Student Theses and Dissertations
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Shixin Liu
Keywords
single-molecule microscopy, chromatin, MeCP2, DNA replication, nucleosome assembly, DNA damage response
Abstract
Beyond the genetic code embedded within a sequence of DNA, the physical features of the double helix as a semi-flexible polymer is a critical dimension that regulates the function of every DNA-binding protein that exists. These proteins can assemble, translocate, and change conformation while bound to DNA and can even alter its physical state, which are often crucial to their physiological roles in the cell. Eukaryotic genomic DNA is also packaged into units called nucleosomes, which offer another layer of physical regulation for proteins operating on chromatin. Previously, tracking these protein behaviors on DNA and chromatin had been technically challenging, dependent on tools that could access short-lived, heterogenous, and out-of-equilibrium interactions. It is now possible to observe these activities in real-time, owing to the advent of single-molecule fluorescence- and force-based techniques that have enabled dynamic and quantitative measurements of protein-chromatin interactions central to nuclear biology. This thesis describes my work, in collaboration with others, at harnessing single-molecule techniques to answer several pressing questions in biology. I hope it serves to highlight the utility and power of single-molecule microscopy for achieving never-before-seen multidimensional, real-time data of protein behavior potentially key to their roles in the cell. Chapter 1 intends to provide an introduction to the tool of choice, including a brief history of the technology and how its been successfully utilized prior in the literature to access mechanistic biology. It then offers a brief presentation into projects discussed in the thesis body, which contain several investigations into a sampling of diverse but all chromatin-related questions, whether surrounding the basic science of disease-prone processes or clinically relevant mutational data. Indeed, the chapter may stand alone to describe the broad utility of correlative single-molecule force and fluorescence microscopy (smCFFM) for investigating nuclear biology. Chapter 2 offers a discussion dedicated towards understanding the function of the epigentic reader, methyl-CpG-binding protein 2 (MeCP2), a protein whose mutations are solely responsible for causing Rett syndrome, a severe neurological disorder. Rett syndrome primarily affects young girls and does not currently have cure. MeCP2 is most well-known for being a reader of CpG methylation on DNA, yet it binds pervasively across the neuronal genome, in part due to its extensive disorder and multivalent binding. It is classically viewed as a transcriptional repressor, but several studies have reported its association with active genes. Finally, there are many clinically reported mutations, but how they lead to dysfunction and disease remain elusive. In sum, MeCP2’s substrate preferences, biological function, and dysregulation in the context of Rett syndrome remain challenging to study and an ongoing mystery for a disease-motivated field. To address these challenges, we studied MeCP2 from a previously-unexamined angle of biophysics. Using smCFFM, we found that MeCP2 uses differential dynamics on DNA and chromatin to specify methylation- and nucleosome-specific functions, which are altered when mutant forms of MeCP2 are present. We also discovered that MeCP2 preferentially binds nucleosomes over bare DNA, contrasting its canonical role as a DNA reader and providing a new therapeutic opportunity to modulate its genomic distribution in vivo. Overall, our study provides a new perspective for understanding MeCP2 function that may help clarify its role in disease. Chapter 3 takes a break from biological investigation and presents a new method for loading nucleosomes across DNA directly within a smCFFM instrument. Beyond packaging, nucleosomes serve as interactive hotspots for many important chromatin-binding proteins. The mainstream method for reconstituting nucleosome templates for single-molecule techniques has been assembling nucleosomes on custom DNA templates by salt dialysis; however, this approach suffers from biases stemming from non-native nucleosome positioning sequences, requirement for high amounts of DNA and octamers, and a lengthy timeline of one to several days of work. To address these disadvantages, we report a new method that utilizes the histone chaperone Nap1 to directly assemble nucleosomes on DNA substrates stably tethered within a smCFFM instrument. In addition to more closely resembling the physiological pathway of nucleosome formation in vivo, this method allows users to reconstitute nucleosomes on non-specific DNA sequences, easily adjust nucleosome density, and utilize significantly fewer amounts of reagents all within minutes. When uniform and specific nucleosome positioning is not needed, this protocol provides a useful way to investigate nucleosome mechanics or protein behavior on chromatin, for which example experiements are also described. Chapter 4 discusses a study on DNA replication, specifically the dynamics of the eukaryotic sliding clamp, proliferating nuclear cell antigen (PCNA). PCNA is an essential ring-shaped protein required for replication in addition to diverse cellular processes including DNA repair, chromatin maintenance, and sister chromatid cohesion. To perform its functions, it must be loaded onto and encircle DNA, which is performed by its canonical loader, replication factor C (RFC). Despite rigorous biochemical studies that have characterized RFC and PCNA’s substrate preferences and mechanism of loading, it had remained unknown how RFC navigates and then loads PCNA to its target DNA sites. To answer this question, we employed smCFFM to visualize RFC-PCNA complexes on varying DNA substrates, which revealed RFC frequently remains bound to PCNA after DNA loading. This is in contrast to the prevailing model in the field hypothesizing RFC is immediately ejected at this step. Additionally, we found that RFC-PCNA complexes are active for fill-in synthesis and can assemble with the lagging strand polymerase δ (Polδ). Finally, we show that this activity is dependent on the BRCT domain of Rfc1 and that deficient PCNA-Polδ fill-in can be rescued by another PCNA-binding partner and flap endonuclease, FEN1. Together our findings show how PCNA-enabled DNA synthesis is regulated by functions of its binding partners that are separate from their own catatlytic activities and assigns a role for the previously elusive RFC BRCT domain. This study is in close collaboration with Michael O’Donnell’s lab at Rockefeller. Chapter 5 examines a unique mycobacterial helicase, Lhr whose activities are required to confer cellular resistance to DNA damage. Specifically, biochemical assays have revealed that Lhr translocates along single-stranded DNA in the 3’ to 5’ direction, unwinding DNA:DNA or RNA:DNA duplexes en route. They have also shown that its helicase and tetramerization activities are dependent on an intact C-terminal domain (CTD), which are all required to confer mycobacterial resistance; however, the mechanisms behind the CTD and what its role is in resistance had remained unknown. We used smCFFM to demonstrate the CTD is required to grip Lhr on to the DNA upon 5’ engagement during ssDNA translocation, which explains its requirement for helicase activity. Hidden previously from bulk experiments, we also found Lhr at the 3’ junction exhibits single-stranded DNA reeling activity. Finally, we show that reeling frequency is increased by an intact CTD, which confers Lhr preference for binding both the 3’ and 5’ junctions as compared to single-stranded DNA. Our findings inform the mechanistic details of Lhr function at DNA and may help explain its role in DNA repair in vivo. This study is in close collaboration with Stewart Shuman’s lab at Memorial Sloan Kettering Cancer Center. Finally, chapter 6 presents a study surrounding the chromatin-binding protein, linker histone H1, which has been well-known for compacting nucleosomes but whose interaction with DNA has remained under-studied. H1 is a highly disordered protein and undergoes liquid-liquid phase separation with chromatin; nevertheless, the biophysical basis and biological relevance of these condensates had remained unknown. Using smCFFM, we found H1 exhibits enhanced phase separation with single-stranded DNA, which relies on multivalent and transient engagement with each other. Cellular imaging led us to propose H1 accumlates on nascent ssDNA that is formed after DNA damage, potentially protecting it from degradation. Our results highlight H1’s multifaceted roles at chromatin and provides a new role for the protein in the context of DNA damage. This study is in close collaboration with Yael David’s lab at Memorial Sloan Kettering Cancer Center.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Chua, Gabriella N. L., "Making Molecular Movies: Using Single-Molecule Techniques to Unveil Hidden Features of Protein-Chromatin Interactions" (2025). Student Theses and Dissertations. 796.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/796
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy