Biochemical Timekeeping Via Reentrant Phase Transitions

Abstract

Appreciation for the role of liquid-liquid phase separation in the functional organization of cellular matter has exploded in recent years. More recently there has been a growing effort to understand the principles of heterotypic phase separation, the demixing of multiple proteins and nucleic acids into a single functional condensate. A phase transition is termed reentrant if it involves the transformation of a system from one state into a macroscopically similar or identical state via at least two phase transitions elicited by variation of a single parameter. Reentrant liquid-liquid phase separation can occur when the condensation of one species is tuned by another. Reentrant phase transitions have been modeled in vitro using protein and RNA mixtures. These biochemical studies reveal two features of reentrant phase separation that are likely important to functional cellular condensates: (1) the ability to generate condensates with layered functional topologies, and (2) the ability to generate condensates whose composition and duration are self-limiting to enable a form of biochemical timekeeping. We relate these biochemical studies to potential cellular examples and discuss how layered topologies and self-regulation may impact key biological processes.

Keywords: RNA; condensates; disordered proteins; phase separation; reentrant phase transitions; transcription.

Figure 1.. Reentrant phase separation of a heterotypic condensate.

For a homotypic condensate (top panel) composed a single RNA binding protein (RBP), a phase transition occurs (red arrow) when [RBP] > C_sat, leading to a transition from a one-phase to a two-phase solution consisting of a light phase depleted of the RBP and a RBP rich dense phase. Increasing the RBP concentrations leads to an increase in the volume fraction of the dense phase, depicted by increased condensate size and number as a function of RBP concentration. An example of a heterotypic condensate (bottom panel) composed of the same RBP and a co-condensing RNA. Here, [RBP] < C_sat and is fixed. Increasing the concentration of RNA drives an initial phase transition (left red arrow) and the formation of heterotypic condensates composed of both the RBP and RNA. As RNA concentration increases, condensates dissolve via a second, reentrant phase transition (right red arrow), leading to a one-phase solution macroscopically similar to the first. At the molecular level, this solution consists of RBP + RNA complexes, and unbound RNA.

Figure 2.. A model for self-limited transcription via reentrant LLPS.

1. An initial enhancer-promoter contact is formed involving the factor YY1, leading to Pol II recruitment and gene activation (right). 2. As more RNA is produced, it entraps YY1 and the high local concentration nucleates a transcriptional condensate rich in YY1 (bottom center). 3. Increasing mRNA production leads to a reentrant phase transition resulting in dissolution of the transcriptional condensate (left). 4. The reduced local YY1 concentration leads to a loss of DNA binding, and the gene is shut off (top). Thus, phase separation peaks after nucleation and wanes as the RNA concentration increases providing temporal regulation of transcription (indicated as a clock).