IF analysis of female iMEFs transiently transfected with Jmjd2b-GFP constructs. Cells were costained with DAPI and the indicated antibodies, and GFP-positive cells were visualized by excitation under a wavelength of 488 nm. (A) Overexpression of the full-length Jmjd2b-GFP construct. (B) Overexpression of various Jmjd2b-GFP mutants. The 1–424 Jmjd2b-GFP truncation, but not the 1–424 (H189A) point mutant, becomes enriched at pericentric heterochromatin (indicated by arrows). This pericentric localization is lost in Suv39h double-null (Suv39h dn) iMEFs.

ChIP analysis of known chromosomal Suv39h targets (major satellites, Line L1, and IAPLTR1) in NIH3T3 Tet off (parental cells) and in the IB21, T2J3, and T2M4 cell lines (see Fig. 2). ChIP was performed with the indicated methyl-lysine histone antibodies and with a GFP antibody. Precipitation of enriched chromatin fragments relative to input material was quantified by real-time PCR. In chromatin from induced T2J3 cells, H3K9me3 signals are nearly abrogated (asterisk). Although there is no alteration for H3K9me3 levels in chromatin from uninduced versus induced T2M4 cells, there are differences in ChIP efficiency across the IAPLTR1 element, as compared with parental, IB21, and T2J3 cells.

Mass spectrometry analysis of H3K9 and H3K36 methylation states in bulk histone preparations. Mouse cell lines (NIH3T3 Tet-off) that inducibly express GFP-tagged full-length Jmjd2b (clone IB21), the 1–424 Jmjd2b truncation (clone T2J3), and the 1–424 (H189A) point mutation (clone T2M4) were either uninduced or induced for 2 d, and then histone H3 was isolated from nuclear extracts. The inducibility of the various clones differs such that in 35% of IB21 cells, in 92% of T2J3 cells, and in none of the T2M4 cells, pericentric H3K9me3 is lost (see Supplementary Fig. S4). (A) Mass spectrometry analysis for H3K9 methylation states using a histone H3 peptide that spans amino acid positions 9–17. (B) Mass spectrometry analysis for H3K36 methylation states using a histone H3 peptide that spans amino acid positions 27–40. H3K36me3 is underrepresented (<1%) in the analyzed cell lines (data not shown).

Turnover of histone H3 and stability of H3K9 methyl groups in T2J3 cells analyzed by metabolic labeling with “heavy” methionine. (A) Histone H3 turnover was analyzed by mass spectrometry of a histone H3 peptide that spans amino acid positions 117–128. (B) Stability of H3K9 methyl groups was analyzed by mass spectrometry of a different histone H3 peptide that spans amino acid positions 9–17. Metabolic labeling results in various combinations of “light” methyl (pre-existing) and “heavy” methyl (newly synthesized) groups. The black bars indicate the percentage of H3 peptide fragments that carry three “light” methyl groups. The yellow bars show the percentage of H3 peptide fragments that carry three “heavy” methyl groups. Intermediate combinations, such as one “light” methyl and one “heavy” methyl group (1/1; white bars) or (2/1; gray bars) or (1/2; light yellow bars), are also indicated.