Structural and Functional Studies of Transcription Through Chromatin

Abstract

Transcription elongation by RNA polymerase II (Pol II) through chromatin presents a formidable challenge, as nucleosomes impede Pol II progression. Despite the nucleosomal barrier, eukaryotic cells routinely achieve high-fidelity gene expression while preserving chromatin structure, ensuring both transcriptional fidelity and epigenetic continuity. This dissertation investigates the molecular mechanisms by which Pol II overcomes the nucleosomal barrier and maintains nucleosome integrity during elongation. Through cryo-electron microscopy and in vitro biochemical approaches, I characterize structural intermediates that reveal how Pol II and associated elongation factors coordinate histone displacement, retention, and reassembly. I describe a nucleosome retention transcription elongation complex in the absence of FACT and a distinct hexasome intermediate stabilized by multiple acidic blocks in the N-terminal domain of SPT6 during FACT-assisted transcription. Together, these structures visualize chaperone-dependent and -independent models of nucleosome retention. Complementary in vitro assays further demonstrate how transcription-generated nucleosomal states modulate the enzymatic activity of transcription-associated chromatin modifiers RNF20/40-UBE2A ubiquitin ligase and MLL1 complex. Together, these findings establish a structural and biochemical framework for how the elongation machinery preserves chromatin architecture while directly coupling nucleosome traversal to the installation of transcription-associated histone modifications.