Quantitative Determination of Binding of ISWI to Nucleosomes and DNA Shows Allosteric Regulation of DNA Binding by Nucleotides

Abstract

The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β1 = 1.3 ± 0.6 nM, and 1/β2 = 13 ± 7 nM2. Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.

Effects of nucleosome stability on remodeler-catalyzed repositioning

“…As before, we determined values of k 2 and k d using estimates of n and k 1 from our previously published DNA translocation studies [41]. These results, shown in Table I, indicate that only values of n ≥ 4 are consistent with the affinity of the ISWI:nucleosome interaction [38]. As mentioned before, it is reasonable to assume that the rate of DNA translocation by ISWI will likely be slower when the DNA is wrapped around the histone octamer (or otherwise interacts with the histone octamer) than when it is free in solution, as was the case in our previously studies [41].…”

“…3 to determine estimates of the associated kinetic parameters. We note that a range of possible values for k d for these models can be determined from the equilbirium dissociation constant K D = 1.3nM we determined for nucleosome binding by ISWI [38]. Assuming an association rate of 10 6 M −1 s −1 to 10 8 M −1 s −1 [40], we estimate that k d can vary between 0.078min −1 and 7.8min −1 .…”

“…In other words, it is possible that experiments designed to analyze the repositioning activity of chromatin remodelers, may instead be reporting primarily on the affinity of histone:DNA interactions within the nucleosome rather than the activity of the remodeler itself. While this may be helpful in providing an alternative tool for measuring DNA:histone interactions (i.e., an alternative probe of histone:DNA interaction free energy [6]), it clearly muddles what can be concluded about the intrinsic repositioning activity of the remodeler and how this activity is influenced by post-translational modifications, the presence of accessory proteins, etc.. To address this concern, we present here a mathematical model for remodeler-catalyzed nucleosome repositioning and demonstrate its application to both previously published and new data using the chromatin remodeler ISWI [35][36][37][38] as a categorical example. We show how this model can reconcile seemingly disparate characterizations of the ISWI's nucleosome repositioning activity, including estimates of the number of processes associated with this reaction (and corresponding rate constants) as well as the influence of substrate and assay design on measurements of this activity.…”

“…1 through Eq. 11 is predicated on the assumption that the affinity of NCP binding by the chromatin remodeler is independent of the concentration of nucleotide (ATP and/or ADP) in solution, as has been observed for ISWI [38]. If the affinity of NCP binding is dependent upon the nucleotide binding, we can replace the dissociation rate constant k d in these equations with the expression shown in Eq.…”

Touch, act and go: landing and operating on nucleosomes

“…Many of these techniques make use of fluorescence, which overcomes limitations of dynamic range posed by other techniques, such as isothermal calorimetry, that is however successfully employed for the analysis of nucleosome-derived peptides. Among these fluorescence-based techniques, we find fluorescence polarization (FP), a versatile in-solution method that allows quantitative and rapid analysis of molecular interactions (Eryilmaz et al, 2009;Lea & Simeonov, 2011;Rossi & Taylor, 2011;Canzio et al, 2013;Al-Ani et al, 2014a;Mattiroli et al, 2014;Pilotto et al, 2015;Taherbhoy et al, 2015). Microscale thermophoresis (MST) also takes advantage of fluorescence detection in solution to measure molecular interactions in a flexible and rapid manner (Schubert et al, 2012;Greer et al, 2014;Zhang et al, 2014).…”