Biophysical Determinants of the Nucleosome as an Activity Center for Chromatin Regulators

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PROJECT SUMMARY/ABSTRACT The fundamental unit of hierarchically organized eukaryotic chromatin is the nucleosome, which contains 147 base pairs of genomic DNA wrapped around an octamer of core histone proteins. Conventionally, nucleosomes have been viewed as DNA packaging units that inhibit gene expression by obstructing the accessibility of DNA to the transcriptional machinery. However, we and others have shown that nucleosomes also serve as potent hotspots which recruit, modulate, and stimulate the activity of various essential chromatin regulators, indicating a new role for nucleosomes in furnishing the genome with a multitude of physical features and interactions which direct protein function. As such, I hypothesize that the physical characteristics and topology of nucleosomes modulate the activity of chromatin regulators, constituting an underappreciated layer of physical parameters encoded within chromatin architecture that govern genomic transactions in the nucleus. These parameters include the shape and composition of the nucleosome core particle as well as the spacing and geometry of contiguous nucleosomes in an array. To test this hypothesis, I propose to use single-molecule fluorescence detection and force manipulation technologies established in my laboratory, which uniquely track real-time transient and heterogeneous molecular interactions, to investigate the physical characteristics of nucleosome topology that determine its capacity to tune the activity of several important classes of chromatin regulators at multiple scales. We will first investigate how the topology of individual nucleosomes directs the DNA targeting activity and cooperation of essential pioneer transcription factors (Aim 1). We will then probe how the geometry of local nucleosomes in an array modulates the engagement, recruitment, and propagation of chromatin- modifying enzymes on chromatin (Aim 2). Finally, we will investigate the biophysical basis of global nucleosome localization and functionalization by energy-consuming molecular machines (Aim 3). Together, these studies will zoom in on the topology of nucleosomes comprising a layer of biophysical parameters encoded within chromatin architecture that regulate genomic transactions in the nucleus. They will contribute evidence towards a new perspective that views nucleosomes as genomic regulators which harness their unique physical features to actively modulate, recruit, and stimulate the activity of chromatin-associated factors, rather than passive DNA packaging units. The proposed investigations will shed light on a nucleosome-focused angle for tackling several long-standing questions about the interplay between chromatin and its regulators and promise to mechanistically inform how disease-associated mutations perturb essential genomic activities, potentially revealing mutation- selective protein-chromatin interfaces that may be therapeutically exploited to treat human disease.