Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions.

Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in single-molecule super-resolution microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression, but several important mechanistic details of this process remain unexplored. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and their binding partners, and we inferred their most likely interaction sites. Our results demonstrate that H3K9 methylation spatially restricts HP1 proteins and their interactors, thereby promoting ternary complex formation on chromatin while simultaneously suppressing off-chromatin binding. As opposed to being an inert platform to direct HP1 binding, our studies propose a novel function for H3K9me in promoting ternary complex formation by enhancing the specificity and stimulating the assembly of HP1-protein complexes in living cells.

Visualizing molecular-scale interactions in living cells is challenging, but advances in single-molecule super-resolution microscopy enable high-resolution imaging of molecular positions of proteins and their motions within cells. HP1 proteins bind to H3K9 methylated histones to form complexes that silence gene expression. Here, we tracked single HP1 proteins and their binding partners to measure when and where they form complexes in live fission yeast cells. Genetic perturbations enabled us to connect their motions to specific changes in their cellular properties. Surprisingly, we noted that HP1 proteins preferentially form ternary complexes with their binding partners at sites of H3K9me. This work proposes a novel function for chromatin and shows how H3K9 methylation spatially restricts HP1-associated complex formation while suppressing off-chromatin binding.

Epigenetic memory is governed by an effector recruitment specificity toggle in Heterochromatin Protein 1.

HP1 proteins are essential for establishing and maintaining transcriptionally silent heterochromatin. They dimerize, forming a binding interface to recruit diverse chromatin-associated factors. Although HP1 proteins are known to rapidly evolve, the extent of variation required to achieve functional specialization is unknown. To investigate how changes in amino acid sequence impacts heterochromatin formation, we performed a targeted mutagenesis screen of the S. pombe HP1 homolog, Swi6. Substitutions within an auxiliary surface adjacent to the HP1 dimerization interface produce Swi6 variants with divergent maintenance properties. Remarkably, substitutions at a single amino acid position lead to the persistent gain or loss of epigenetic inheritance. These substitutions increase Swi6 chromatin occupancy in vivo and altered Swi6-protein interactions that reprogram H3K9me maintenance. We show how relatively minor changes in Swi6 amino acid composition in an auxiliary surface can lead to profound changes in epigenetic inheritance providing a redundant mechanism to evolve HP1-effector specificity.

Identification of a novel, tunable interface in the S.pombe HP1 protein, Swi6, that underpins epigenetic inheritance.

HP1 proteins bind dynamically to H3K9 methylation and are essential for establishing and maintaining transcriptionally silent epigenetic states, known as heterochromatin. HP1 proteins can dimerize, forming a binding interface that interacts with and recruits diverse chromatin-associated factors. HP1 proteins rapidly evolve through sequence changes and gene duplications, but the extent of variation required to achieve functional specialization is unknown. To investigate how changes in amino acid sequence impact epigenetic inheritance, we performed a targeted mutagenesis screen of the dimerization and protein interaction domain of the S.pombe HP1 homolog Swi6. We discovered that substitutions mapping to an auxiliary motif in Swi6 outside the dimerization interface can lead to complete functional divergence. Specifically, we identified point mutations at a single amino acid residue that resulted in either persistent gain or loss of function in epigenetic inheritance without affecting heterochromatin establishment. These substitutions increase Swi6 chromatin occupancy in vivo and alter Swi6-protein interactions that selectively affect H3K9me inheritance. Based on our findings, we propose that relatively minor changes in Swi6 amino acid composition can lead to profound changes in epigenetic inheritance, underscoring the remarkable plasticity associated with HP1 proteins and their ability to evolve new functions.

Human DNA ligases I and III have stand-alone end-joining capability, but differ in ligation efficiency and specificity.

Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.

HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization.

HP1 proteins traverse a complex and crowded chromatin landscape to bind with low affinity but high specificity to histone H3K9 methylation (H3K9me) and form transcriptionally inactive genomic compartments called heterochromatin. Here, we visualize single-molecule dynamics of an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. By tracking single Swi6 molecules, we identify mobility states that map to discrete biochemical intermediates. Using Swi6 mutants that perturb H3K9me recognition, oligomerization, or nucleic acid binding, we determine how each biochemical property affects protein dynamics. We estimate that Swi6 recognizes H3K9me3 with ~94-fold specificity relative to unmodified nucleosomes in living cells. While nucleic acid binding competes with Swi6 oligomerization, as few as four tandem chromodomains can overcome these inhibitory effects to facilitate Swi6 localization at heterochromatin formation sites. Our studies indicate that HP1 oligomerization is essential to form dynamic, higher-order complexes that outcompete nucleic acid binding to enable specific H3K9me recognition.

Investigating Mitotic Inheritance of Histone Modifications Using Tethering Strategies.

The covalent and reversible modification of histones enables cells to establish heritable gene expression patterns without altering their genetic blueprint. Epigenetic mechanisms regulate gene expression in two separate ways: (1) establishment, which depends on sequence-specific DNA- or RNA-binding proteins that recruit histone-modifying enzymes to unique genomic loci, and (2) maintenance, which is sequence-independent and depends on the autonomous propagation of preexisting chromatin states during DNA replication. Only a subset of the vast repertoire of histone modifications in the genome is heritable. Here, we describe a synthetic biology approach to tether histone-modifying enzymes to engineer chromatin states in living cells and evaluate their potential for mitotic inheritance. In S. pombe, fusing the H3K9 methyltransferase, Clr4, to the tetracycline-inducible TetR DNA-binding domain facilitates rapid and reversible control of heterochromatin assembly. We describe a framework to successfully implement an inducible heterochromatin establishment system and evaluate its molecular properties. We anticipate that our innovative genetic strategy will be broadly applicable to the discovery of protein complexes and separation-of-function alleles of heterochromatin-associated factors with unique roles in epigenetic inheritance.

Response and remediation actions following the detection of Naegleria fowleri in two treated drinking water distribution systems, Louisiana, 2013-2014.

Naegleria fowleri causes the usually fatal disease primary amebic meningoencephalitis (PAM), typically in people who have been swimming in warm, untreated freshwater. Recently, some cases in the United States were associated with exposure to treated drinking water. In 2013, a case of PAM was reported for the first time in association with the exposure to water from a US treated drinking water system colonized with culturable N. fowleri. This system and another were found to have multiple areas with undetectable disinfectant residual levels. In response, the water distribution systems were temporarily converted from chloramine disinfection to chlorine to inactivate N. fowleri and reduced biofilm in the distribution systems. Once >1.0 mg/L free chlorine residual was attained in all systems for 60 days, water testing was performed; N. fowleri was not detected in water samples after the chlorine conversion. This investigation highlights the importance of maintaining adequate residual disinfectant levels in drinking water distribution systems. Water distribution system managers should be knowledgeable about the ecology of their systems, understand potential water quality changes when water temperatures increase, and work to eliminate areas in which biofilm growth may be problematic and affect water quality.

Concise Modular Synthesis of Thalassotalic Acids A-C.

The novel N-acyldehydrotyrosine analogues known as thalassotalic acids A-C were isolated from a marine bacterium by Deering et al. in 2016. These molecules were shown to have tyrosinase inhibition activity and thus are an attractive set of molecules for further study and optimization. To this end, a concise and modular synthesis has been devised and executed to produce thalassotalic acids A-C and two unnatural analogues. This synthesis has confirmed the identity and inhibitory data of thalassotalic acids A-C, more potent synthetic analogues (IC50 = 65 µM), and provides a route for further structure-activity relationship studies to optimize these molecules.