Bulk-level maps of pioneer factor binding dynamics during Drosophila maternal-to-zygotic transition

Gene regulation by transcription factors (TFs) binding cognate sequences is of paramount importance. For example, the TFs Zelda (Zld) and GAGA factor (GAF) are widely acknowledged for pioneering gene activation during zygotic genome activation (ZGA) in Drosophila . However, quantitative dose/response relationships between bulk TF concentration and the DNA binding, an event tied to transcriptional activity, remain elusive. Here, we map these relationships during ZGA: a crucial step in metazoan development. To map the dose/response relationship between nuclear concentration and DNA binding, we performed raster image correlation spectroscopy, a method that can measure concentration and binding of fluorescent molecules. We found that, although Zld concentration increases over time, its DNA binding in the transcriptionally active regions decreases, consistent with its function as an activator for early genes. In contrast, GAF DNA binding is nearly linear with its concentration, which sharply increases during the major wave, implicating it in the major wave. This study provides key insights into the properties of the two factors and puts forward a quantitative approach that can be used for other TFs to study transcriptional regulation.