Remodeling and Repositioning of Nucleosomes in Nucleosomal Arrays.

ATP-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit or evict nucleosomes or reposition them along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate.

Structural Architecture of the Nucleosome Remodeler ISWI Determined from Cross-Linking, Mass Spectrometry, SAXS, and Modeling.

Chromatin remodeling factors assume critical roles by regulating access to nucleosomal DNA. To determine the architecture of the Drosophila ISWI remodeling enzyme, we developed an integrative structural approach that combines protein cross-linking, mass spectrometry, small-angle X-ray scattering, and computational modeling. The resulting structural model shows the ATPase module in a resting state with both ATPase lobes twisted against each other, providing support for a conformation that was recently trapped by crystallography.

Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail.

ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously.

Nucleosome spacing generated by ISWI and CHD1 remodelers is constant regardless of nucleosome density.

Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density.

No need for a power stroke in ISWI-mediated nucleosome sliding.

Nucleosome remodelling enzymes of the ISWI family reposition nucleosomes in eukaryotes. ISWI contains an ATPase and a HAND-SANT-SLIDE (HSS) domain. Conformational changes between these domains have been proposed to be critical for nucleosome repositioning by pulling flanking DNA into the nucleosome.

The ATPase domain of ISWI is an autonomous nucleosome remodeling machine.

ISWI slides nucleosomes along DNA, enabling the structural changes of chromatin required for the regulated use of eukaryotic genomes. Prominent mechanistic models imply cooperation of the ISWI ATPase domain with a C-terminal DNA-binding function residing in the HAND-SANT-SLIDE (HSS) domain. Contrary to these models, we show by quantitative biochemical means that all fundamental aspects of nucleosome remodeling are contained within the compact ATPase module of Drosophila ISWI.

Probing the conformation of the ISWI ATPase domain with genetically encoded photoreactive crosslinkers and mass spectrometry.

We present a strategy for rapidly gaining structural information about a protein from crosslinks formed by genetically encoded unnatural amino acids. We applied it to ISWI, a chromatin remodeling enzyme involved in chromatin assembly, DNA replication and transcription. ISWI is part of the vast Snf2 family of helicase-related proteins, many of which constitute the catalytic cores of chromatin remodeling complexes.