Person:

Hwang, William

Eukaryotic genomes are packaged as chromatin, which restricts access to the DNA by critical processes such as DNA replication, repair, and transcription. As a result, eukaryotic cells rely on ATP-dependent chromatin remodeling enzymes (remodelers) to alter the position, structure, and composition of nucleosomes. Understanding the mechanism and regulation of remodeling requires detailed information about transient intermediates of the remodeling process--a challenge ideally suited for single-molecule approaches. In particular, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure nanometer-scale distance changes between strategically placed donor and acceptor dyes to monitor nucleosome translocation in real-time. The mechanism(s) by which remodelers use the free energy of ATP hydrolysis to disrupt histone-DNA contacts and reposition nucleosomes are not well understood. Using smFRET, we show that remodeling by ISWI enzymes begins with a 7 base-pair (bp) step followed by subsequent 3 bp steps toward the exit-side of the nucleosome. These multi-bp steps are actually compound steps composed of 1 bp elementary steps. We discover that DNA movement on the entry side lags behind exit side translocation, which is contrary to previously proposed models. Based on our results, we propose a new integrated mechanism for nucleosome translocation by ISWI enzymes. In the physiological context, remodelers are highly regulated. We study the regulation of human ACF, a prototypical ISWI complex, by critical features of the nucleosomal substrate. First, we dissect how the nucleosome translocation cycle is affected by the linker DNA length and histone H4 tail. Next, we introduce mutations/deletions into conserved enzyme domains to determine the mechanism by which linker length information sensed by the Acfl accessory subunit is allosterically transmitted to the Snf2h catalytic subunit. Interestingly, we find that Acfl modulates the activity of Snf2h indirectly by interacting with the H4 tail in a linker-length dependent fashion. While the majority of our experiments focus on observing changes in nucleosome position, we also develop strategies for site-specific labeling of ISWI enzymes and demonstrate their use in the study of dynamic enzyme-substrate interactions and enzyme conformational changes.

ISWI-family remodelling enzymes regulate access to genomic DNA by mobilizing nucleosomes1. These ATP-dependent chromatin remodellers promote heterochromatin formation and transcriptional silencing1 by generating regularly-spaced nucleosome arrays2-5. The nucleosome-spacing activity arises from regulation of nucleosome translocation by the length of extranucleosomal linker DNA6-10, but the underlying mechanism remains unclear. Here, we studied nucleosome remodelling by human ACF, an ISWI enzyme comprised of a catalytic subunit, Snf2h, and an accessory subunit, Acf12,11-13. We found that ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome. Mutation of AutoN, an auto-inhibitory domain within Snf2h that bears sequence homology to the H4 tail14, abolished the linker-length sensitivity in remodelling. Addition of exogenous H4-tail peptide or deletion of the nucleosomal H4 tail also diminished the linker-length sensitivity. Moreover, the accessory subunit Acf1 bound the H4-tail peptide and DNA in a manner that depended on its N-terminal domain, and lengthening the linker DNA in the nucleosome reduced the proximity between Acf1 and the H4 tail. Deletion of the N-terminal portion of Acf1 (or its homologue in yeast) abolished linker-length sensitivity in nucleosome remodeling and led to severe growth defects in vivo. Taken together, our results suggest a mechanism for nucleosome spacing where linker DNA sensing by Acf1 is 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.

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.