UCSF

A fundamental mode of gene regulation in eukaryotes is to alter access of the nuclear machinery to DNA through packaging into chromatin. Human ACF, a member of the ISWI-family of chromatin remodeling enzymes, is an ideal model system to study the basic mechanism of moving nucleosomes because it is a small complex and makes one class of products, evenly spaced nucleosomes, thought to be important for heterochromatin formation. ACF kinetically distinguishes between different flanking DNA lengths on either side of a nucleosome, moving the nucleosome toward the longer

flanking DNA faster than toward the shorter DNA.

Using electron microscopy and enzymatic assays, we observe that ACF can bind and function as a cooperative homodimer to move nucleosomes. The dimeric partners bind the nucleosome near the N-terminal tails of H4 and face in opposing directions. This unusual architecture raises new questions about how the protomers collaborate rather than compete in a tug of war. We observe nucleotide-dependent changes in contacts of the enzyme with the two H4 tails. In the presence of ADP, the enzyme complex contacts one H4 tail, whereas with a nucleotide analogue thought to mimic an activated ATP state, both H4 tails are immobilized. These conformational states support a model for allosteric communication between the dimeric partners.

We next focused on how the enzyme interprets and integrates two critical components of the nucleosome that we term `substrate cues:' the H4 tail and flanking DNA. We find that the H4 tail and flanking DNA stimulate remodeling activity nonadditively, suggesting that the two cues may either function in two different rate limiting steps, or that the two cues function in a coupled manner in one rate limiting step. Using spin-labeled ATP, we observe that the H4 tail but not flanking DNA is important for formation of a restricted conformation of the nucleotide-binding pocket. The H4 tail and flanking DNA both have larger effects on nucleosome remodeling than on ATP hydrolysis. The H4 tails and flanking DNAs play synergistic roles on remodeling and may be important for coupling ATP hydrolysis to nucleosome remodeling.