In the first part of the project, the SG protein G3BP1 and RNA were used to form minimal SGs. The resulting compartments were biophysically characterised and partitioning of client proteins was studied. I discovered that the partitioning of some client proteins depends on the type of RNA used in the minimal SGs. This suggests that recruitment is at least partially determined by the RNA binding preference and that protein-protein interactions play a less important role than previously thought. This data was part of a manuscript published in the journal Cell which was accompanied by a press release describing the findings to the general public.
The metabolite lipoamide had previously been shown to slow down SG solidification in vivo and to affect the biophysical properties of FUS droplet in vitro. I used lipoamide to also study its effect on reconstituted minimal SGs. Even though G3BP1 is essential for SG formation and lipoamide affects ageing of SGs inside cells, it had no effect on G3BP1 droplets. However, it increased partitioning of FUS into the minimal SGs. This demonstrates that even though G3BP1 is essential for SG formation, their overall properties can be dictated by other components. Targeting those components can in turn change SG properties. This information is important to design treatment strategies against neurodegenerative diseases.
In the second part of the project, I reconstituted a different type of membrane-less compartment: TF condensates. For this, I purified the pioneer transcription factor (TF) Klf4 and analysed its condensation behaviour in the presence and absence of DNA. In collaboration with a biophysics and a theoretical physics group, we were able to show that Klf4 condenses on DNA at physiological concentrations using a mechanism that is different from phase separation. This was surprising but can explain several cellular observations that were previously puzzling.
In addition, we could show that Klf4 condensates localise to specific sequences on the DNA. We can now explain how TFs are able to distinguish functional regions from single binding sites that occur at other locations. Furthermore, we discovered that condensation is advantageous for correct localisation as compared to simple DNA adsorption: only in this way, TFs can robustly localise over a wide concentration range. This study was published on a pre-print server for early dissemination and is currently being revised for Nature Physics.
Both parts of the project give valuable insight into general condensate biology and are relevant for basic research that concerns membrane-less compartments. In addition, the data can be exploited by medically oriented researchers to design new treatment strategies against diseases that involve SGs and TF condensates. This includes neurodegenerative diseases, cancer, and developmental defects. Furthermore, we are in contact with Dewpoint Therapeutics, a start-up company that is screening small molecules for interference with liquid condensates.
