ZELDA · Investigating the mechanism of spatiotemporal control over genome activation by Zelda in an in vitro reconstituted system
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2019-01-01 → 2020-12-31
- EU contribution
- €159,461
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Investigating the mechanism of spatiotemporal control over genome activation by Zelda in an in vitro reconstituted system
How transcription factors (TFs) interact with DNA in order to regulate gene expression is a central question in cell biology. Most TFs contain large disordered regions, whose exact role in transcriptional regulation is not well understood. Recent work suggest that disordered regions of TFs could facilitate phase separation together with other factors critical for transcriptional initiation. In this project we investigated how a pioneering transcription factor Zelda, interacts with DNA using in vitro assays. We discovered that DNA triggers condensation of Zelda and that this behavior is distinct from classical liquid-liquid phase separation. Through experiments on optical tweezer combined with fluorescent microscopy we found that Zelda and other pioneer factor Klf4 undergo surface condensation. We also provided a theoretical framework for this phenomenon and showed how it leads to a controlled size of transcriptional condensates, consistent with the previously observed in vivo data.
Data: CORDIS, © European Union
Project objective
A long-standing question in biology is how do transcription activators and repressors exert their function over large batteries of specific targets spread throughout the genome in a timely and coordinated way. An excellent model system to study this process is zygotic genome activation (ZGA) that happens during development of every animal and hallmarks initiation of transcription from the zygotic genome. During ZGA, developmental cues such as morphogen gradients are converted into coordinated and robust spatiotemporal patterns of gene expression. I will study the mechanisms that govern this process by focusing on transcriptional activator Zelda – a key protein that orchestrates ZGA in Drosophila melanogaster. Using in vitro assays on purified recombinant proteins, I will investigate Zelda’s interactions with DNA, chromatin, RNA and transcription factors and examine the capability of such reconstituted system to recapitulate key aspects of gene regulation such as local clustering of regulatory DNA regions and transcription factors. I will examine structural and biophysical properties of Zelda, test its propensity to undergo liquid-liquid phase separation and explore the consequences it has for its interactions with its binding partners and its function. The in vitro approach will be combined with in vivo experiments in Drosophila using tagged wild-type and mutant variants. This combined approach will give us novel insights into mechanisms governing zygotic genome activation as well as general rules of transcriptional regulation. Local clustering of enhancer and promoters emerges as a general principle of transcriptional regulation. Understanding the mechanisms that could drive formation of these micro-domains is therefore of paramount importance, not only to basic biology, but also for development of new strategies to treat diseases caused by transcriptional dysregulation.
Original text from CORDIS.
Participants
- MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV · MUNCHENCoordinatorGermany
Links
- View on CORDIS
- DOI: 10.3030/798297
- https://www.mpi-cbg.de/research-groups/current-groups/anthony-hyman/research-focus/
Data: CORDIS, © European Union