The Formation of Phase-Separated Transcription Hubs and their Function in Transcription Regulation

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.

About us

The Brugues lab is known for combining theory with the development and use of quantitative methods to characterize living matter. Using single molecule microscopy, polarization microscopy, microrheology and theory, we have recently shown that the mitotic spindle—a self-organizing protein machinery that segregates chromosomes during cell division—behaves like an active liquid droplet that assembles through an autocatalytic process. Building on our expertise in Xenopus laevis egg extracts and previous reconstitution efforts we have developed an assay to reconstitute functional nuclei in encapsulated extracts using microfluidics (synthetic nuclei). These nuclei self-organize in extract upon the addition of pre-engineered synthetic chromosomes (such as large plasmids or BACs), properly assemble a nuclear membrane, lamina, and chromatin, and are capable of nuclear import and export. Thus, synthetic nuclei provide a powerful simplified system to study nuclear organization with total control of sequences, DNA to cytoplasm ratio, labeling of proteins, and addition or depletion of components. As a cell-free system, the extract allows excellent imaging, and the combination of high resolution 3D imaging with fluorescence correlation spectroscopy allows to measure dynamics and local protein concentrations. Additionally, we are developing several strategies to transcriptionally activate our synthetic nuclei in space at any given time. Remarkably, addition of transcription factors in the synthetic nuclei is sufficient to create micron-sized transcription hubs in the nucleus.

The Vastenhouw lab is known for its important contributions to our understanding of transcriptional control in embryos. We take advantage of the onset of transcription during embryogenesis to uncover general principles of transcription regulation. Most animals go through a phase in which the genome is inactive, and embryos rely on the products their mothers provided them. Transcription begins during the maternal to zygotic transition, when maternally loaded RNAs are degraded and the zygotic genome engages in transcription. In zebrafish, the first genes are transcribed as early as 64-cell stage, with miR-430 being the earliest and most abundantly transcribed gene (Heyn et al., 2014). We and others have previously shown that miR-430 transcription creates two isolated microenvironments of transcriptional activity. These are characterized by high concentrations of elongating RNA polymerase and the exclusion of inactive chromatin. These transcription hubs precede all other transcription and therefore provide us with an ideal system to analyze transcription hubs in vivo. To follow transcriptional activity at high resolution in embryos, we have developed an in vitro system for genome activation using dissociated cells. In combination with the use of fluorescently labeled antigen-binding fragments (Fabs), this allows us to analyze the requirements for the formation of phase-separated transcription hubs at high spatial resolution in vivo.