Genome-Wide Quantitative Mapping Early Embryonic Enhancer Activity in Drosophila by Starr-Seq and Meta-Analysis of Multi-Omic Data(2/2)

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.