Project Details
Projekt
The formation of phase-separated transcription hubs and their function in transcription regulation
Subject Area
Cell Biology
Biophysics
Biophysics
Term
from 2019 to 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 419139183
Transcription hubs of transcription factors and RNA polymerase II have been observed in tissue culture cells and embryos. These transcription hubs are assembled through a phase separation mechanism driven by the interaction between DNA, transcription factors, and nascent RNA. Although the transcription factors that form these hubs typically have many binding sites in the genome, they only form a limited number of hubs in any given nucleus. This raises the question what determines whether the transcriptional machinery forms a transcription hub, and how the formation of a hub affects transcriptional output of the gene(s) associated with it.Here, we will use a combination of biology and physics to address these questions. We have previously discovered that the onset of transcription in zebrafish embryos is accompanied by the formation of two isolated transcription hubs, which provides us with an excellent system to study transcription hubs in vivo. Complementary to this, we have established an assay for the reconstitution and analysis of transcriptionally active synthetic nuclei made of tunable DNA and transcription factor content. This allows for quantitative analysis of the dynamics of transcription hub formation.In Aim 1, we will determine whether transcription is required for the formation of transcription hubs. Although transcription hubs have been associated with transcribed genes, it is unclear whether transcription is a pre-requisite for hub formation. Here, we will take advantage of the two transcription hubs that precede all other transcriptional activity in zebrafish embryos to determine what comes first, transcription or transcription hubs. In Aim 2, we will determine the conditions that lead to phase separated transcription hubs. Transcription hubs are activated by transcription factors that typically have many binding sites in the genome. How the transcriptional machinery is associated only at specific gene loci is not understood. Here, we will use both zebrafish embryos and synthetic nuclei to obtain a phase diagram of transcription hubs as a function of the number of transcription factor binding sites, gene copy number, and the overall concentration of transcription factors. In Aim 3, we will determine the effect of transcription hubs on transcriptional output. The functional relevance of transcription hubs has not yet been investigated. Given that mechanisms governed by phase-separation are highly sensitive and responsive, we propose that transcription hubs may explain the non-linear response of transcription to the binding of transcription factors that is often observed for developmentally important genes. Here, we will test this hypothesis by comparing transcriptional output for genes that do and genes that do not generate transcription hubs. Taken together, these studies will reveal the function of phase separation in organizing transcriptional activity in space and time.
DFG Programme
Priority Programmes
