Date of Award
Doctor of Philosophy (PhD)
The eukaryotic genome is organized in many length scales, reflecting the intricacy associated with evolution of complex biological processes. This organization serves to exert spatiotemporal control of many DNA-transacting processes such as gene expression. Despite emerging progress, the biophysical mechanism underpinning eukaryotic genome organization remains an outstanding question in the field. In this thesis, I describe mechanistic insights on genome organization and its regulation through leveraging single-molecule biophysical techniques. In Chapter 2, I characterize the dynamic interplay of Sox2 and H1 DNA binding activity. Both families constitute large classes of chromatin and DNA binding proteins that have been historically thought to be antagonistic regulators of each other, but the underlying mechanism is not well understood. Using single-molecule fluorescence-based approach, I show that Sox2 and H1 regulate each other’s loading rate on bare DNA and nucleosomes in a concentrationdependent fashion. In particular, H1 promotes the Sox2’s loading rate at low concentration but inhibits Sox2’s loading rate at higher concentration. Together, these findings highlight the potential importance of tuning protein concentrations in the regulation of gene expression. In Chapter 3, I characterize the mechanical effects on DNA from biomolecular condensation, which has recently emerged as an important mechanism of gene regulation. In particular, I investigate how Sox2, which constitutes an important pioneer factor implicated in the maintenance of pluripotency, forms co-condensates with DNA and chromatin components. The described results present three conceptual advances to the field: 1) protein:DNA co-condensation can generate high forces, up to ~7 pN, comparable to other reported cellular forces, 2) the intrinsically disordered regions (IDRs) are dispensable for condensate formation but necessary for high force generation, and lastly, 3) chromatin components, such as nucleosomes and linker histone H1, attenuate the force generating capacity of Sox2 condensates and reduce their mechanical effects on DNA via colocalization. The results add to the growing body of studies that the chromatin architecture can function as a mechanical sink that regulates cellular forces. In Chapter 4, I visualize the DNA compaction activity of the structural maintenance of chromosome (SMC) complex 5/6, an important ATPase implicated in regulating DNA repair and replication. Despite emerging insights on the SMC5/6 complex’s cellular function, the molecular mechanism behind the complex’s DNA binding activity is not well understood. Using singlemolecule fluorescence method, I present data showing the SMC5/6 complex can compact DNA in a tether-like mechanism without the requirement for ATP hydrolysis. Thus, this work adds a novel perspective towards understanding the molecular mechanism of the SMC5/6 complex. Together, the thesis below contributes novel mechanistic insights towards understanding genome organization and regulation. I reveal unique modes of DNA compaction spanning from transcription factors to ATPase molecular motor as well as associated regulatory mechanism. Due to the implications in diverse molecular pathways, aberrant regulation of genome organization underpins many disease processes. Thus, these findings help establish a molecular basis towards understanding many disease mechanisms, which can be potentially exploited for therapeutic avenues.
Nguyen, Tuan, "Mechanical Manipulation of Eukaryotic Chromatin by DNA-Binding Proteins" (2023). Student Theses and Dissertations. 719.