Effects of nucleosome stability on remodeler-catalyzed repositioning

Abstract

Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself.

Remodeler Catalyzed Nucleosome Repositioning: Influence of Structure and Stability

“…This process then repeats in units of three basepairs translocated per translocation cycle. This model is consistent with the results of more recent ensemble experiments [92], theoretical analysis [93], and with the formation of DNA loops in Isw1-nucleosome complexes being allosterically regulated through nucleotide binding by Saccharomyces cerevisiae Isw1 [49]. In an alternative 'ratchet' model for nucleosome repositioning by ISWI, the ATPase domain of the ISWI complex is responsible for all mechanical processes associated with nucleosome repositioning [73].…”

“…Indeed, since most studies of chromatin remodeler function involve NCP substrates reconstituted using the Widom 601 DNA sequence [9], it is possible that experiments intended to assess the repositioning activity of chromatin remodelers, may instead report primarily on the affinity of histone:DNA interactions within the nucleosome rather than the activity of the remodeler. This proposition is further supported by the observation that the rate of nucleosome repositioning by Xenopus laevis ISWI depends upon the sequence of the nucleosomal DNA, with faster repositioning occurring with DNA sequences with lower affinity for histone binding [93]. Indeed, if the DNA translocation associated with nucleosome repositioning requires the chromatin remodeler to compete with the histones for binding the nucleosomal DNA [49,53,87], then it should be more difficult for chromatin remodelers to reposition nucleosomes reconstituted with DNA sequences with higher affinity for histone binding [93].…”

“…This proposition is further supported by the observation that the rate of nucleosome repositioning by Xenopus laevis ISWI depends upon the sequence of the nucleosomal DNA, with faster repositioning occurring with DNA sequences with lower affinity for histone binding [93]. Indeed, if the DNA translocation associated with nucleosome repositioning requires the chromatin remodeler to compete with the histones for binding the nucleosomal DNA [49,53,87], then it should be more difficult for chromatin remodelers to reposition nucleosomes reconstituted with DNA sequences with higher affinity for histone binding [93]. It is therefore not surprising that several studies have shown that the sequence of nucleosomal DNA influences the nucleosome repositioning activity of chromatin remodelers [92,[100][101][102][103].…”

“…However, since nucleosome repositioning requires the chromatin remodeler to break and reform contacts between the histones and the nucleosomal DNA in order for DNA translocation to occur, disrupting the interactions between the histones and the nucleosomal DNA does provide an energetic barrier to nucleosome repositioning. This barrier, if sufficiently large, could result in multiple rounds of unsuccessful attempts at nucleosome repositioning and the associated DNA translocation for each successful repositioning event [92,93]. This hypothesis could also explain the poor template commitment reported for human Snf2H [98] as well as the observation that Xenopus laevis ISWI repositions nucleosomes through a random walk mechanism [92].…”

“…It is worth noting that the nucleosome repositioning activity of Saccharomyces cerevisiae INO80 has been reported to be independent of the sequence of nucleosomal DNA [104], but a lack of determination of the stoichiometry of INO80 binding to the NCP substrates used in these experiments creates some ambiguity in the interpretation of those results, especially for nucleosome substrates containing long lengths of DNA flanking the NCP [50,92]. While a dependence of nucleosome repositioning on the sequence of nucleosomal DNA may be helpful in providing an alternative tool for measuring DNA-histone interactions (i.e., an alternative probe of histone:DNA interaction free energy [10]), it clearly muddles what can be concluded about the intrinsic repositioning activity of the remodeler and how this activity is influenced and modulated [93].…”