Pioneer factor activity in transcription and DNA replication

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PROJECT SUMMARY Shortly after fertilization, a dramatic reprogramming of the genome and transcriptome occurs, enabling the embryo to develop quickly and robustly. This is especially exemplified in the Drosophila embryo, which within a two-hour period undergoes 13 mitotic cycles, cellularizes the blastoderm, 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. Moreover, although it was observed that DNA replication occurs before transcription, the mechanisms that control this timing are not known. Previously, we demonstrated that a single factor, Zelda, acts globally to activate early-expressed genes. We found that Zelda binding to CAGGTAG sites across the genome lowers nucleosome barriers at enhancers, thereby facilitating the binding of other key transcription factors. Zelda and these other factors are present in sub-nuclear “hubs” (discrete foci) that were seen to colocalize at enhancers. We aim to use high-resolution microscopy to investigate the which structural features of Zelda mediate hub formation. In addition, we are interested in the regulatory mechanisms that control the timing of transcription and DNA replication so that conflicts between the two processes are avoided. We propose that Zelda plays a dual pioneering role: 1) to open chromatin for the Origin Recognition Complex to load and subsequent formation of the Pre-replication Complex, which occludes the transcriptional machinery until origins have fired, and 2) to open newly formed chromatin for transcription to initiate. Our goal is to first define origins of replication in early embryos, and to characterize Zelda's role in origin licensing and transcriptional initiation. Our studies will lend into how these two fundamental processes of DNA replication and transcription are coordinated during genome activation.