Abstract/introPioneer factors have the exquisite ability to engage their target sites at nucleosomal DNA, which leads to a local remodeling of chromatin and the establishment of a transcriptional competence. However, the direct impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains elusive. In Drosophila embryos, the maternally deposited activator Zelda (Zld) exhibits key pioneer factor properties and indeed regulates the awakening of the zygotic genome. The analysis of thousands of endogenous Zld bound regions in various genetic contexts, as well as the study of isolated synthetic enhancers with static approaches, led to the proposal that Zld could act as a quantitative developmental timer. Here we employ quantitative live imaging methods and mathematical modeling to directly test the effect of Zld on temporal coordination in gene activation and on mitotic memory. Using an automatic tracking software, we quantified the timing of activation in hundreds of nuclei and their progeny in Drosophila embryos. We demonstrate that increasing the number of Zld binding sites accelerates the kinetics of transcriptional activation regardless of their past transcriptional state. In spite of its known pioneering activities, we show that Zld is not a mitotic bookmarker and is neither necessary nor sufficient to foster mitotic memory. Fluorescent recovery after photo-bleaching and fluorescent correlation spectroscopy experiments reveal that, Zld is highly dynamic and exhibits transient binding to chromatin. We propose that Zld low binding rates could be compensated for by local accumulation of Zld in nuclear microenvironments in vivo, thus allowing rapid and coordinated gene activation.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/282426 doi: bioRxiv preprint first posted online Mar. 14, 2018; 2
Results and Discussion
Zelda fosters temporal transcriptional coordinationIn the blastoderm embryo, two redundant enhancers control snail (sna) gene expression, a proximal (primary) and a distal (shadow enhancer) 1,2 . Both enhancers are bound by Zelda (Zld) at early nuclear cycles 3,4 . Expression driven by the long intact shadow enhancer (sna-shadow) leads to rapid transcriptional activation, precluding analysis of the impact of Zld on fine-tuning the timing of this activation ( Figure S1, A-B). We therefore used a previously described truncated version of the sna shadow enhancer (snaE) that leads to a stochastic activation, compatible with mitotic memory tracking 5 .To follow transcriptional dynamics in a quantitative manner, we created a series of enhancer