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Elucidating the mechanism of the ISWI family of chromatin remodeling complexes

  • Author(s): Yang, Janet Gloria
  • Advisor(s): Narlikar, Geeta J
  • et al.
Abstract

Chromatin structure regulates the accessibility of DNA in several crucial nuclear processes, including transcription, replication and recombination. The ISWI-family of chromatin remodeling complexes translationally repositions histone octamers on DNA. One member of this family, the ATP-dependent chromatin assembly factor (ACF), generates regularly spaced nucleosomes in vitro and is required for transcriptional silencing in vivo.

To dissect ACF mechanism, we used a FRET based approach to follow the movement of DNA with respect to the histone octamer in real time. We find that ACF generates at least one transient intermediate in which part of the DNA has moved across the histone octamer. The rate of ACF remodeling is sensitive to the length of the linker DNA such that shortening the linker DNA below 60 bp progressively slows down both ATP hydrolysis and remodeling. As a result, ACF generates and maintains nucleosome spacing by constantly moving nucleosomes towards longer flanking DNA faster than shorter flanking DNA. ACF thus creates a dynamic equilibrium between different nucleosome positions such that the centered position is most highly populated.

To understand how ACF can rapidly sample both sides of a nucleosome to accomplish bidirectional movement, my collaborators and I employed a myriad of techniques to conclude that ACF functions as a dimeric molecular motor. We found that ACF binds nucleosomes in a cooperative manner, and that this oligomeric state is required for back and forth remodeling of the nucleosome. Additionally, the ATPase subunit energetically couples different nucleotide states with distinct reaction intermediates, and thus we observe that the physical interactions with the nucleosome change as a function of the ATP reaction cycle. Lastly, we found that ACF utilizes ATP in three distinct ways, and that translocation is characterized by distinct pauses during movement of DNA relative to the histone octamer.

In summary, these data provide a mechanistic basis for the directionality of nucleosome repositioning and explain the centering activity of ACF. Futhermore, the dimeric architecture of ACF allows for the rapid and processive bidirectional nucleosome movement required for nucleosome spacing.

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