Structure and Dynamics of Dinucleosomes Assessed by Atomic Force Microscopy

Dynamics of nucleosomes and their interactions are important for understanding the mechanism of chromatin assembly. Internucleosomal interaction is required for the formation of higher-order chromatin structures. Although H1 histone is critically involved in the process of chromatin assembly, direct internucleosomal interactions contribute to this process as well. To characterize the interactions of nucleosomes within the nucleosome array, we designed a dinucleosome and performed direct AFM imaging. The analysis of the AFM data showed dinucleosomes are very dynamic systems, enabling the nucleosomes to move in a broad range along the DNA template. Di-nucleosomes in close proximity were observed, but their population was low. The use of the zwitterionic detergent, CHAPS, increased the dynamic range of the di-nucleosome, facilitating the formation of tight di-nucleosomes. The role of CHAPS and similar natural products in chromatin structure and dynamics is also discussed. 1. Introduction The formation of nucleosomes is the first stage of DNA packing into chromatin, followed by the assembly of the “beads-on-a-string” nucleosomal array into compact chromatin fibers (e.g., [1] and references therein). H1 histone is a key player in the formation of the 30-nm-thick fibers (e.g., [2]); however, interactions between the nucleosomal particles contribute to the assembly process as well. Although the molecular mechanisms behind the formation of higher-order chromatin structures remain unclear, work employing model systems has shown that in vitro reconstituted nucleosome arrays containing only DNA and core histone proteins undergo the same initial salt-dependent condensations as native chromatin [1, 3]. In solutions containing physiological concentrations of mono- and divalent cations, nucleosome arrays spontaneously fold into structures with the same hydrodynamic shape as the 30-nm-diameter chromatin fiber [1, 4]. This implies that the primary protein determinants defining these structures reside within the core histone proteins. Indeed, in the early crystallography work of Luger et al. [5], it was shown that the K16 to N25 segment of H4 makes extensive contacts with an H2A-H2B dimer of an adjacent particle. This H4 region, K16 to N25, makes multiple hydrogen bonds and salt bridges between its basic side chains (K16, R19, K20, R23) and acidic side chains of H2A (E56, E61, E64, D90, E91, E92) and H2B (E110). These structure-based predictions were supported with studies [6–8] of recombinant core mutants. They showed that interaction between the H4 N-terminal domain