Identification and maturation of newly synthesized fungal histone H3

Abstract

Reversed‐phase hplc of histone H3 in Ustilago maydis, Saccharomyces cerevisiae and Schizosaccharomyces pombe revealed the existence of a distinct form, ~5–20% of total histone H3 depending on strains and culture conditions, with delayed column elution, i.e. a more hydrophobic character. This form was more highly labeled by methionine than bulk H3, consistent with increased levels of lysine methylation and protein hydrophobicity. It does not contain non‐acetylated histone H3 and, in U.maydis, and has higher steady‐state acetylation levels than bulk H3. In asynchronous cultures, acetylation of replacement and of replication‐dependent H3 variants was more than 3‐, and 2‐fold higher, respectively, than bulk H3. Lysine pulse label incorporated exclusively into this form, identifying it as newly synthesized histone H3. Consistent with this, its abundance decreased in cycloheximide. Cell fractionation demonstrated that it does not represent a cytoplasmic pool. Lysine pulse‐chase labeling in U.maydis demonstrated a discrete chase into bulk, mature histone H3 with a half‐life of 30 min, complete within the 150 min cell doubling time. Western blotting and mass spectrometry are used to identify sites of acetylation and methylation in new H3 and changes during histone H3 maturation.Research supported by Missouri Life Sciences Research Board #13254