Structural and Functional Characterization of the Chd1 Chromatin Remodeler

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ABSTRACT Chromatin remodelers are ATP-dependent DNA translocases that catalyze disassembly, reassembly, and repositioning of nucleosomes throughout eukaryotic genomes. As evidenced from multiple types of cancer and developmental disorders associated with remodeler mutations, chromatin remodeling is essential for normal growth and development. This proposal aims to address core mechanistic questions of remodeler action and regulation, using the Chd1 chromatin remodeler as a model system. Recent studies revealed that chromatin remodelers shift nucleosomes using a twist defect mechanism. In this process, remodelers couple distinct nucleotide-dependent conformations of the ATPase motor to create and then eliminate DNA distortions (twist defects) that stimulate the nucleosome to transiently absorb and then release an extra bp at the remodeler binding site. Currently, it is unknown how remodelers create twist defects. Aim 1 of this proposal seeks to identify key residues and elements of Chd1 necessary for distorting DNA into twist defects, which should allow for a mechanistic understanding of this central process. Another question addressed by this proposal is how remodeler ATPases are regulated. For Chd1, nucleosome sliding activity is coupled to DNA outside the nucleosome, with a requirement for flanking DNA on the “entry” side and preference for little or no DNA on the “exit” side. For Chd1, flanking DNA controls sliding activity through a DNA-binding domain that is coupled to autoinhibitory elements. A central question addressed by Aim 2 of this proposal is how stability and interactions of autoinhibitory elements are controlled by distinct domain arrangements on the nucleosome. Experiments are designed to reveal kinetic transitions of remodeler rearrangements on the nucleosome. Finally, a major unanswered question is how two remodelers bound to the same nucleosome affect activity of each other. Chd1 has been shown to bind to nucleosomes in a 2:1 ratio, with the DNA-binding domain interacting in trans with the chromo-ATPase, yet the significance of these interactions is unknown. Aim 3 tests the hypothesis that two opposing remodelers bound to the same nucleosome antagonize each other. Together, these studies will provide new mechanistic insights into how remodelers alter nucleosome structure, autoregulate action of their central ATPase motor, and coordinate and potentially control other remodelers on the nucleosome.