Dynamic multifactor hubs interact transiently with sites of active transcription inDrosophilaembryos

Abstract

The regulation of transcription requires the coordination of numerous activities on DNA, yet it remains poorly understood how transcription factors facilitate these multiple functions. Here we use lattice lightsheet microscopy to integrate singlemolecule and highspeed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key patterning factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high offrates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid dependent transcription. Based on our observations we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets. 1 15 20 25 30 35

Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences

“…Our data on replication compartments, as well as other recent studies from our group (Mir et al 2017(Mir et al , 2018Chong et al 2018) demonstrate that there are multiple routes to establish regions with high local concentrations of specific factors inside the cell. These studies prompted us to critically reexamine the current evidence for LLPS in vivo.…”

“…One cellular system in particular where current enthusiasm for LLPS has vastly outpaced the evidence is in transcription regulation mediated by enhancers, where it has been emphatically postulated by many to be dependent on a process of phase separation (Hnisz et al 2017;Boija et al 2018;Cho et al 2018;Lu et al 2018;Sabari et al 2018;Shrinivas et al 2018;Guo et al 2019;Nair et al 2019). Single-molecule experiments tracking the behavior of clusters of molecules, thought to be located at enhancers or other active DNA elements, highlight the problems of this particular interpretation (Cisse et al 2013;Liu et al 2014;Mir et al 2017Mir et al , 2018Boehning et al 2018). The observation that the clusters themselves appear and disappear with extremely short half-lives and do so heterogeneously throughout the nucleus is inconsistent with our current understanding of the formation of thermodynamically driven LLPS condensates.…”

“…It is for the reasons outlined above that in our recent studies we have very purposefully avoided using the terms LLPS/condensate to describe the formation in vivo of transient local high-concentration biomolecules, in favor of the more agnostic term "hubs" (Mir et al 2017(Mir et al , 2018Boehning et al 2018;Chong et al 2018). This distinction is more than simply a semantic difference; as carefully outlined above, they represent distinct molecular mechanisms.…”

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).…”

Functional genomic approaches to elucidate the role of enhancers during development

“…However, we currently lack a clear understanding of what functional properties phase separation may confer, and how phase separation should be defined and experimentally validated in biological systems (Mir, Bickmore, Furlong, & Narlikar, ). Indeed, several recent studies have highlighted transcriptional hubs that resemble phase‐separated condensates but appear to be driven by independent mechanisms (McSwiggen et al, ; Mir et al, ). At present, it remains unclear whether phase separation has functional roles in gene regulation, or whether the appearance of phase‐separated condensates is simply a consequence of transient protein–protein interactions (Chong et al, ).…”