Single and double box HMGB proteins differentially destabilize nucleosomes.

Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A-H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.

Accurate nanoscale flexibility measurement of DNA and DNA-protein complexes by atomic force microscopy in liquid.

The elasticity of double-stranded DNA (dsDNA), as described by its persistence length, is critical for many biological processes, including genomic regulation. A persistence length value can be obtained using atomic force microscopy (AFM) imaging. However, most AFM studies have been done by depositing the sample on a surface using adhesive ligands and fitting the contour to a two-dimensional (2D) wormlike chain (WLC) model. This often results in a persistence length measurement that is different from the value determined using bulk and single molecule methods. We describe a method for obtaining accurate three-dimensional (3D) persistence length measurements for DNA and DNA-protein complexes by using a previously developed liquid AFM imaging method and then applying the 3D WLC model. To demonstrate the method, we image in both air and liquid several different dsDNA constructs and DNA-protein complexes that both increase (HIV-1 Vpr) and decrease (yeast HMO1) dsDNA persistence length. Fitting the liquid AFM-imaging contour to the 3D WLC model results in a value in agreement with measurements obtained in optical tweezers experiments. Because AFM also allows characterization of local DNA properties, the ability to correctly measure global flexibility will strongly increase the impact of measurements that use AFM imaging.

DNA bridging and looping by HMO1 provides a mechanism for stabilizing nucleosome-free chromatin.

The regulation of chromatin structure in eukaryotic cells involves abundant architectural factors such as high mobility group B (HMGB) proteins. It is not understood how these factors control the interplay between genome accessibility and compaction. In vivo, HMO1 binds the promoter and coding regions of most ribosomal RNA genes, facilitating transcription and possibly stabilizing chromatin in the absence of histones. To understand how HMO1 performs these functions, we combine single molecule stretching and atomic force microscopy (AFM). By stretching HMO1-bound DNA, we demonstrate a hierarchical organization of interactions, in which HMO1 initially compacts DNA on a timescale of seconds, followed by bridge formation and stabilization of DNA loops on a timescale of minutes. AFM experiments demonstrate DNA bridging between strands as well as looping by HMO1. Our results support a model in which HMO1 maintains the stability of nucleosome-free chromatin regions by forming complex and dynamic DNA structures mediated by protein-protein interactions.

Basic N-terminus of yeast Nhp6A regulates the mechanism of its DNA flexibility enhancement.

HMGB (high-mobility group box) proteins are members of a class of small proteins that are ubiquitous in eukaryotic cells and nonspecifically bind to DNA, inducing large-angle DNA bends, enhancing the flexibility of DNA, and likely facilitating numerous important biological interactions. To determine the nature of this behavior for different HMGB proteins, we used atomic force microscopy to quantitatively characterize the bend angle distributions of DNA complexes with human HMGB2(Box A), yeast Nhp6A, and two chimeric mutants of these proteins. While all of the HMGB proteins bend DNA to preferred angles, Nhp6A promoted the formation of higher-order oligomer structures and induced a significantly broader distribution of angles, suggesting that the mechanism of Nhp6A is like a flexible hinge more than that of HMGB2(Box A). To determine the structural origins of this behavior, we used portions of the cationic N-terminus of Nhp6A to replace corresponding HMGB2(Box A) sequences. We found that the oligomerization and broader angle distribution correlated directly with the length of the N-terminus incorporated into the HMGB2(Box A) construct. Therefore, the basic N-terminus of Nhp6A is responsible for its ability to act as a flexible hinge and to form high-order structures.