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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 coordinate to target chromatin regulatory complexes. DOI: http://dx.doi.org/10.7554/eLife.17667.001

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output., National Cancer Institute (U.S.) (DP2CA195769)

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells – 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. DOI: http://dx.doi.org/10.7554/eLife.17056.001, eLife digest Huntington's disease belongs to a group of human genetic disorders in which faulty proteins cause nerve cells to progressively die. All proteins are made from building blocks called amino acids, and these diseases are collectively called PolyQ expansion diseases because the faulty proteins usually have an abnormally long stretch that contains many repeats of an amino acid called glutamine. The stretches of glutamines make these mutant proteins stick to each other, which means that they aggregate in the diseased cells. Researchers have proposed several mechanisms to explain how aggregates of one such mutant protein, called mutant Huntingtin (mHtt), might contribute to Huntington's disease. One possibility is that mHtt accumulates in the nucleus of the cell – which houses most of the cell’s DNA – and interferes with the proteins that are required to switch genes on or off. Preventing these gene regulatory proteins from carrying out their role could disrupt the normal pattern of gene activity. However, working out if this is the case would require researchers being able to follow how mHtt forms aggregates and interferes with normal processes in living cells. Li et al. have now used high-resolution, high-speed microscopy to directly track the movement of individual mHtt proteins in living mouse cells in real time. The analysis showed that PolyQ protein fragments of mHtt behaved in three distinct ways – they diffused, briefly clustered or stably aggregated. Large stable aggregates of mHtt created decoy-like traps that interfered with the behavior of the gene regulatory proteins. Using computer-aided simulations, Li et al. then showed that that these molecular traps make the proteins take much longer to find their true target genes. Further studies in nerve cells suggested that this phenomenon disrupted the normal pattern of gene activity. Next, Li et al. also used the live cell imaging system to look at which gene regulatory proteins were sequestered in the mHtt aggregates. This approach identified proteins that are important regulators involved in a number of neurological disorders. These proteins included some with long stretches of glutamines and unexpectedly others in which the glutamines were interspersed with other amino acids. Aggregates of mHtt therefore appear to affect a much broader range of proteins than previously thought. Together, the results provide insight into how mHtt forms aggregates and disrupts the finely balanced mechanisms that control gene activity in nerve cells. Future studies could explore the general principles that determine which proteins interact with mHtt aggregates. This could help reveal the faulty processes that underlie Huntington's disease and other neurodegenerative disorders. DOI: http://dx.doi.org/10.7554/eLife.17056.002

Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription. DOI: http://dx.doi.org/10.7554/eLife.04236.001, eLife digest Stem cells in an embryo have the potential to become any type of cell in the body. When a cell begins to specialize, it loses this ability and can only become a limited number of cell types. These transitions are caused by changes in gene expression. Proteins called transcription factors bind to DNA to switch different genes on or off as cells become specialized. One such transcription factor, called Sox2, binds to particular DNA sequences in the cell's nucleus to encourage nearby genes to be expressed at the right levels and keep a stem cell unspecialized. However, how these binding sites are positioned throughout the three-dimensional space inside the nucleus was unknown, as was the likelihood of Sox2 finding and binding to these sites. Now Liu et al. have taken advantage of advanced microscopes to observe the interaction between Sox2 and its binding site in the nucleus of living embryonic cells. This three-dimensional imaging technology is powerful enough to capture images of individual molecules; and Liu et al. attached fluorescent tags to Sox2 to make it easier to watch them in action. By making a series of time-lapse movies, it was revealed that instead of being evenly scattered in the nucleus, Sox2's binding sites are grouped together to form individual clusters; these clusters preferably occupy spaces in the nucleus that are likely enriched for active genes. Liu et al. suggest that the clustering of Sox2 binding sites makes it more difficult for a Sox2 protein to find these sites at first, but much easier to find when the Sox2 protein is near to the cluster. Thus, the uneven positioning of the binding sites for transcription factors may provide an additional layer of control over gene expression. In the future, it would be important to map Sox2's binding sites while visualizing the activities of single genes in living cells. This would improve our understanding of how the structural organization of the contents of the nucleus can influence the correct timing of specific patterns of gene expression. DOI: http://dx.doi.org/10.7554/eLife.04236.002

The body produces proteins by transcribing DNA (genes) to make messenger RNA, which is then translated to make a protein. Transcription begins when an enzyme called RNA polymerase binds to the DNA and catalyzes the process by which genetic information from the double helix is copied to a complementary RNA transcript, which subsequently becomes the messenger RNA. Because a living cell usually contains only one or a few copies (alleles) of a given gene, molecular fluctuations play a crucial role in cellular transcription. Therefore, studying transcription kinetics at the level of single molecules may provide critical insights into how cells deal with—or even take advantage of—molecular fluctuations. A number of different single-molecule techniques can be used to follow transcription, but these techniques are often relatively slow compared to transcription in living cells, or they suffer from other problems such as only being able to study one step in the transcription process. Now, Zhang, Revyakin et al. have systematically devised a technique called ‘fastFISH’ that is fast enough to track the production of single RNA molecules directly and instantaneously. FastFISH builds on an existing technique called FISH—short for fluorescence in situ hybridization—in which fluorescent molecules are attached to single strands of DNA or RNA. These single strands pair with specific regions of complementary DNA or RNA molecules, and they can be visualized with a fluorescence microscope. However, conventional FISH is a ‘snap-shot’ technique that is not suitable for making real-time observations under physiological conditions. FastFISH relies on single strands of fluorescently labeled DNA and RNA that bind to complementary strands of DNA or RNA extremely quickly, even under physiological conditions, because they contain only three of the four ‘regular’ nucleotides that make up DNA or RNA. As a proof of principle, Zhang, Revyakin et al. used fastFISH to study the kinetics of transcription by the bacteriophage T7 RNA polymerase and were able to measure multiple stages of the transcription cycle in a single-molecule experimental setup. By allowing each stage of transcription to be tracked in real-time at the level of single-molecules, fastFISH will permit a more in-depth analysis of the factors that regulate how genes are expressed as proteins in our cells. Moreover, the ability to design single-strand probes that bind rapidly to DNA and RNA targets could have many additional applications, including new strategies for more efficient gene silencing.