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Activation mechanisms of SWI2/SNF2 family ATP-dependent Chromatin Remodeling Enzymes

  • Author(s): Gamarra, Nathan Ian
  • Advisor(s): Narlikar, Geeta J
  • et al.
Abstract

Eukaryotic genomes are packaged into chromatin: a highly heterogeneous structure composed of nucleic acids and proteins. This packaging controls access to the underlying DNA sequences and, as a result plays a critical role in nearly all genomic processes. The primary molecular structure of chromatin is the nucleosome: ~147 bp of DNA wrapped around a core of histone proteins. Both the location and status of nucleosomes in the genome are critical for the proper packaging of chromatin. As a result, cells have evolved several sophisticated molecular machines to disrupt or modify nucleosomes to achieve specific packaging states. A critical member of these machines are the SWI2/SNF2 superfamily of ATP-dependent chromatin remodeling enzymes, which are DNA translocases that harness the energy of ATP hydrolysis to physically disrupt nucleosomes. Because of their central role in control nucleosome structure throughout the genome, remodelers play roles in virtually all DNA-dependent process, but the precise mechanisms of how remodelers disrupt nucleosomes and how this disruption is coupled to other molecular events remains very poorly understood. In this thesis we focus on understanding the remodeling mechanisms of two subfamilies of SWI2/SNF2 remodelers that slide nucleosomes: INO80 and ISWI. To understand how these and other SWI2/SNF2 ATP-dependent remodelers might cooperate with nuclear machinery to enable biological processes, we first review our broad understanding of remodeling mechanism as it compares to another molecular motor that disrupts nucleosomes: RNA polymerase. We then speculate on how these two distinct families cooperate to accomplish transcription on chromatin templates. After this, we set out to uncover elements of nucleosome that control remodeler activity and identify a conserved surface of the nucleosome known as the acidic patch that is required to activate both ISWI and INO80 family remodelers. Using a combination of biochemical and biophysical assays, we show that this surface activates remodeling by these two families after they bind the nucleosome. For the ISWI remodeler SNF2h, the acidic patch activates remodeling by serving as a landing pad for the binding of autoinhibitory domains while INO80 uses a separate mechanism. We then solve the near-atomic CryoEM structure of SNF2h bound to the nucleosome. Unexpectedly, we find that SNF2h binding in an activated state asymmetrically distorts the histone core of the nucleosome and that this may be important in regulating the activity of the enzyme. Finally, we test the hypothesis that by measuring remodeling activity of nucleosomes with site-specific restraints in the histone core. We find that specifically restraining histone dynamics in locations across all 4 histone proteins inhibits SNF2h-mediated nucleosome sliding. Taken together, these results suggest that remodelers rely on the structure and dynamics of both the DNA and protein components of the nucleosome to accomplish their activities.

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