The Drosophila pioneer factor Zelda modulates the nuclear microenvironment of a Dorsal target enhancer to potentiate transcriptional output

Abstract

1 SummaryConnecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal/ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (DL), a homologue of the NF-κB family of transcriptional activators found in humans [1,2]. A second maternally deposited factor, Zelda (ZLD), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors such as DL [3][4][5][6][7][8][9]. Here we utilized the MS2 live imaging system to evaluate the expression of the DL target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that ZLD modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of DL at the site of transcription, suggesting that transcription factor "hubs" induced by ZLD [10] functionally regulate transcription. ResultsOur study focused on the DL target gene sog as its expression domain spans a large dynamic range of the DL gradient, allowing us to examine how ZLD potentiates DL activity across the dorsal/ventral axis. Previous experiments have demonstrated that the lateral stripe of sog expression narrows dramatically in zelda null embryos [5,11] (Figure 1A,B), and that progressively removing ZLD DNA binding sites from the sog shadow enhancer shrinks the domain of activation of reporter genes in a linear manner [7]. In order to understand how ZLD alters transcription at different points along the DL gradient, we revisited these constructs with the aim of visualizing transcription in real time by adding 24 MS2 loops to the 5' end of the lacZ reporter. Since previously utilized MS2 loops [12][13][14][15] contained potential ZLD binding sites [16], we revised the MS2v5 [17] sequence to make a ZLD binding site-free non-repetitive version, referred to as MS2v5(-TAG) (see Methods and Table S1).Constructs also contained either the sog distal (shadow) enhancer [18,19] with its three native canonical ZLD binding sites, CAGGTAG (hereafter referred to as "3TAG"), or without these sites (hereafter referred to as 2 "0TAG") ( Figure 1C; see Table S2 for enhancer sequences; [7]). The narrowing effect of removing ZLD binding sites was confirmed by in situ hybridization ( Figure 1D,E).By crossing these transgenic reporter lines to females with a nanos promoter-driven MCP-GFP fusion transgene [14], we visualized the transcriptional activation of each reporter as fluorescent foci. These embryos also carry an H2Av(histone 2A variant)-RFP transgene [20], allowing us to track nuclear cycles and record transcriptional ac...

Context dependent activation and repression of enhancers by Hunchback binding sites in Drosophila embryo

“…5A, B). This observation also agrees with a study in which Zld binding sites were added to the snail enhancer, substantially accelerating enhancer activation [49].…”

Multimodal transcriptional control of pattern formation in embryonic development

“…For example, one recent study has demonstrated that the limited readout time imposed by short nuclear cycles in early Drosophila development places strict constraints on the kinds of regulatory architecture that could be responsible for driving observed patterns of hunchback gene expression (73). Other recent work has indicated that the pioneer factor Zelda plays a key role in regulating both the timing and probability of transcriptional activation following mitosis (74,75). Our work complements these previous observations by exploring yet another aspect of the interplay between timing and transcriptional regulation.…”

GAF is essential for zygotic genome activation and chromatin accessibility in the earlyDrosophilaembryo

“…S4E). Zld is known to facilitate the binding of multiple different transcription factors (Twist, Dorsal, and Bicoid), likely by forming dynamic subnuclear hubs (Yáñez-Cuna et al 2012;Xu et al 2014;Foo et al 2014;Mir et al 2018;Dufourt et al 2018;Yamada et al 2019). We therefore examined GAF localization upon depletion of maternal zld using RNAi driven in the maternal germline by matα-GAL4-VP16 in a background containing sfGFP-GAF(N) (Sun et al 2015).…”