyze.During early Drosophila embryogenesis, a key activator, Zelda, was found responsible for the zygotic genome activation (ZGA) during marternal-to-zygotic transition (Liang, et al., 2008). Zelda uses a simple strategy through the binding to TAG sites not only to collectively and selectively activate batteries of genes essential for shaping early development. Although many important roles of Zelda in transcriptional regulation have been demonstrated, the biochemical and structural features that make Zld a master regulator of the embryonic genome remain largely unknown. Unlike the zebrafish which uses a group of key factors, how a single factor like Zelda executes multi-mechanisms on genome activation remains elusive. To tackle this question, we will use Cryo-EM for Zelda structural analysis to understand the relationship between its structure and function. Moreover, ChIP-seq analysis of Zelda showed that Zelda bounds to more than ten thousand of genomic regions, but the in vivo relevance is still undetermined, due to lacking comprehensive mapping of functional enhancer activities. This study aims to modify and use STARR-seq (self-transcribing active regulatory region sequencing) to quantitatively map whole-genome enhancer activities at the present or absent of Zelda. With this and meta-analysis of pre-established related datasets, we will be able to discover the bona fide signatures of ZGA enhancers.Specific Aims:Aim 1. To analyze the functional structure of Zelda by cryo-EM and genetic assay.Aim 2. To map quantitative genome-wide enhancer activity of early Drosophila embryos by STARR-seq.Aim3. To reconstruct Zelda-responsive cis-regulatory landscape by meta-analysis.
