In contrast, we could find no significant differences in replication timing or distribution of Me2K9H3 between Suv39hdn and HM1. epitope at the nuclear and nucleolar periphery and colocalized with sites of DNA synthesis primarily in mid-S phase. Me3K9H3 Methoxamine HCl decorated late-replicating pericentric heterochromatin in mouse cells and sites of DAPI-dense intranuclear heterochromatin in human and hamster cells that replicated throughout S phase. Disruption of the Suv39h1,2 or G9a methyltransferases in murine embryonic stem cells resulted in a redistribution of methyl epitopes, but did not alter the overall spatiotemporal replication program. These results demonstrate that mono-, di-, and trimethylated states of K9H3 largely occupy distinct chromosome domains. INTRODUCTION Histone methylation has emerged as a primary epigenetic mark, central to the regulation of local and global chromatin structure. In particular, lysine 9 methylation of histone H3 (MeK9H3) has been correlated with both local and global repression of transcription and the formation of large constitutive heterochromatin domains. The complexity that can Methoxamine HCl be achieved with this one modification alone is quite remarkable. K9H3 residues can be either mono-, di- or trimethylated Methoxamine HCl (Waterborg, 1993 ; Santos-Rosa 2002 ; Peters 2003 ; Rice 2003 ; Wang 2003 ; Zhang 2003 ). At least five methyltransferases have been shown to methylate K9H3: Suv39h1 and Suv39h2 (Rea 2000 ; Peters 2001 ), G9a (Tachibana 2001 ), ESET/SETDB1 (Schultz 2002 ), and EuHMTase1 (Ogawa 2002 ). These enzymes have different affinities for the un-, mono- or dimethylated states and produce different methylation states; some may act only on previously methylated lysines, whereas others may carry out de novo methylation. In vivo, these enzymes differentially methylate histones in euchromatin versus heterochromatin, and knockout mice for Suv39h1,2 and G9a have very different phenotypes, suggesting that methyltransferases may be differentially targeted to specific chromatin contexts for regulatory purposes. Efforts to determine whether these different methylation claims reside stably at different chromatin sites have not produced a definite picture. Imunolocalization experiments in mouse cells have revealed that the majority of Me3K9H3 is definitely localized to the prominent clusters of pericentromeric heterochromatin (chromocenters) that are observed in this varieties, whereas the mono- and dimethylation claims could not become resolved (Peters 2003 ; Rice 2003 ). Here, we have localized the epitopes for each of these antibodies relative to sites of coordinated DNA synthesis (replicon clusters) at different times during S phase. These 1-Mb segments of coordinately replicated DNA constitute probably one of the most recognizable subnuclear devices of chromosome corporation (Jackson and Pombo, 1998 ; Ma 1998 ; Dimitrova and Gilbert, 1999 ; Zink 1999 ; Leonhardt 2000 ; Sadoni 2004 ). Because specific classes of chromosomal domains replicate at particular instances, the combination of spatial and temporal separation provides an added dimensions of resolution to the subnuclear localization of specific epitopes (Wu 2004 ). Furthermore, because cells exploit the spatio-temporal separation of replication to assemble different types of chromatin at different times during S phase (Bozhenok 2002 ; Gilbert, 2002 ; Zhang 2002 ; McNairn and Gilbert, 2003 ), one can infer a relationship between spatiotemporal localization and function. We find that mono-, di-, and trimethylated K9H3 can be distinguished from the spatiotemporal properties of the domains in which they reside, indicating that they are mainly targeted to different chromosomal domains. These results strongly suggest that each of the three claims of MeK9H3 serve unique functions within the nucleus. MATERIALS AND METHODS Cell Tradition C127 (mouse mammary tumor), CHOC400, HeLa S3 were all cultivated in DMEM (Existence Systems, Rockville, MD) with appropriate health supplements at 37C in 5% CO2. Medium for C127 was supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT). Medium for CHOC 400 was supplemented with 5% FCS and 1% nonessential amino acids (Life Systems). Medium for HeLa S3 was supplemented with 10% cosmic calf serum (Hyclone). Sera cells were cultivated on GMEM (Existence Systems) supplemented with 5% newborn calf serum (NCS; Hyclone), 5% FCS, 1% nonessential amino acid, 2 mM l-glutamine, 0.1 mM -mercaptoethanol, and 500 U/ml leukemia inhibitor element (LIF; Chemicon, Temecula, CA). Sera cells were subcultured every day and new medium was constantly changed on the same day time as the experiment. dn (double null) Sera cells were constructed by first focusing on the solitary, X-linked Suv39h1 allele, followed by sequential Epha2 focusing on of the two.