Comments

"The Mediator complex is basically a central regulator/ coordinating scaffold for enhancers that beckons Pol II to transcribe DNA to RNA, but doesn’t bind to DNA itself. I think of it as a rotating log that floats around, but doesn’t touch the DNA. This log has peg holes, and the peg holes can change size and position, leaving room for various enhancers (the pegs). The size and position of the peg holes will continue to change as new enhancers bind. When the peg holes are filled ‘favorably’ then Pol II is finally recruited to the DNA, initiating transcription. This review says that the Mediator complex is composed of protein modules that are flexible, and that the flexibility (ie. Peg holes that change size and position) is conferred by the IDRs within those modules. Transcription factors also have IDRs, particularly in the regions where they bind the Mediator. So, to go back to the log analogy, the pegs and the logs physically diffuse and concentrate between/ among each other. Some of theoretical open questions that Mediator IDRs suggest will be closed as better crystal structures of the Mediator complex and its modules are resolved and made available. Condensate biologists may also link colocalization/ kinetics experiments to transcriptional regulation experiments to say more about this. What’s interesting to me at this point, is the matrix representation of transcription and translation that one can picture: The Mediator-directed ‘scaffold’ exists around DNA and drives the production of RNA, which now either degrades, remains, or is translated into protein. These newly translated proteins may serve as enhancers themselves, now circling back to regulate the Mediator complex from which they came from. The cell is basically a meshwork/ soup/ jungle of RNA and protein that comes together and separates in real time, in response to unique cellular contexts. The Mediator complex encourages/ directs this cyclical cascade of RNA-protein interactions that support cells. What’s interesting to consider is whether Mediators in separate cells are ‘aware’ of each other, and coordinate processes to support neighboring cells? If these processes are coordinated through cells, are they coordinated to support the health of the cells, or the health of the Mediators themselves? Perhaps there are peripheral RNAs or proteins in the same cell, or neighboring cells, that are modulating the group-conformation of the Mediators within single cells or across microenvironments? Is there a way that diffusing the Mediator complex (or modules within it), which is highly disordered and displays a condensate phenotype according to this review, in one cell type, would produce favorable effects in a heterogenous environment?"

The Mediator complex as a master regulator of transcription by RNA polymerase II

"Sumoylation is a post-translational modification that organizes and coordinates systematic processes within cells. One of these processes that people are interested in is targeted protein degradation. E3 ligases are ultimately responsible for depositing SUMO proteins onto target proteins, and scientists are hoping that the process of identifying sumoylated proteins (target proteins), and then tuning SUMO deposition will enable programmed upregulation and downregulation of these targets to conquer disease. Because sumoylation (and ubiquitinoylation) is reversible, the goal would be to somehow sustain this phenotype. However, it’s probably more nuanced than this- there is likely a threshold within the SUMO-cycle that needs to be met in order to activate proteolytic degradation of target proteins. This review says that SUMO encourages LLPS among proteins known to be sumoylated. So, why not run a dose-response curve that induces ‘spot formation’ with these target proteins, in disease-relevant cells. You will likely observe the ‘dose window’ where spot formation begins, intensifies, and then disappears altogether. Then, running closer doses within this window and fixing cells on timepoints would help to understand the ‘time+concentration’ combinations that would trigger degradation of target proteins. Bigger, conceptual questions are 1) Do we want sustained or sporadic spot formation? 2) Does sustained spot formation even mean that degradation of target proteins will take place? 3) Is it possible that LLPS is occluding the observation/ access of SUMOd proteins by proteases? 4) Sumo is also an additive process, so is it possible that spot area/ intensity, rather than formation, will lead us in the right direction? 5) Are there peripheral proteins that may modulate SUMO-LLPS dynamics that would be worth monitoring in relation to target proteins?"

Signalling mechanisms and cellular functions of SUMO

"NANOG is a transcription factor that can reestablish ground-state pluripotency in stem cells. The authors of this paper sought to investigate the LLPS dynamics that resulted from amino acid-level differences in NANOG c-termini, that may drive this effect. Changing all tryptophans to alanines (WT to W8A) resulted in smaller monomeric assemblies of NANOG, which was not favorable for droplet formation. Additionally, FRAP experiments confirmed that these larger WT assemblies recover from photobleaching more slowly than W8A. CHIP-seq experiments show that these amino acid changes influence the regions of chromatin DNA recognized by WT and W8A, which connects these findings to the actual biology of pluripotency control. From this paper it is worth considering whether a similar strategy of systematically substituting amino acids in c-termini of other core transcription factors (ie. OCT4, SOX2), or other proteins with amyloid-like behaviors, would deliver favorable LLPS events, and subsequently unlock the doors to multi-factor ESC fate control."

NANOG prion-like assembly mediates DNA bridging to facilitate chromatin reorganization and activation of pluripotency

"The general idea is that the patterns of charge differences in proteins will influence the kinetics of their phase separation-guided reassembly. Charge blocks are stretches of amino acids with similar charge, and ‘blockiness’ is the alternation of blocks of opposite charge (+/-). If we make these blocks shorter, then droplet formation is impeded, and droplets that previously existed dissolve/ diffuse. Hyperphosphorylation during mitosis, lengthens the blocks, which encourages droplet formation. Tuning the lengths, the total number, sequence, and likely stochasticity of these kinds of blocks will likely result in favorable LLPS dynamics/ arrangements for diseases with known condensate MOA."

Cell cycle-specific phase separation regulated by protein charge blockiness