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.

There are extensive long-distance chromosomal interactions in eukaryotic genomes, but to what extent these interactions affect gene expression is not clear. Recent works have identified several cases where clustering of co-regulated genes leads to enhanced gene expression in budding yeast. Similar phenomenon was also observed in mammalian cells. These results challenge widely held views of gene regulation in yeast and further our understanding of how the 3D organization of the genome contribute to gene regulation in eukaryotes.

Numerous intra- and inter-chromosomal contacts have been mapped in eukaryotic genomes, but it remains challenging to link these 3D structures to their regulatory functions. To establish the causal relationships between chromosome conformation and genome functions, we  develop a method, Chemically Induced Chromosomal Interaction (CICI), to selectively perturb the chromosome conformation at targeted loci. Using this method, long-distance chromosomal interactions can be induced dynamically between intra- or inter-chromosomal loci pairs, including the ones with very low Hi-C contact frequencies. Measurement of CICI formation time allows us to probe chromosome encounter dynamics between different loci pairs across the cell cycle. We also conduct two functional tests of CICI. We perturb the chromosome conformation near a DNA double-strand break and observe altered donor preference in homologous recombination; we force interactions between early and late-firing DNA replication origins and find no significant changes in replication timing. These results suggest that chromosome conformation plays a deterministic role in homology-directed DNA repair, but not in the establishment of replication timing. Overall, our study demonstrates that CICI is a powerful tool to study chromosome dynamics and 3D genome function.

Numerous intra-and inter-chromosomal contacts have been mapped in eukaryotic genomes, but it remains challenging to link these 3D structures to their regulatory functions. To establish the causal relationships between chromosome conformation and genome functions, we need a method that allows us to selectively perturb the conformation at targeted loci. Here, we developed a method in budding yeast, Chemically Induced Chromosomal Interaction (CICI), to engineer long-distance chromosomal interactions selectively and dynamically. We implemented CICI at multiple intra-and inter-chromosomal loci pairs and showed that CICI can form in >50% of cells, even between loci with very low Hi-C contact frequencies. CICI formation is slower at these low Hi-C sites, revealing the dynamic nature of the Hi-C signals.As a functional test, we forced the interaction between mating-type locus (MAT) and HMR and observed significant change in donor preference during mating-type switching, showing that chromosome conformation plays an important role in homology-directed DNA repair. Overall, these results demonstrate that CICI is a powerful tool to study chromosome dynamics and the 3D genome function.

The MET3 promoter (MET3pr) inserted into the silenced chromosome in budding yeast can overcome Sir2-dependent silencing upon induction and activate transcription in every single cell among a population. Despite the fact that MET3pr is turned on in all the cells, its activity still shows very high cell-to-cell variability. To understand the nature of such “gene expression noise,” we followed the dynamics of the MET3pr-GFP expression inserted into ribosomal DNA (rDNA) using time-lapse microscopy. We found that the noisy “on” state is comprised of multiple substable states with discrete expression levels. These intermediate states stochastically transition between each other, with “up” transitions among different activated states occurring exclusively near the mitotic exit and “down” transitions occurring throughout the rest of the cell cycle. Such cell cycle dependence likely reflects the dynamic activity of the rDNA-specific RENT complex, as MET3pr-GFP expression in a telomeric locus does not have the same cell cycle dependence. The MET3pr-GFP expression in rDNA is highly correlated in mother and daughter cells after cell division, indicating that the silenced state in the mother cell is inherited in daughter cells. These states are disrupted by a brief repression and reset upon a second activation. Potential mechanisms behind these observations are further discussed.