Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases

The cell cycle is
a highly regulated and evolutionary conserved
process that results in the duplication of cell content and the equal
distribution of the duplicated chromosomes into a pair of daughter
cells. Histones are fundamental structural components of chromatin
in eukaryotic cells, and their post-translational modifications (PTMs)
benchmark DNA readout and chromosome condensation. Aberrant regulation
of the cell cycle associated with dysregulation of histone PTMs is
the cause of critical diseases such as cancer. Monitoring changes
of histone PTMs could pave the way to understanding the molecular
mechanisms associated with epigenetic regulation of cell proliferation.
Previously, our lab established a novel middle-down workflow using
porous graphitic carbon (PGC) as a stationary phase to analyze histone
PTMs, which utilizes the same reversed-phase chromatography for gradient
separation as canonical proteomics coupled with online mass spectrometry
(MS). Here, we applied this novel workflow for high-throughput analysis
of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively,
we identified 1133 uniquely modified canonical histone H3 N-terminal
tails. Consistent with previous findings, histone H3 phosphorylation
increased significantly during the mitosis (M) phase. Histone H3 variant-specific
and cell-cycle-dependent expressions of PTMs were observed, underlining
the need to not combine H3.1 and H3.2 together as H3. We confirmed
previously known H3 PTM crosstalk (e.g., K9me–S10ph) and revealed
new information in this area as well. These findings imply that the
combinatorial PTMs play a role in cell cycle control, and they may
serve as markers for proliferation.