Chromatin was collected by centrifugation at 14,000 g for 10 min at 4C. three methyl marks are each regulated differentially during development and their detection on western blots does not overlap with their detection on chromosomes. Monomethylated H4-K20 is detected on condensed chromosomes throughout development, while di-, and trimethylated H4-K20 is detected on metaphase chromosomes at specific stages. Our results suggest that the detection of methylated H4-K20 on chromosomes may reveal chromatin packaging rather than the distribution of the methyl marks. and were found to suppress position effect variegation, indicating that both genes function in silencing gene expression (Schotta et al., 2004, Karachentsev et al., 2005). Monomethylation is also essential for the normal progression through the cell cycle. Imaginal discs from mutants lacking PR-Set7 have only ~25% as many cells as wild-type discs and the cells are larger (Karachentsev et al., 2005). The monomethyl mark is stable over several cell generations, because in homozygous animals the enzyme cannot be detected on western blots from the 1st instar larval stage onward. On the other hand, monomethylated H40-K20 can be detected on salivary glands of early third instar larvae and disappears only in late third instar (Karachentsev et al., 2002). Histone methylation was originally thought to be a stable modification controlling gene expression, LDHAL6A antibody with the marks set at specific stages of development, and inherited from mother to daughter cells. But now lysine demethylases have been identified, suggesting that the histone methylation marks are reversible and that the marks may have a AG-17 more transient function (Shi et al., 2004; Trojer and Reinberg, 2006). We chose a developmental approach to determining the regulation and distribution of the three methylated states of the same lysine of histone H4. We investigated if the modifications are stable during development and the cell cycle, if their distribution on chromosomes is linked, and if different cell types show distinct patterns of distribution. MATERIALS AND METHODS Cell culture and synchronization HeLa cells (ATCC) were grown in DMEM supplemented with 10% fetal bovine serum (Invitrogen). To arrest cells in G1, cells were treated with 2 mM thymidine (Sigma) for 16 h, released into fresh media for 8 h, and blocked AG-17 again by addition of 0.4 mM mimosine overnight (Sigma). Cells were released into fresh media and time points were taken every 2.5 h (Rice et al., 2002). At each time point, 105 cells were used for western blot analysis. Drosophila S2 cells (ATCC) were grown in SFM (Gibco) supplemented with 10% fetal bovine serum. Protein analysis Histones were fractionated from cultured cells as described in Schwartz and Ahmad (2005). Briefly, cells were lysed in buffer containing 0.5% NP-40, 0.4 M NaCl, 5 mM 2-mercaptoethanol, 10 mM Tris HCl pH 8, protease inhibitors. Chromatin was collected by centrifugation at 14,000 g for 10 min at 4C. The supernatant (containing soluble histones) was dialyzed AG-17 against 10 mM Tris HCl pH 8. Samples were mixed with SDS loading buffer and separated on 14% PAGE. Cell fractionation Drosophila S2 cells were pelleted and resuspended in small volume of cold 4C, 10 mM Tris HCl pH 8, 5 mM 2-mercaptoethanol, and protease inhibitor cocktail. Cells were mechanically lysed with few strokes in Dounce homogenizer. Nuclei were collected by centrifugation at 14,000 g for 10 min at 4C. The supernatant and pellet were separately collected. Samples were mixed with SDS loading buffer and separated on 8% PAAG. Western blot was probed with 1:1000 dilution of anti-DrosPRSet7 antibodies published previously (Karachentsev et al., 2005). Western.