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Investigating the mechanism of spatiotemporal control over genome activation by Zelda in an in vitro reconstituted system

Periodic Reporting for period 1 - ZELDA (Investigating the mechanism of spatiotemporal control over genome activation by Zelda in an in vitro reconstituted system)

Reporting period: 2019-01-01 to 2020-12-31

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.
In the course of the projected I investigated interactions between a pioneer transcription factor Zelda, and nucleic acids in an in vitro reconstituted system in order to gain insights into molecular biology of zygotic genome activation. In the process, I successfully purified and characterized several variants of Zelda containing various fluorescent tags and mutations and performed numerous biochemical assays as well as experiments in flies. In the process, I have established collaborations that led to a novel description of a pre-wetting phenomenon on DNA by a pioneer transcription factor. Some of these results have been published on bioRxiv (https://doi.org/10.1101/2020.09.24.311712) and are currently under review in Nature Physics. The bulk of the work will be submitted for publication in 2021. The results will then be disseminated to the research audience as well as the broader public.
The field of biological phase separation has been rapidly expanding providing a new angle on many poorly understood phenomena in cell biology. Transcriptional regulation has been proposed to be regulated through phase separation driven partially by the disordered domains of transcription factors. However, the data published so far does not fully support the liquid-liquid phase separation model of transcriptional control. The surface condensation model which is the result of this project provides a new concept for how the transcriptional condensates form in cells. We believe that this will pave way to better understand the basic biophysical mechanism that govern gene regulation during development as well as in disease, This could ultimately lead to novel approaches for drug design.
ZELDA findings