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

Summary Gene regulation by transcription factors (TFs) binding cognate sequences is of paramount importance in development and homeostasis 1–3 . 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 a crucial step in metazoan development: the transcriptional activation of the zygotic genome. In Drosophila , zygotic genome activation (ZGA) begins with the transcription of a handful of genes during the minor wave of ZGA, followed by the major wave when thousands of genes are transcribed 4–7 . The TF Zelda (Zld) has the ability to bind nucleosomal DNA 8,9 and subsequently to facilitate the binding of other TFs 10–16 : the two defining features of a special class of TFs known as pioneer factors 17–19 . The maternally encoded TF GAGA factor (GAF) also possesses pioneer-like properties 13,14,20–23 . 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 24–29 . 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.