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Updated: Apr 28, 2026
Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF
Published on: January 12, 2024
Tracking histone variant nucleosomes across the human cell cycle using biophysical, biochemical, and cytological
Marcin P Walkiewicz1, Minh Bui, Delphine Quénet
1Chromatin Structure and Epigenetic Mechanisms Unit, Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, 20892, USA.
None:
Histone variants such as H3.3, macroH2A, H2A.Z, and CENP-A are important epigenetic modifiers of the chromatin state in eukaryotic genomes. The centromeric histone H3 variant CENP-A/CENH3 epigenetically marks centromeres and is required for assembly of the kinetochore complex, a region of the chromosome that is responsible for proper genome segregation during mitosis. Several diverse techniques using biochemical, cell biology, and biophysical approaches have been utilized to study the nature of the CENP-A nucleosome across the cell cycle. In this chapter, we describe methods for CENP-A nucleosome purification and separation of CENP-A from other core histones using traditional SDS-PAGE and more resolving techniques such as Triton acid urea (TAU) and two-dimensional gels. We also discuss methods for observation of CENP-A on chromatin fibers using immunofluorescence. Finally, we provide a detailed description of analysis of chromatin structures using atomic force microscopy.
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