Title: Regulation of Chromatin Remodeling
Description: ATP-dependent chromatin remodeling enzymes (remodelers) regulate access to genomic DNA by reading epigenetic marks such as histone modifications and using the energy of ATP hydrolysis to assemble, reposition, disassemble, and modify the composition of nucleosomes. The catalytic activity of remodeling enzymes is highly regulated by various substrate characteristics including extranucleosomal linker DNA, histone modifications, and linker histones. In this thesis, we developed and employed a synergistic combination of single-molecule biophysical techniques and biochemical approaches to elucidate the mechanisms underpinning the regulation of chromatin remodelers by these substrate features.

The imitation switch (ISWI)-family of remodelers promotes heterochromatin formation and transcriptional silencing by generating regularly-spaced nucleosome arrays. It was previously known that this nucleosome-spacing activity arises from the dependence of nucleosome translocation on the length of linker DNA, but the underlying mechanism remains unclear. We studied nucleosome remodeling by the human ATP-dependent chromatin assembly and remodeling factor (ACF), an ISWI enzyme composed of a catalytic subunit, Snf2h, and an accessory subunit, Acf1. The H4 tail bears significant sequence homology to an autoinhibitory domain, AutoN, present in the catalytic subunit. The presence of an unmodified H4 tail is thought to stimulate catalytic activity by competing with AutoN for a binding site on the ATPase. Our results suggest a mechanism for nucleosome spacing where linker DNA is sensed by the N-terminus of Acf1 and allosterically transmitted to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing AutoN to inhibit the ATPase activity of Snf2h. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to compete AutoN off the ATPase and thereby activating ACF. This intricate signal transduction between the accessory and catalytic subunits coupled to two distinct substrate features may be a paradigm for this entire class of critical enzymes.

Most of our knowledge on the function of chromatin remodeling complexes has been gleaned from studies using nucleosomes with only core histones. In contrast, physiological chromatin is replete with linker histones, e.g., H1, at a prevalence of approximately one linker histone per nucleosome in differentiated eukaryotic cells. The complex formed by a core nucleosome and linker histone is known as a chromatosome. There has been significant disagreement among prior investigations on the effects of linker histones on remodeling activity, with some studies reporting varying degrees of general repression while others describe qualitative changes in remodeling outcomes. Using single-molecule fluorescence resonance transfer (smFRET), we provided the first direct observation of intact chromatosome remodeling by ACF. Furthermore, we discovered that the presence of linker histones changes the remodeling outcomes of the linker DNA-insensitive SWI/SNF enzymes by preventing nucleosome translocation past the DNA edge.