Abstract
We employ a stable isotope strategy wherein both histones and their methylations are labeled in synchronized human cells. This allows us to differentiate between old and new methylations on pre-existing versus newly synthesized histones. The strategy is implemented on K79 methylation in an isoform-specific manner for histones H3.1, H3.2, and H3.3. Although levels of H3.3K79 monomethylation are higher than that of H3.2/H3.1, the rate of establishing the K79 methylation is the same for all three isoforms. Surprisingly, we find that pre-existing "old" histones continue to be K79-monomethylated and -dimethylated at a rate equal to the newly synthesized histones. These observations imply that some degree of positional "scrambling" of K79 methylation occurs through the cell cycle.
Highlights
Histones are subject to myriad post-translational modifications
The information obtained can broadly be divided into the global and the local; antibodies to a particular modification can provide a global read-out of levels across the genome, and chromatin immunoprecipitation (ChIP) of a particular modification followed by quantitative PCR can give highly local information [7]
Mass spectrometric analysis of epitope-tagged H3 has confirmed that the majority of (H3-H4)2 tetramers remain intact during replication, a small proportion of H3.3-containing tetramers are split during replication-dependent nucleosome incorporation [18]
Summary
Histones are subject to myriad post-translational modifications. The most prominent among these modifications are acetylation, methylation, phosphorylation, and ubiquitination. Fractions containing H3.1, H3.2, and H3.3 were ϳ70% old histones at the 12 h time point (supplemental Fig. 2).
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