Properties of repression condensates in living Ciona embryos

Abstract

Recent studies suggest that transcriptional activators and components of the pre-initiation complex (PIC) form higher order associations—clusters or condensates—at active loci. Considerably less is known about the distribution of repressor proteins responsible for gene silencing. Here, we develop an expression assay in living Ciona embryos that captures the liquid behavior of individual nucleoli undergoing dynamic fusion events. The assay is used to visualize puncta of Hes repressors, along with the Groucho/TLE corepressor. We observe that Hes.a/Gro puncta have the properties of viscous liquid droplets that undergo limited fusion events due to association with DNA. Hes.a mutants that are unable to bind DNA display hallmarks of liquid–liquid phase separation, including dynamic fusions of individual condensates to produce large droplets. We propose that the DNA template serves as a scaffold for the formation of Hes condensates, but limits the spread of transcriptional repressors to unwanted regions of the genome.

ERK signaling dissolves ERF Repression Condensates in Living Embryos

“…S2A). This behavior is similar to previous observations for Hes.a where the removal of the Gro-interaction motif (WRPW) abolished droplet formation (9). Based on these observations we designate the Cterminal 160 residues of Ci-ERF as a droplet forming domain (Fig.…”

“…2B). It was previously shown that mutations in the DNA binding of Hes.a resulted in condensates exhibiting definitive liquid-like properties (9). We similarly observe that a K83Q mutation in the ERF ETS domain forms fewer, larger puncta, that retain association with Gro (Fig.…”

“…S4, Supplemental Movies 6,7). When treated with U0126, mutant ERF condensates continued to grow and fuse to sizes over 1uM, considerably larger than wild-type condensates (Figs 4, S4, Supplemental Movies 8,9). These observations suggest that ERF condensate growth is limited by both DNA binding and ERK phosphorylation.…”

“…Hes.a/Gro condensates are stable throughout the cell cycle when expressed in living Ciona embryos, finally dissolving at the onset of mitosis (9). These observations point towards a simple mechanism for transcriptional repression, whereby Hes.a/Gro condensates exclude transcriptional activators at silent loci (9).…”

“…Dechorionated Ciona zygotes were electroporated as previously described (9). All experiments were replicated at least in triplicate using different batches of Ciona eggs.…”