Differential modulation of the topography of the nucleosomal barrier to transcription by epigenetic modifications revealed by high resolution optical tweezers

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¹Institute for Quantitative Biosciences-QB3, University of California, Berkeley, USA
²Department of Physics, University of California, San Diego, USA
³Department of Physics, University of California, Berkeley, USA
⁴Department of Biochemistry and Molecular Biology, Colorado State University, USA
⁵Department of Biological Sciences, University of Pittsburgh, USA

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. Modulation of this barrier by epigenetic modifications plays an important role in transcriptional regulation. However, understanding of the molecular basis for this regulation requires a high-resolution characterization of how epigenetic modifications affect the barrier topography and the dynamics of transcription through the nucleosome. In this work, we designed a single molecule optical tweezers assay to obtain topographic and transcriptional maps of canonical, H2A.Z-modified, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. Epigenetic modifications have distinct effects on the barrier, with uH2B heightening it and H2A.Z distributing it over a wider region of the nucleosome. The transcriptional dynamics through the nucleosome and how they are affected by epigenetic modifications closely resemble the topography of the mechanical barrier, indicating that the mechanical barrier alone, rather than specific Pol II-nucleosome interactions, is sufficient to form the observed transcriptional barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.