Temporal Control of Transcription by Zelda in living Drosophila embryos

Abstract

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 No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint .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<promoter<MS2-yellow transgenes, with a unique minimal promoter (sna promoter) and a MS2-yellow reporter ( Figure 1A). Upon transcription, MS2 loops are rapidly bound by the maternally provided MCP-GFP, which allows tr...

Dynamic multifactor hubs interact transiently with sites of active transcription inDrosophilaembryos

“…Based on previous observations (Hannon, Blythe, and Wieschaus 2017) that Bcd binding in more posterior nuclei is dependent on Zld, we examined the distribution of Bcd binding in nuclei lacking Zld and found that Bcd hubs no longer form ) . Our preliminary experiments with fluorescently tagged Zld revealed that it also forms hubs (distinct clusters of Zld were also recently reported by (Dufourt et al 2018) ).…”

“…Recently, a highly clustered spatial distribution of Zld was reported using confocal microscopy (Dufourt et al 2018) , but the temporal dynamics of these clusters have not been examined due to the technical limitations of confocal microscopy. We thus performed high resolution 4D imaging using LLSM of Zld in developing embryos.…”

Remembering the past: Mitotic bookmarking in a developing embryo

“…So far, clear evidences of passive supports of memory are lacking. In a recent work, by monitoring transcriptional activation in living early Drosophila embryos, it was shown that experiencing transcription prior to mitosis does not always lead to a rapid post-mitotic activation [11] . With a mesodermal enhancer, memory of active transcriptional states is unequivocally occurring [12] , while with a dorsal ectoderm enhancer, memory is not detected [11] .…”

“…However, little is known regarding the functional importance of pioneer factors for mitotic memory in developing multicellular organisms. Intriguingly, the pioneer factor Zelda, an essential activator of the early zygotic genome in Drosophila embryos, is not retained during mitosis and does not contribute to mitotic memory [11] . This could reflect that mitotic bookmarking is not a general feature of all pioneer factors, or alternatively, that Zelda is not a canonical pioneer factor.…”

“…A rather general class of mathematical models of memory can be based on Markov chains, used to explain transcriptional memory in Drosophila embryos [11] ( Figure 3 ). Originally, Markov chains were used to describe memoryless processes.…”