Structural insights into chromatin remodeling by ISWI during active ATP hydrolysis.

Chromatin remodelers utilize the energy of adenosine triphosphate (ATP) hydrolysis to slide nucleosomes, regulating chromatin structure and gene activity in cells. In this work, we report structures of imitation switch (ISWI) bound to the nucleosome during active ATP hydrolysis and remodeling, revealing conformational transitions of the remodeling motor across the adenosine triphosphatase (ATPase) cycle. The DNA strands were distorted accordingly, showing one full base-pair bulge and a loss of histone contact at the site of motor binding in the adenosine diphosphate* (ADP*) and apo* (unbound) states. We also identified several important elements for regulation of the remodeling activity. Notably, an enzyme conformation exiting the remodeling cycle reveals a linker DNA–sensing brake mechanism. Together, our findings elucidate a multistate model of ISWI action, providing a comprehensive mechanism of DNA translocation and regulation underpinning chromatin remodeling. Editor's summary: Chromatin remodelers are motor proteins that slide nucleosomes along genomic DNA, playing a key role in the regulation of chromatin structure and gene activity. Sia et al. used cryo–electron microscopy to capture snapshots of the chromatin remodeler ISWI in actively translocating a nucleosome. This work provides a molecular movie to illustrate how the remodeler couples the energy of ATP hydrolysis to reposition the nucleosome, and sheds light on the mechanism of ISWI regulation to prevent undue remodeling. —Di Jiang INTRODUCTION: DNA wraps around histone octamers to form nucleosomes, chromatin's basic units. Adenosine triphosphate (ATP)–dependent chromatin remodelers—switch/sucrose nonfermentable (SWI/SNF); imitation switch (ISWI); chromodomain, helicase, DNA binding (CHD); and inositol requiring 80 (INO80)—reposition nucleosomes to regulate chromatin structure and function. SWI/SNF creates accessible chromatin, whereas ISWI senses linker DNA length and spaces nucleosomes evenly. These enzymes share highly conserved motor domains, which are at the heart of the chromatin remodeling reaction. Recent cryo–electron microscopy (EM) studies revealed staggered DNA translocations of the nucleosome, with a ½-bp rather than full 1-bp DNA bulge at the site bound by the motor. When and how the other strand is translocated remains unknown. Moreover, to prevent excessive chromatin remodeling, the translocation activity is negatively regulated, the mechanism of which remains enigmatic. RATIONALE: The previous studies were presumably constrained by the absence of actual translocation so that only three states of the remodeling reaction are captured. By using ATP to sustain the translocation reaction, we enabled the motor to traverse all potential stages of the remodeling cycle. To catch the actions in various states, we collected datasets with different concentrations of ATP and determined nine high-resolution cryo-EM structures of ISWI bound to the nucleosome during active translocation. RESULTS: The structures of the canonical ATP- and adenosine diphosphate (ADP)–bound states were determined at resolutions of 2.3 to 2.6 Å, which provide clear views of the motor domains and the interaction with the DNA. In particular, we found that motifs V and VI relay the signal from the bound nucleotides to the nucleosome, underlying the DNA bulge and translocation upon ATP hydrolysis. In the ADP* state, we observed the full 1-bp DNA distortion. The tracking strand movement results in a loss of the DNA-histone contact at SHL2.5. The complexes in the unbound apo and apo* states adopt conformations similar to that of the ADP and ADP* states, respectively. In the ADP+ and ADP*+ states, we identified a C-terminal positive element (PosC) and an arginine-tyrosine anchor motif (RYA), both of which promote the ISWI activity. In the ADPS state, the motor adopts a globally open conformation, but the DNA slips back to a relaxed state, suggesting that the energy of ATP hydrolysis is dissipated without being converted to DNA translocation. In the ADPB state, we identified a newly formed regulatory element, the Brake helix. Brake binds to the Gating helix, which is induced to extend more, resulting in abnormal opening of the enzyme and ISWI inhibition. This mechanism of Brake-mediated inhibition is important for linker DNA sensitivity of ISWI. CONCLUSION: Together, these findings lead to a multistate remodeling model that includes an inner core of the remodeling cycle and an outer regulatory layer. The proposed inner cycle provides a unifying mechanism of DNA translocation for chromatin remodeling. The outer regulation layer is ISWI specific, shedding light on the mechanism of linker DNA sensing by ISWI. Mechanism of chromatin remodeling by ISWI.: Chromatin remodeler ISWI slides nucleosomes to generate regularly spaced nucleosome arrays. By using electron microscopy, we caught the actions of ISWI in actively translocating the nucleosome and determined the structures of the complex in the ADP, ATP, ADP*, apo, apo*, ADP+, ADP*+, ADPS, and ADPB states at resolutions ranging from 2.3 to 3.0 Å. The structures reveal the conformational changes of ISWI and the altered interactions with the nucleosome in different states. These findings lead to a multistate model of ISWI action, providing a comprehensive mechanism of DNA translocation and regulation underpinning chromatin remodeling. [ABSTRACT FROM AUTHOR]