HMGB1 is an architectural DNA binding protein that plays an important role in modulating chromatin compaction, facilitating protein factor binding, and numerous other nuclear processes. In this thesis, I explored HMGB1 movement and binding dynamics on DNA and nucleosomes to better understand how HMGB1 manages its interaction with these nucleic acids and nucleoprotein complexes.

Within the nucleus, HMGB1 is capable of rapidly navigating through the complexities of the chromatin environment. However, it is unclear what interaction mechanisms facilitates HMGB1s high mobility within the nucleus. In Chapter 2, I explored the roles that each of the individual HMGB1 domain’s play in modulating its movement speed, how varying the density and length of the immobilized DNA influenced its movement characteristics and examined the implications that electrostatic interactions have on driving HMGB1 movement on the surface of a microscopy slide using single molecule Total Internal Reflection Fluorescence Microscopy (TIRF-M). I discovered that each HMGB1’s domains work together to modulate its movement speed, that reducing the density or length of the immobilized DNA drastically reduced its presence on the surface of the microscopy slide, and that electrostatic interactions were sufficient to facilitate HMGB1’s movement between its ligands. The culmination of these findings provides a framework for describing the mechanisms that allow for HMGB1 to move between DNA and ultimately contribute to our understanding of how this protein interacts with its environment.

Nuclear HMGB1 can function as an architectural factor by operating as a DNA chaperone to facilitate the binding of nearby transcription factors to their cognate DNA binding sites. Currently, it is known that HMGB1 is capable of interacting with the nucleosome by binding to the linker DNA at the entry/exit junctions and that its negatively charged C-terminal tail is capable of interacting with the histone H3 N-terminal tail. However, it is unclear how these interactions facilitate HMGB1’s positioning onto the nucleosome and the influences it has on its binding kinetics. In Chapter 3, I used single molecule TIRF-M to study the on/off binding kinetics of HMGB1 on nucleosome particles. HMGB1 exhibited on/off binding behaviors on nucleosomes; however, this data was not reproducible across multiple replicates. In an attempt to isolate the irreproducibility issue, I simplified the system by examining HMGB1 on/off binding on the free nucleosomal. 157bp DNA construct. Like with the nucleosome dataset, HMGB1 on/off binding kinetics was inconsistent between experimental replicates. Since HMGB1 on/off binding was irreproducible on free DNA, I decided to examine HMGB1 on/off binding on a shorter 32bp DNA construct. However, issues with reproducibility persisted even in this minimal DNA system. It is unclear whether the reproducibility issues is a result of protein function or the system used, and that the results of this investigation remains inconclusive.