Tetrameric organization of vertebrate centromeric nucleosomes.
Mitosis ensures equal genome segregation in the eukaryotic lineage. This process is facilitated by microtubule attachment to each chromosome via its centromere. In centromeres, canonical histone H3 is replaced in nucleosomes by a centromere-specific histone H3 variant (CENH3), providing the unique epigenetic signature required for microtubule binding. Due to recent findings of alternative CENH3 nucleosomal forms in invertebrate centromeres, it has been debated whether the classical octameric nucleosomal arrangement of two copies of CENH3, H4, H2A, and H2B forms the basis of the vertebrate centromere. To address this question directly, we examined CENH3 [centromere protein A (CENP-A)] nucleosomal organization in human cells, using a combination of nucleosome component analysis, atomic force microscopy (AFM), and immunoelectron microscopy (immuno-EM). We report that native CENP-A nucleosomes contain centromeric alpha satellite DNA, have equimolar amounts of H2A, H2B, CENP-A, and H4, and bind kinetochore proteins. These nucleosomes, when measured by AFM, yield one-half the dimensions of canonical octameric nucleosomes. Using immuno-EM, we find that one copy of CENP-A, H2A, H2B, and H4 coexist in CENP-A nucleosomes, in which internal C-terminal domains are accessible. Our observations indicate that CENP-A nucleosomes are organized as asymmetric heterotypic tetramers, rather than canonical octamers. Such altered nucleosomes form a chromatin fiber with distinct folding characteristics, which we utilize to discriminate tetramers directly within bulk chromatin. We discuss implications of our observations in the context of universal epigenetic and mechanical requirements for functional centromeres.
Scanning Probe Microscopy Unit, Biomedical Engineering and Physical Science Shared Resource, National Institute for Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA.
Emilios K
EK
Dimitriadis
Christian
C
Weber
Rajbir K
RK
Gill
Stephan
S
Diekmann
Yamini
Y
Dalal
D001324:Autoantigens / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D002503:Centromere / Q000737:chemistry* / Q000648:ultrastructure; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D018625:Microscopy, Atomic Force; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D009707:Nucleosomes / Q000737:chemistry* / Q000648:ultrastructure
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D009707:Nucleosomes