Project Summary In eukaryotic cells, the packaging of genomic DNA into nucleosomes represents the basic barrier to transcription by RNA polymerase II (Pol II). The dynamic modulation of the magnitude of this barrier and the degree of chromatin compaction constitute fundamental mechanisms of regulation of gene expression. Having previously characterized the magnitude and mechanical properties of the nucleosomal barrier and their effect on Pol II transcription, we aim to investigate the modulation of this barrier and its regulation of genome accessibility to Pol II by environmental and cellular factors. We will do this by combining single-molecule optical tweezers/fleezers (SM-OT) methods with high-resolution single particle cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and quantitative in vivo fluorescence imaging. We have determined that nucleosomes display a trimodal distribution of unwrapping forces corresponding to alternative nucleosome states that we hypothesize represent different barriers to transcription by Pol II. We will investigate this intrinsic plasticity of nucleosomes and its modulation by factors that promote their interconversion, including histone variants, post-translational modifications, and specific transcriptional regulators (pioneer factors, elongation factors, and histone chaperones). Next, we will use our SM-OT assay to investigate how specific transcriptional regulators (e.g., FACT, Spt4/5, Chd1) affect the dynamics of Pol II as it transcribes through the nucleosomal barrier. We will also explore how histone PTMs, that have either activating or repressive roles on transcription, modify the molecular trajectories and dynamics of individual Pol II molecules. Moreover, we will study how Pol II crosses hexasomes and tetrasomes, both generated during moderate and intense transcription and that have been proposed to play important roles in vivo. We will structurally characterize the nucleosome remodeling during passage of Pol II by cryo-ET both in the presence or absence of transcription regulators. In eukaryotic cells, gene expression requires the passage of Pol II through arrays of nucleosomes organized as chromatin. These arrays can adopt higher-order and more compacted structures that impose a greater barrier to transcription by Pol II. Therefore, to understand how nucleosome arrays regulate genome accessibility, we will study their mechanical stability using SM-OT unwrapping assays and characterize the energetics of internucleosome interactions in these structures. We will also investigate the impact of nucleosomal arrays on the transcription dynamics of individual Pol II using SM-OT in the absence and presence of transcription regulators, and under different conditions that modulate chromatin compaction. We will compare the dynamics of Pol II obtained in these in vitro experiments, with its dynamics in vivo. To this end, we will introduce the strong nucleosome position sequence 601 into Dros...