The Mechanism Of Nucleosome Spacing By A Dimeric Chromatin Remodeling Enzyme

Transcriptional regulation and cellular differentiation involve changes in the structure, composition and post-translational modification state of chromatin, the material that packages the eukaryotic genome. Changes to chromatin structure require the action of chromatin remodeling enzymes, protein complexes that use the energy of ATP hydrolysis to move, disassemble or deposit nucleosomes. Despite their central importance to chromatin biology, the mechanism by which chromatin remodelers break histone-DNA contacts and reposition nucleosomes remains largely unknown. Our work focuses on ACF, a human chromatin remodeler that establishes even spacing between adjacent nucleosomes. ACF spaces nucleosomes by sensing the length of linker DNA flanking each nucleosome and preferentially moving toward the side with longer DNA. Recently, we have shown that two copies of ACF bind each nucleosome, one on either side, suggesting a model in which the two motors take turns translocating in one direction or the other. These findings raise two unanswered questions: 1) How is the core DNA translocase activity of ACF regulated in response to linker DNA? and 2) How do two copies of ACF coordinate their activities to allow directional movement of a single nucleosome? To address these questions we have studied SNF2h, the motor subunit of ACF, using biochemical, spectroscopic and structural approaches. We have identified a conformational change in the linker DNA-binding domain of SNF2h that is coupled to its catalytic cycle. Moreover, we have used protein engineering and targeted mutagenesis to probe the communication between two motors bound to the same nucleosome. We will present our latest progress toward understanding how the two motor subunits respond to linker DNA and coordinate their actions to enable nucleosome spacing.