PROJECT SUMMARY Shortly after fertilization, a dramatic reprogramming of the transcriptome occurs whereby maternally deposited RNAs are degraded and zygotic RNAs are synthesized, enabling the embryo to develop quickly and robustly. This is especially exemplified in the Drosophila embryo, which within a two-hour period undergoes cleavage, cellularizes the blastoderm, determines sex and X-chromosome dosage, patterns the body plan, and gets ready for gastrulation. While the gene networks underlying these processes have been well studied, it is not clear how they are collectively initiated, a process referred to as zygotic genome activation. In the previous grant period we demonstrated that a single factor, Zelda (zinc finger early drosophila activator), acts globally to activate early-expressed genes, either solely or together with the pattern-forming transcription factors. We found that Zelda binds enhancers across the genome, which have intrinsically-high nucleosome occupancy because the underlying sequences are favorable for nucleosome formation. We further showed that Zelda lowers this nucleosome barrier, thus facilitating the binding of other factors and thus increasing expressivity of downstream target genes. But how does Zelda fulfill this ?pioneering? role? We hypothesize that Zelda first ?scouts out? regions of high nucleosome occupancy, then upon binding to CAGGTAG motifs, displaces nucleosomes just enough for other factors to access the genome. In this way Zelda imparts ?enhancer competence?, a new concept in how developmental programs are initiated. We also found additional novel features of Zelda that set it apart from other factors. First, Zelda plays two different molecular roles on two types of target genes ? as a direct transcriptional activator or as an indirect potentiator (other factors required). We propose that promoter-specificity dictates which role Zelda plays. Second, Zelda has two DNA binding domains that bind different motifs, the canonical CAGGTAG motif and a newly identified G-rich motif. What is the function of this novel motif? In this grant, we aim to determine how Zelda functions mechanistically at multiscale levels - from different protein domains, to unique transcription factor roles, to global chromatin interactions. We have strong supporting evidence for each aim. In Aim 1, we will use in vitro DNA binding assays to assess how Zelda interacts with chromatin, and innovative live-imaging techniques to study how Zelda regulates transcriptional dynamics during genome activation. Aim 2 tests the hypothesis that promoter- specificity underlies which molecular role Zelda will play using a series of promoter-swap assays. Aim 3 investigates the function of the novel DNA binding domain using a deletion mutant without the domain in genomic assays (ChIP-seq and RNA-seq) to determine its role in target gene transcription. We will also use DNA binding assays to reveal interactions between the G-rich motif and CAGGTAG. If achieved these aims will impact current thinking of how transcriptional programs are initiated in development and human health.