EPR Spectroscopy Shows That the H4 Tail of the Nucleosome Influences the Nucleotide-Binding Pocket of Acf

Abstract

ATP-dependent chromatin remodeling enzymes help to determine the structural and transcriptional state of chromatin in the cell by repositioning nucleosomes. ACF, a member of the ISWI class of remodeling enzymes, spaces nucleosomes on DNA with a defined periodicity, which promotes folding of chromatin into higher-order transcriptionally silent structures. The mechanism by which these molecular motors couple the energy of ATP hydrolysis to breaking histone-DNA contacts is not well understood. To position a nucleosome appropriately, ACF must recognize and integrate two critical types of substrate cues, the length of DNA flanking the nucleosome on either side, and the acetylation state of the histone H4 tails. Acetylation of histone H4 at lysine 16, a mark typically associated with transcriptionally active chromatin, inhibits ACF function, thus potentially allowing ACF to distinguish chromatin destined for silencing from actively transcribed regions. How ACF integrates and uses these two critical substrate cues is still a mystery. Previous work has shown that these two cues stimulate ATP hydrolysis but have minimal effects on ground state binding of ACF. One prediction then is that the H4 tail and flanking DNA stabilize ACF-nucleosome interactions in specific activated ATP states. Using EPR spectroscopy of spin-labeled ATP analogues bound to the ATPase subunit of ACF, we find that the H4 tail of the nucleosome causes more restricted mobility of the spin probe in the ATP-analog state, SLADP∗BeFx. This restricted mobility surprisingly also requires the presence of nucleosomal DNA but not flanking DNA. Thus the H4 tail in the presence of nucleosomal DNA directly affects the environment of the nucleotide-binding pocket. EPR spectroscopy provides direct support for the hypothesis that substrate cues for directing the appropriate activity of ACF are read in an activated ATP state.