When Force Met Fluorescence: Single-Molecule Manipulation and Visualization of Protein-DNA Interactions.

Myriad DNA-binding proteins undergo dynamic assembly, translocation, and conformational changes while on DNA or alter the physical configuration of the DNA substrate to control its metabolism. It is now possible to directly observe these activities-often central to the protein function-thanks to the advent of single-molecule fluorescence- and force-based techniques. In particular, the integration of fluorescence detection and force manipulation has unlocked multidimensional measurements of protein-DNA interactions and yielded unprecedented mechanistic insights into the biomolecular processes that orchestrate cellular life. In this review, we first introduce the different experimental geometries developed for single-molecule correlative force and fluorescence microscopy, with a focus on optical tweezers as the manipulation technique. We then describe the utility of these integrative platforms for imaging protein dynamics on DNA and chromatin, as well as their unique capabilities in generating complex DNA configurations and uncovering force-dependent protein behaviors. Finally, we give a perspective on the future directions of this emerging research field. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Differential dynamics specify MeCP2 function at methylated DNA and nucleosomes.

Methyl-CpG-binding protein 2 (MeCP2) is an essential chromatin-binding protein whose mutations cause Rett syndrome (RTT), a leading cause of monogenic intellectual disabilities in females. Despite its significant biomedical relevance, the mechanism by which MeCP2 navigates the chromatin epigenetic landscape to regulate chromatin structure and gene expression remains unclear. Here, we used correlative single-molecule fluorescence and force microscopy to directly visualize the distribution and dynamics of MeCP2 on a variety of DNA and chromatin substrates. We found that MeCP2 exhibits differential diffusion dynamics when bound to unmethylated and methylated bare DNA. Moreover, we discovered that MeCP2 preferentially binds nucleosomes within the context of chromatinized DNA and stabilizes them from mechanical perturbation. The distinct behaviors of MeCP2 at bare DNA and nucleosomes also specify its ability to recruit TBLR1, a core component of the NCoR1/2 co-repressor complex. We further examined several RTT mutations and found that they disrupt different aspects of the MeCP2-chromatin interaction, rationalizing the heterogeneous nature of the disease. Our work reveals the biophysical basis for MeCP2's methylation-dependent activities and suggests a nucleosome-centric model for its genomic distribution and gene repressive functions. These insights provide a framework for delineating the multifaceted functions of MeCP2 and aid in our understanding of the molecular mechanisms of RTT.

Students' Perspective on Scientific Training.

How should lab heads train their students and what is the role of degree-granting institutions in this process? Chua, Vandana, and Hsieh discuss the needs of graduate students and ways to cultivate purposeful, personalized mentee training in the lab.

Single-stranded nucleic acid binding and coacervation by linker histone H1.

The H1 linker histone family is the most abundant group of eukaryotic chromatin-binding proteins. However, their contribution to chromosome structure and function remains incompletely understood. Here we use single-molecule fluorescence and force microscopy to directly visualize the behavior of H1 on various nucleic acid and nucleosome substrates. We observe that H1 coalesces around single-stranded DNA generated from tension-induced DNA duplex melting. Using a droplet fusion assay controlled by optical tweezers, we find that single-stranded nucleic acids mediate the formation of gel-like H1 droplets, whereas H1-double-stranded DNA and H1-nucleosome droplets are more liquid-like. Molecular dynamics simulations reveal that multivalent and transient engagement of H1 with unpaired DNA strands drives their enhanced phase separation. Using eGFP-tagged H1, we demonstrate that inducing single-stranded DNA accumulation in cells causes an increase in H1 puncta that are able to fuse. We further show that H1 and Replication Protein A occupy separate nuclear regions, but that H1 colocalizes with the replication factor Proliferating Cell Nuclear Antigen, particularly after DNA damage. Overall, our results provide a refined perspective on the diverse roles of H1 in genome organization and maintenance, and indicate its involvement at stalled replication forks.

Cytosine-Based TET Enzyme Inhibitors.

DNA methylation is known as the prima donna epigenetic mark for its critical role in regulating local gene transcription. Changes in the landscape of DNA methylation across the genome occur during cellular transition, such as differentiation and altered neuronal plasticity, and become dysregulated in disease states such as cancer. The TET family of enzymes is known to be responsible for catalyzing the reverse process that is DNA demethylation by recognizing 5-methylcytosine and oxidizing the methyl group via an Fe(II)/alpha-ketoglutarate-dependent mechanism. Here, we describe the design, synthesis, and evaluation of novel cytosine-based TET enzyme inhibitors, a class of small molecule probes previously underdeveloped but broadly desired in the field of epigenetics. We identify a promising cytosine-based lead compound, Bobcat339, that has mid-µM inhibitor activity against TET1 and TET2, but does not inhibit the DNA methyltransferase, DNMT3a. In silico modeling of the TET enzyme active site is used to rationalize the activity of Bobcat339 and other cytosine-based inhibitors. These new molecular tools will be useful to the field of epigenetics and serve as a starting point for new therapeutics that target DNA methylation and gene transcription.