Nucleated transcriptional condensates amplify gene expression

Abstract

Liquid-liquid phase separation is thought to underly gene transcription, through the condensation of the large-scale nucleolus, or in smaller assemblies known as transcriptional hubs or condensates. However, phase separation has not yet been directly linked with transcriptional output, and our biophysical understanding of transcription dynamics is poor. Here, we utilize an optogenetic approach to control condensation of key FET-family transcriptional regulators, particularly TAF15. We show that amino acid sequence-dependent phase separation of TAF15 is enhanced significantly due to strong nuclear interactions with the C-terminal domain (CTD) of RNA Pol II. Nascent CTD clusters at primed genomic loci lower the energetic barrier for nucleation of TAF15 condensates, which in turn further recruit RNA Pol II to drive transcriptional output. These results suggest a model in which positive feedback between key transcriptional components drives intermittent dynamics of localized phase separation, to amplify gene expression.Cells organize complex biochemical reactions through compartmentalization into various organelles. Many of these organelles are membrane-less condensates, which form through liquid-liquid phase separation (LLPS), in which biomolecules condense into dynamic liquid droplets [1][2][3][4] . Nuclear condensates are thought to play key roles in regulating the flow of genetic information and include a diverse set of structures ranging from large scale assemblies such as nuclear speckles and the nucleolus, to structures that form on smaller length scales such as Cajal bodies, promyelocytic leukemia (PML) bodies, and gems.The concept that LLPS can drive transcriptional activity has recently received intense attention. The nucleolus is a large nuclear condensate which assembles around actively transcribing ribosomal RNA (rRNA) loci 5,6 , and is thought to facilitate the processing and maturation of rRNA into pre-ribosomal particles. Smaller transcriptional condensates have been proposed to assemble throughout the genome to facilitate transcription of non-ribosomal genes through the RNA polymerase II (Pol II) machinery [7][8][9][10] . These nanoscale transcriptional condensates have been hypothesized to facilitate enhancer-promoter interactions [11][12][13] , possibly through the generation of localized force through surface-tension driven droplet coalescence 14 ..

Biomolecular Condensates and Cancer

“…Compartmentalization allows for high local concentrations of biomolecules and their substrates and exclusion of other molecules that are not functionally relevant ( Figure 3A) (Gibson et al, 2019;Li et al, 2020). In addition, condensates are nonstoichiometric assemblies of factors involved in shared processes, so, for example, a condensate at the promoter of a gene can assemble multiple RNA polymerase molecules, thereby producing a burst of transcription (Cho et al, 2018;Guo et al, 2019;Kwon et al, 2013;Sabari et al, 2018;Wei et al, 2020). Condensates can form and dissolve in short time frames, which provides the cell with a means to produce transient compartments, thereby releasing biomolecules for use elsewhere when they are no longer needed at a specific location .…”

“…Condensates often contain components that can anchor the body to a specific location in the cell. For example, nuclear condensates can form with proteins that bind to specific DNA or RNA sequences, and cytoplasmic condensates can form at sites on the plasma membrane ( Figure 3B) (Boija et al, 2018;Case et al, 2019a;Huang et al, 2019;Shrinivas et al, 2019;Su et al, 2016;Wei et al, 2020). Transcriptional condensates form at specific enhancer and promoter elements by virtue of selective transcription factor (TF) binding (Boija et al, 2018;Shrinivas et al, 2019).…”

Transcription activation is enhanced by multivalent interactions independent of phase separation

“…LLPS arises via transient multivalent interactions of intrinsically disordered protein domains, creating an exclusionary local protein-RNA environment (21)(22)(23)(24). The formation of liquid-like protein droplets could have a number of important functional implications to (i) enhance TF binding site occupancy and residence time, (ii) mediate the recruitment of co-activators, (ii) reduce target site search time (25), (iv) contribute to TF target site selection (26) and (v) enhance transcription (27,28). However, alternative mechanisms to phase separation exist for the assembly of transcriptionally active or silenced subcompartments that include classical (cooperative) chromatin binding and formation of well-defined multi-subunit protein complexes (18,(29)(30)(31)(32).…”

3 ′-5 ′ crosstalk contributes to transcriptional bursting

“…More recently, it has been found that, in proximity to active genes, the PolIIs are incorporated in membrane-less droplets, maintained by liquid-liquid phase separation (LLPS) from the rest of the nucleus, with the net effect of locally increasing the population of factors involved in initiation; when PolII is liberated from this domain, transcription can be initiated [23][24][25][26][27][28]. LLPS also provides an explanation for the hitherto enigmatic action-at-a-distance type of gene regulation by distal enhancers, as the nuclear condensates are indeed able to restructure the genome, albeit results on LLPS are relatively preliminary at this stage [29,30].…”