As superfamily 2 (SF2)-type translocases, chromatin remodelers are expected to use an inchworm-type mechanism to walk along DNA. Yet how they move DNA around the histone core has not been clear. Here we show that a remodeler ATPase motor can shift large segments of DNA by changing the twist and length of nucleosomal DNA at superhelix location 2 (SHL2). Using canonical and variant 601 nucleosomes, we find that the Saccharomyces cerevisiae Chd1 remodeler decreased DNA twist at SHL2 in nucleotide-free and ADP-bound states, and increased twist with transition state analogs. These differences in DNA twist allow the open state of the ATPase to pull in ~1 base pair (bp) by stabilizing a small DNA bulge, and closure of the ATPase to shift the DNA bulge toward the dyad. We propose that such formation and elimination of twist defects underlie the mechanism of nucleosome sliding by CHD-, ISWI-, and SWI/SNF-type remodelers.

Chromatin remodelers are ATP-dependent enzymes that are critical for reorganizing and repositioning nucleosomes in concert with many basic cellular processes. For the Chromodomain Helicase DNA Binding Protein 1 (Chd1) remodeler, nucleosome sliding has been shown to depend on DNA flanking the nucleosome, transcription factor binding at the nucleosome edge, and the presence of the histone H2A/H2B dimer on the entry side. Here we report that Chd1 is also sensitive to the sequence of DNA within the nucleosome, and slides nucleosomes made with the 601 Widom positioning sequence asymmetrically. Kinetic and equilibrium experiments show that poly(dA:dT) tracts perturb remodeling reactions if within one and a half helical turns of superhelix location 2 (SHL2), where the Chd1 ATPase engages nucleosomal DNA. These sequence-dependent effects do not rely on the Chd1 DNA-binding domain and are not due to differences in nucleosome affinity. Using site-specific cross-linking, we show that internal poly(dA:dT) tracts do not block engagement of the ATPase motor with SHL2, yet they promote multiple translational positions of DNA with respect to both Chd1 and the histone core. We speculate that Chd1 senses the sequence-dependent response of DNA as the remodeler ATPase perturbs the duplex at SHL2. These results suggest that the sequence sensitivity of histones and remodelers occurs at unique segments of DNA on the nucleosome, and therefore can work together or in opposition to determine nucleosome positions throughout the genome.

Chromatin remodelers are essential for establishing and maintaining the placement of nucleosomes along genomic DNA. Yet how chromatin remodelers recognize and respond to distinct chromatin environments surrounding nucleosomes is poorly understood. Here, we use Lac repressor as a tool to probe how a DNA-bound factor influences action of the Chd1 remodeler. We show that Chd1 preferentially shifts nucleosomes away from Lac repressor, demonstrating that a DNA-bound factor defines a barrier for nucleosome positioning. Rather than an absolute block in sliding, the barrier effect was achieved by altered rates of nucleosome sliding that biased redistribution of nucleosomes away from the bound Lac repressor site. Remarkably, in addition to slower sliding toward the LacO site, the presence of Lac repressor also stimulated sliding in the opposite direction. These experiments therefore demonstrate that Chd1 responds to the presence of a bound protein on both entry and exit sides of the nucleosome. This sensitivity to both sides of the nucleosome allows for a faster and sharper response than would be possible by responding to only the entry side, and we speculate that dual entry/exit sensitivity is also important for regularly spaced nucleosome arrays generated by Chd1 and the related ISWI remodelers.

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chromosome structure have broad implications in studying effects of the geometry of nucleus on higher-order genome organization and nuclear functions. Here we describe a multi-chromosome constrained self-avoiding chromatin model for studying ensembles of structural genome models to understand the folding principles of budding yeast genome. We successfully generated a large number of model genomes of yeast under different geometrical constraints and found that spatial confinement of cell nucleus and molecular crowding in the nucleus are key determinants of the folding behavior of yeast chromosomes. Furthermore, the relative positioning of chromosomes and the interactions between them are found to be due to presence of nuclear landmarks such as centromere tethering to spindle pole body. In eukaryotic cells, DNA is wrapped around histone octamers, forming nucleosomes. In the centromere, the region of the chromosome that links sister chromatids, histone H3 is replaced by CENP-A (Centromere protein A). Since CENP-A chromatin is the point of contact between the microtubules/kinetochore complex and the rest of the chromosome, these regions endure very high forces. Whereas canonical nucleosomes unwrap at 3pN and disassemble at 15-20pN of force, the estimated forces applied by microtubili are much higher. To investigate how CENP-A nucleosomes respond to externally applied forces and torque, we studied how CENP-A chromatin responds to defined stretching forces and torque generated by magnetic tweezers. With this technique, a single DNA molecule containing a few (up to 10) nucleosomes is tethered between a glass surface and a magnetic bead. By applying stretching forces at constant negative and positive supercoiling, the forces needed for disassembly of canonical H3 and CENP-A nucleosomes can be obtained and compared. By measuring the DNA end-to-end length as a function of applied rotations before and after CENP-A nucleosomes are removed from the DNA, the linking number of the nucleosomes can be determined. Interestingly, while inducing supercoiling at constant low (<0.5pN) forces, unlike canonical nucleosomes, CENP-A nucleosomes appear to respond to the applied torque. By further analyzing the structural stability of CENP-A nucleosomes under stretching force and torque, we hope to unravel the mechanism by which CENP-A nucleosomes resists disassembly during mitosis in vivo. Chromatin remodelers are essential for establishing and maintaining the placement of nucleosomes along genomic DNA. However, how remodelers respond to transcription factors and other bound factors that might influence chromatin organization is poorly understood. Here we use the Lac repressor to investigate how the Chd1 remodeler responds to a protein bound to the edge of a nucleosome. We found that Lac repressor effectively provided a barrier for nucleosome sliding by Chd1. This barrier did not require an absolute block in sliding, but instead was achieved through the bidirectional movement of nucleosomes, with a higher preference for sliding nucleosomes...