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Histone H3 lysine 9 methylation (H3K9me) epigenetically silences gene expression by forming heterochromatin. Proteins called HP1, which contain specialized reader domains, bind to H3K9me and recruit factors that regulate epigenetic silencing. Though these interactions have been identifiedin vitro, we do not understand how HP1 proteins specifically and selectively bind to heterochromatin-associated factors within the nucleus. Using fission yeast as a model system, we measured the single-molecule dynamics associated with two archetypal HP1 paralogs, Swi6 and Chp2, and inferred how they form complexes with their interacting partners: Epe1, a putative H3K9 demethylase; Clr3, a histone deacetylase; and Mit1, a chromatin remodeler. Through a series of genetic perturbations that affect H3K9 methylation and HP1-mediated recruitment, we were able to track altered diffusive properties associated with each HP1 protein and its binding partner. Our findings show that the HP1-interacting proteins we investigated only co-localize with Swi6 and Chp2 at sites of H3K9me. When H3K9me is absent, Epe1 and Swi6 exhibit diffusive states consistent with off-chromatin interactions. Our results suggest that histone modifications like H3K9 methylation are not simply inert binding platforms but rather, they can shift the balance of HP1 complex assembly toward a predominantly chromatin-bound state. By inferring protein-protein interactions based on the altered mobilities of proteins in living cells, we propose that H3K9 methylation can stimulate the assembly of diverse HP1-associated complexes on chromatin.SIGNIFICANCE STATEMENTDuring differentiation, epigenetic silencing is essential for preserving cellular identity. Establishing and maintaining epigenetic silencing depends on histone H3 lysine 9 methylation, which HP1 proteins recognize and bind with low micromolar affinity and millisecond-scale kinetics. HP1 proteins also recruit diverse histone modifiers to maintain gene silencing. HP1 protein biochemistry has revealed whatcanhappen, but the state-of-the-art in this field includes little about whatdoeshappen in the complex and crowded environment of the nucleus. Using single-molecule tracking of HP1 proteins and their binding partners, we identified the rules that govern their complex formation in the native chromatin context, and we found that chromatin— previously thought to be an inert platform—enhances complex formation between HP1 and its binding partners.

Complex carbohydrates shape the gut microbiota and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii (Rb) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron (Bt). We analyzed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (Δsus Bt) can grow within the halo. Furthermore, when these Δsus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.Impact StatementDietary intake of complex carbohydrates including resistant starch benefits human health by supporting the growth of keystone species that increase the microbiome diversity via cross-feeding. For instance, Ruminococcus bromii (Rb) is a specialist in degrading resistant starch, and the byproducts of this degradation process are used by bacterial species. In this study, we show that Bacteroides thetaiotaomicron (Bt) cross-feeds on the glucose released during potato starch degradation by Rb. We also show that proximity to Rb cells that are releasing sugars is important for the growth of the cross-fed Bt in solid media. Additionally, we find a longer phenotype for Bt cells grown on solid media in high glucose conditions, indicating that, like other bacteria, human gut bacteria undergo significant nutrient-dependent morphological adaptations.

HP1 proteins bind with low affinity but high specificity to sites of histone H3 lysine 9 methylation (H3K9me) in the genome. HP1 binding to H3K9me compartmentalizes the genome into transcriptionally inactive heterochromatin and actively transcribed euchromatin. A characteristic feature of HP1 proteins is their dynamic and rapid turnover from sites of heterochromatin formation. How low-affinity H3K9me recognition enables HP1 proteins to rapidly and efficiently traverse a complex and crowded chromatin landscape on the millisecond timescale remains a paradox. Here, we visualize the real-time motions of an HP1 homolog, the fission yeast protein Swi6, in its native chromatin environment. By analyzing the motions of Swi6 with high spatial and temporal resolution, we map individual mobility states that are directly linked to discrete biochemical intermediates. We find that nucleic acid binding titrates Swi6 away from sites of heterochromatin formation, whereas increasing the valency of chromodomain-mediated H3K9me recognition promotes specific chromatin localization. We propose that Swi6 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for protein turnover. Our high-resolution biophysical studies provide a comprehensive framework for in vivo biochemistry and reveal how the competing biochemical properties of Swi6 affect H3K9me recognition in living cells.

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory (SPT) dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of SPT datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.Statement of SignificanceSuper-resolution microscopy allows researchers access to the motions of individual molecules inside living cells. However, due to experimental constraints and unknown interactions between molecules, rigorous conclusions cannot always be made from the resulting datasets when model fitting is used. SMAUG (Single-Molecule Analysis by Unsupervised Gibbs sampling) is an algorithm that uses Bayesian statistical methods to uncover the underlying behavior masked by noisy datasets. This paper outlines the theory behind the SMAUG approach, discusses its implementation, and then uses simulated data and simple experimental systems to show the efficacy of the SMAUG algorithm. Finally, this paper applies the SMAUG method to two model living cellular systems—one bacterial and one mammalian—and reports the dynamics of important membrane proteins to demonstrate the usefulness of SMAUG to a variety of systems.

The replisome is the multi-protein complex responsible for faithful replication of chromosomal DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in liveBacillus subtiliscells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following both damage-independent and damage-dependent replication fork stress. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replisomal proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process, and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results show that DnaE provides a limited contribution to chromosomal replication and repair.