We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photo-physical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.

Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate ‘hijacking’ of transcriptional condensates in various developmental and disease contexts.

Recent Hi-C measurements have revealed numerous intra- and inter-chromosomal interactions in various eukaryotic cells. To what extent these interactions regulate gene expression is not clear. This question is particularly intriguing in budding yeast because it has extensive long-distance chromosomal interactions but few cases of gene regulation over-a-distance. Here, we developed a medium-throughput assay to screen for functional long-distance interactions that affect the average expression level of a reporter gene as well as its cell-to-cell variability (noise). We ectopically inserted an insulated MET3 promoter (MET3pr) flanked by ~1kb invariable sequences into thousands of genomic loci, allowing it to make contacts with different parts of the genome, and assayed the MET3pr activity in single cells. Changes of MET3pr activity in this case necessarily involve mechanisms that function over a distance. MET3pr has similar activities at most locations. However, at some locations, they deviate from the norm and exhibit three distinct patterns including low expression / high noise, low expression / low noise, and high expression / low noise. We provided evidence that these three patterns of MET3pr expression are caused by Sir2-mediated silencing, transcriptional interference, and 3D clustering. The clustering also occurs in the native genome and enhances the transcription of endogenous Met4-targeted genes. Overall, our results demonstrate that a small fraction of long-distance chromosomal interactions can affect gene expression in yeast.

Transcription factors (TFs) bind to specific sequences in DNA to regulate transcription. Despite extensive measurements of TFs’ dissociation constant (Kd) in vitro, their apparent Kdin vivo are usually unknown. LacI, a bacterial TF, is often used to artificially recruit proteins onto eukaryotic genomes. As LacI binds tightly to its recognition site (LacO) in vitro with a Kd about 10 picomolar (pM), it is often assumed that LacI also has high affinity to LacO in vivo. In this work, we measured LacI binding in living yeast cells using a fluorescent repressor operator system and found an apparent Kd of ∼0.6 μM, four orders of magnitude higher than that in vitro. By genetically altering (i) GFP-LacI structure, (ii) GFP-LacI stability, (iii) chromosome accessibility and (iv) LacO sequence, we reduced the apparent Kd to <10 nM. It turns out that the GFP tagging location and the fusion protein stability have a large effect on LacI binding, but surprisingly, chromosome accessibility only plays a mild role. These findings contribute to our quantitative understanding of the features that affect the apparent Kd of TF in cells. They also provide guidance for future design of more specific chromosomal recruitment through high-affinity TFs.