Eukaryotic genomes are packaged with octameric protein particles, consisting of two copies each of histones H2A, H2B, H3 and H4, which wrap nearly two turns of DNA to form nucleosomes . Since the discovery of nucleosomes in the early 1970s, a variety of techniques have been applied to their study. However, both ultrastructural technologies (e.g. crystallography and electron microscopy) and biochemical analyses (e.g. nuclease assays and sedimentation) have been limited in their scope, because they cannot simultaneously assay structure and dynamics. In recent years, progress has been made in applying new technologies that have the potential to bridge the gap between static ultrastructural features and dynamic physiological processes in the study of chromatin. These technologies, which include scanning confocal fluorescence microscopy , molecular tweezers [3, 4] and atomic force microscopy (AFM) [5, 6], have provided remarkable insights into the behavior of individual nucleosomes. The combination of single-molecule resolution, solution biochemistry and observation of native macromolecular complexes has made AFM especially attractive for studying nucleosomes.
A fundamental level of distinction between nucleosome types is provided by the incorporation of alternative variants of histones H2A and H3 . At chromosomal sites for spindle fiber attachment at mitosis, histone H3 is replaced with a centromere-specific variant (CenH3, CENP-A in humans) to form the specialized nucleosomes that comprise centromeric chromatin . CenH3s in a variety of eukaryotes are found to be absolutely essential for the organization of the kinetochore, which is the only chromosomal structure required for mitosis and meiosis . Therefore, CenH3 nucleosomes are thought to provide the molecular foundation for assembly of the kinetochore at mitosis .
Until recently, the structure of CenH3 nucleosomes was presumed to be the same as that for canonical H3 nucleosomes. However, our recent study showed that CenH3 nucleosomes consist of half the DNA and protein of octameric nucleosomes, with one molecule each of CenH3, H4, H2A and H2B . In that study, we applied AFM to complement detailed biochemical characterization, and found that CenH3 chromatin consists of particles that are half the height of bulk octameric nucleosomes. However, it has been argued that the tetrameric particles that we observed might be non-nucleosomal intermediates in the assembly of CenH3 chromatin . Given the novelty of tetrameric nucleosomes in eukaryotic biology , and the potential for controversy , it is important to directly test alternative interpretations of our observations.
To address the possibility that we have identified an immature intermediate in CenH3 nucleosome assembly as opposed to a mature form, we adapted the purification of the CenH3 nucleosomes to include a DNA-binding step, so that only stable nucleosomes are purified rather than soluble assembly intermediates. In addition, we have applied recognition imaging, wherein an anti-CenH3 antibody is covalently coupled to the AFM tip. Recognition imaging provides highly efficient and specific identification of epitopes within nucleosomes . This strategy has allowed us to show directly that by far the most predominant form of CenH3 particles isolated from chromatin is tetrameric in height, which is inconsistent with them being assembly intermediates. Our application of single-epitope recognition imaging to centromeric nucleosomes also illustrates the potential of recognition imaging by AFM for analyzing minute amounts of specialized chromatin fractions isolated from the nucleus.