NUCLEOSOME EPITOPES INFLUENCE THE ACTIVITY OF CHROMATIN REMODELERS

The association of DNA and histone proteins in the eukaryotic nucleus, called chromatin, impacts diverse aspects of biology including transcription, replication, and cell differentiation. The fundamental unit of chromatin is the nucleosome, in which DNA coils around a symmetric octamer of histones. Chromatin organization is maintained and modulated by molecular motors called chromatin remodelers, which guide the assembly, disassembly and repositioning of nucleosomes. As essential regulators of chromatin structure, chromatin remodelers are sensitive to different features of their nucleosome substrates, and in this dissertation I report on my work to characterize the actions of two chromatin remodelers called Chd1 and SNF2h. By operating on alternating sides of the symmetric nucleosomes, both Chd1 and SNF2h can sense asymmetry in DNA flanking nucleosomes, and slide nucleosomes back and forth to form evenly spaced nucleosome arrays. Analogous to this spacing behavior, in vitro, Chd1 and SNF2h slide mononucleosomes away from DNA ends. Besides differences in flanking DNA lengths, nucleosomes can also contain asymmetries in histone content, mutations or modifications, and here I address how these asymmetries affect remodeler activity. Previously, the two-fold nucleosomal symmetry has complicated this endeavor, but I discovered a method for generating well-defined and oriented asymmetric nucleosomes containing histone mutations on one specific side. Using this tool, I demonstrated that asymmetric disruption of certain nucleosomal epitopes disfavors activity from one side of the nucleosome, promoting unidirectional sliding toward DNA ends. In addition to sliding nucleosomes, Chd1 can unwrap DNA from the nucleosome edge. Using fluorescence techniques, I showed that the nucleotide state of Chd1 and the DNA sequence influence the degree of unwrapping. I also explored various fluorescence techniques for monitoring nucleosome sliding, and here I describe how competing fluorescence effects and nucleosome unwrapping influence interpretations of sliding experiments. Finally, in collaboration with a single-molecule group, we leveraged these techniques and my experience with nucleosomes to developed a scheme to simultaneously monitor DNA movement on both sides of the nucleosome. An exciting outcome of this work was our discovery that Chd1 pulls between 1-4 bp of DNA onto the nucleosome prior to DNA exiting the other side