Single-epitope recognition imaging of native chromatin.
BACKGROUND
Direct visualization of chromatin has the potential to provide important insights into epigenetic processes. In particular, atomic force microscopy (AFM) can visualize single nucleosomes under physiological ionic conditions. However, AFM has mostly been applied to chromatin that has been reconstituted in vitro, and its potential as a tool for the dissection of native nucleosomes has not been explored. Recently we applied AFM to native Drosophila chromatin containing the centromere-specific histone 3 (CenH3), showing that it is greatly enriched in smaller particles. Taken together with biochemical analyses of CenH3 nucleosomes, we propose that centromeric nucleosomes are hemisomes, with one turn of DNA wrapped around a particle consisting of one molecule each of centromere-specific CenH3, H4, H2A and H2B.


RESULTS
Here we apply a recognition mode of AFM imaging to directly identify CenH3 within histone core particles released from native centromeric chromatin. More than 90% of these particles were found to be tetrameric in height. The specificity of recognition was confirmed by blocking with a CenH3 peptide, and the strength of the interaction was quantified by force measurements. These results imply that the particles imaged by AFM are indeed mature CenH3-containing hemisomes.


CONCLUSIONS
Efficient and highly specific recognition of CenH3 in histone core particles isolated from native centromeric chromatin demonstrates that tetramers are the predominant form of centromeric nucleosomes in mature tetramers. Our findings provide proof of principle that this approach can yield insights into chromatin biology using direct and rapid detection of native nucleosomes in physiological salt concentrations.
Wang
Hongda
H
Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.|State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China.
Dalal
Yamini
Y
Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.|National Cancer Institute, Laboratory of Receptor Biology and Gene Expression, National Institutes of Health, Bethesda, MD 20892, USA.
Henikoff
Steven
S
Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.|Howard Hughes Medical Institute, Jones Bridge Road, Chevy Chase, MD 20815-6789, USA.
Lindsay
Stuart
S
Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.