Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging (original) (raw)
Related papers
Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation
Journal of Biological Chemistry
Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region ...
eLife, 2016
The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinat...
Epigenetics & chromatin, 2017
The association of active transcription regulatory elements (TREs) with DNAse I hypersensitivity (DHS[+]) and an 'open' local chromatin configuration has long been known. However, the 3D topography of TREs within the nuclear landscape of individual cells in relation to their active or inactive status has remained elusive. Here, we explored the 3D nuclear topography of active and inactive TREs in the context of a recently proposed model for a functionally defined nuclear architecture, where an active and an inactive nuclear compartment (ANC-INC) form two spatially co-aligned and functionally interacting networks. Using 3D structured illumination microscopy, we performed 3D FISH with differently labeled DNA probe sets targeting either sites with DHS[+], apparently active TREs, or DHS[-] sites harboring inactive TREs. Using an in-house image analysis tool, DNA targets were quantitatively mapped on chromatin compaction shaped 3D nuclear landscapes. Our analyses present evidence ...
Formation of correlated chromatin domains at nanoscale dynamic resolution during transcription
Nucleic Acids Research, 2018
Intrinsic dynamics of chromatin contribute to gene regulation. How chromatin mobility responds to genomic processes, and whether this response relies on coordinated chromatin movement is still unclear. Here, we introduce an approach called Dense Flow reConstruction and Correlation (DFCC), to quantify correlation of chromatin motion with sub-pixel sensitivity at the level of the whole nucleus. DFCC reconstructs dense global flow fields of fluorescent images acquired in real-time. We applied our approach to analyze stochastic movements of DNA and histones, based on direction and magnitude at different time lags in human cells. We observe long-range correlations extending over several m between coherently moving regions over the entire nucleus. Spatial correlation of global chromatin dynamics was reduced by inhibiting elongation by RNA polymerase II, and abolished in quiescent cells. Furthermore, quantification of spatial smoothness over time intervals up to 30 s points to clear-cut boundaries between distinct regions, while smooth transitions in small (<1 m) neighborhoods dominate for short time intervals. Rough transitions between regions of coherent motion indicate directed squeezing or stretching of chromatin boundaries, suggestive of changes in local concentrations of actors regulating gene expression. The DFCC approach hence allows characterizing stochastically forming domains of nuclear activity.
Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin
Nature, 2017
Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein del...
Binding and unbinding of transcription regulators at operator sites constitute a primary mechanism for gene regulation. While many cellular factors are known to regulate their binding, little is known on how cells can modulate their unbinding for regulation. Using nanometer-precision single-molecule tracking, we study the unbinding kinetics from DNA of two metal-sensing transcription regulators in living Escherichia coli cells. We find that they show unusual concentration-dependent unbinding kinetics from chromosomal recognition sites in both their apo and holo forms. Unexpectedly, their unbinding kinetics further varies with the extent of chromosome condensation, and more surprisingly, varies in opposite ways for their apo-repressor versus holo-activator forms. These findings suggest likely broadly relevant mechanisms for facile switching between transcription activation and deactivation in vivo and in coordinating transcription regulation of resistance genes with the cell cycle.
Cell systems, 2017
Interaction networks between chromatin complexes and long noncoding RNAs have become a recurrent theme in epigenetic regulation. However, technical limitations have precluded identification of RNA binding motifs for chromatin-associated proteins. Here, we add a denaturation step to UV-crosslink RNA immunoprecipitation (dCLIP) and apply dCLIP to mouse and human chromobox homolog 7 (CBX7), an RNA binding subunit of Polycomb repressive complex 1 (PRC1). In both species, CBX7 predominantly binds 3' UTRs of messenger RNAs. CBX7 binds with a median RNA "footprint" of 171-183 nucleotides, the small size of which facilitates motif identification by bioinformatics. We find four families of consensus RNA motifs in mouse, and independent analysis of human CBX7 dCLIP data identifies similar motifs. Their mutation abolishes CBX7 binding in vitro. Pharmacological intervention with antisense oligonucleotides paradoxically increases CBX7 binding and enhances gene expression. These dat...
2017
Gene expression states influence the three-dimensional conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features, which define promoter activation states or CTCF binding sites. Single cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fibre fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.
Cell reports, 2017
How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lo...
ABSTRACTHow transcription factors (TFs) navigate the complex nuclear environment to assemble the transcriptional machinery at specific genomic loci remains elusive. Using single-molecule tracking, coupled with machine learning, we examined the mobility of multiple transcriptional regulators. We show that H2B and ten different transcriptional regulators display two distinct low-mobility states. Our results indicate that both states represent dynamic interactions with chromatin. Ligand activation results in a dramatic increase in the proportion of steroid receptors in the lowest mobility state. Mutational analysis revealed that only chromatin interactions in the lowest mobility state require an intact DNA-binding domain as well as oligomerization domains. Importantly, these states are not spatially separated as previously believed but in fact, individual H2B and TF molecules can dynamically switch between them. Together, our results identify two unique and distinct low-mobility states...