(A) Summary of modified forms of histone H3 N-terminal peptides, as detected by HILIC-MS/MS, from Tetrahymena micronuclei of hour 5 conjugating cells. As in main Figure 1D, each row represents a uniquely modified H3 N-terminal peptide species (residues 1–50 for H3S and 7–50 for H3F), and each dotted horizontal line beneath a bolded residue represents a site where the specified modification was identified. No modifications were detected on residues 28–50 on H3S and H3F. The relative abundance of each peptide, or multiple isobaric peptides identified within the same spectrum (grouped by brackets on the right), is highlighted by the number of asterisks. For example, peptides with four asterisks are more abundant than peptides with one asterisk. Red asterisks highlight the dually modified H3K23me3/H3K27me3 state enriched during early conjugation. Forms of the H3S(1–50) peptide with four, five, and six methyls were detected, but due to the isobaric character of each group of peptides and the low levels of these species it was not possible to determine the locations of the methyl modifications. Also, an H3F(7–50) peptide with one acetylation and one phosphorylation modification was detected by accurate mass, but no MS/MS spectra were acquired and therefore location of these modifications could not be determined. Abbreviations: MonoMe, monomethylation; DiMe, dimethylation; TriMe, trimethylation; Acetyl, acetylation; Phos, phosphorylation. (B) Co-immunofluorescence staining of conjugating Tetrahymena (at nuclear differentiation stage) using α-H3K23me3 and α-H3K9me3 antibodies. Red arrowheads point to micronuclei and open red arrowheads point to new developing macronuclei. The parental macronucleus (Old MAC) is marked by an ‘X’ in the cartoon and by dotted circles in the immunofluorescence images.