doi:10.1038/sj.emboj.7600314. the strongest HOE 33187 upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimensions of chromatin regulation important in the maintenance of pluripotency. INTRODUCTION Human embryonic stem cells (hESCs) are an increasingly powerful tool for regenerative medicine. They recapitulate, counterparts, ESCs proliferate rapidly and are able to form the three embryonic germ layers (1). This highly self-renewing and HOE 33187 pluripotent state is usually sustained by a unique epigenetic scenery, consisting of transcription factors, chromatin remodeling complexes, and histone modifications that provide the transcriptional plasticity required for quick response to differentiation cues (2). Histone H3 lysine 4 and 27 trimethylations (H3K4me3 and H3K27me3, respectively) are key histone modifications that are involved in transcriptional regulation (3, 4). H3K4me3 near transcriptional start sites (TSSs) marks regions of active transcription or transcriptional readiness (5). H3K27me3 modification, in contrast, is usually a well-established unfavorable regulator of gene expression that repels transcriptional activators and attracts chromatin repressors that promote chromatin compaction (6). Genomic regions that host both histone marks, so-called bivalent domains, were first observed in ESCs, primarily near promoters of genes with developmental functions (7,C9). Significant effort has gone into understanding the biological role of bivalency; the consensus is usually that, in ESCs, it represses transcription but poises genes for quick expression during lineage commitment (10). Although this proposition is not yet supported with direct evidence, it has become obvious that bivalent domains are essential for maintaining ESC pluripotency and self-renewing capacity (10). Despite the extensive availability of genome-wide maps of these histone marks in pluripotent and committed cells, it is not understood how they contribute to faithful reestablishment of transcriptional status after cell division. Compelling questions remain, including the detailed localization of H3K4me3 and H3K27me3 during mitosis, whether these histone marks are gained/lost exclusively during mitosis, and perhaps more importantly, whether they constitute bivalent domains that are retained after cells exit mitosis. Here, we show that dynamic cell cycle control of H3K4 methylation/demethylation of bivalent genes represents a new dimensions to chromatin regulation that advances understanding of how the pluripotent histone modification landscape contributes to maintenance of hESC identity. We developed a new method for isolating real populations of hESCs at the G2, mitosis (M), and G1 phases of the cell cycle and used these phase-specific populations to map the genome-wide distribution of bivalent domains (H3K4me3/H3K27me3) throughout the pluripotent cell cycle. Consistent with a pivotal developmental function, we demonstrate that bivalent genes enriched with H3K4me3 during mitosis are maximally upregulated following induction of hESC differentiation, and subsequently, H3K4me3 on these genes becomes cell cycle impartial. Finally, we show that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. MATERIALS AND METHODS hESC culture and differentiation. The H9 hESC collection from WiCell Research Institute (Madison, WI) was managed on hESC-qualified Matrigel (BD Bioscience; catalog no. 354277) in mTeSR-1 medium (Stemcell Technologies; catalog no. 05850) or essential E8 medium (Life HOE 33187 Technologies; catalog no. A1517001), as recommended by the supplier. Cells were expanded every 5 to 6 days, using nonenzymatic passaging according to WiCell Research Institute standard protocols. To generate PAX6 cells, undifferentiated ESCs were incubated in mTeSR-1 medium supplemented with 10 M retinoic acid (RA) (Sigma-Aldrich; catalog no. R2625-50MG) for 5 days. The treatment started 1 day after plating of the cells, and medium was changed every day. hESC research was approved by the Institutional Embryonic Stem Cell Research Oversight Committee at the University or college of Vermont. Cell sorting. Pure populations of cells at the G2, mitosis, or G1 phase of the cell cycle were isolated by fluorescence-activated cell sorting (FACS), taking advantage of differences in DNA content to distinguish cells in G2/M from cells in G1 and the unique presence of histone H3 serine 28 phosphorylation (H3S28p) in mitosis to discriminate cells in G2 from those in M phase (Fig. 1A and ?andC).C). As indicated in the physique legends, both nocodazole-synchronized and untreated cells were sorted using the procedure explained here. After fixation, cells were permeabilized for 10 min using a moderate permeabilization/wash buffer made up of saponin (BD Bioscience; catalog no..2D). H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimensions of chromatin regulation important in the maintenance of pluripotency. INTRODUCTION Human embryonic stem cells (hESCs) are an increasingly powerful tool for regenerative medicine. They recapitulate, counterparts, ESCs proliferate rapidly and are able to form the three embryonic germ layers (1). This highly self-renewing and pluripotent state is sustained by a unique epigenetic landscape, consisting of transcription factors, chromatin remodeling complexes, and histone modifications that provide the transcriptional plasticity required for quick response to differentiation cues (2). Histone H3 lysine 4 and 27 trimethylations (H3K4me3 and H3K27me3, respectively) are key histone modifications that are involved in transcriptional regulation (3, 4). H3K4me3 near transcriptional start sites (TSSs) marks regions of active transcription or transcriptional readiness (5). H3K27me3 modification, in contrast, is usually a well-established unfavorable regulator of gene expression that repels transcriptional activators and attracts chromatin repressors that promote chromatin compaction (6). Genomic regions that host both histone marks, so-called bivalent domains, were first observed in ESCs, primarily near promoters of genes with developmental functions (7,C9). Significant effort has gone into understanding the biological role of bivalency; the consensus is usually that, in ESCs, it represses transcription but poises genes for quick expression during lineage commitment (10). Although this proposition is not yet supported with direct evidence, it has become obvious that bivalent domains are essential for maintaining ESC pluripotency and self-renewing capacity (10). Despite the extensive availability of genome-wide maps of these histone marks in pluripotent and committed cells, it is not understood how they contribute to faithful reestablishment of transcriptional status after cell division. Compelling questions remain, including the detailed localization of H3K4me3 and H3K27me3 during mitosis, whether these histone marks are gained/lost exclusively during mitosis, and perhaps more importantly, whether they constitute bivalent domains that are retained after cells exit mitosis. Here, we show that dynamic cell cycle control of H3K4 methylation/demethylation of bivalent genes represents a new dimensions to chromatin regulation that advances understanding of how the pluripotent histone modification landscape contributes to maintenance of hESC identity. We developed a new method for isolating real populations of hESCs at the G2, mitosis (M), and G1 phases of the cell cycle and used these phase-specific populations to map RACGAP1 the genome-wide distribution of bivalent domains (H3K4me3/H3K27me3) throughout the pluripotent cell cycle. Consistent with a pivotal developmental function, we demonstrate that bivalent genes enriched with H3K4me3 during mitosis are maximally upregulated following induction of hESC differentiation, and subsequently, H3K4me3 on these genes becomes cell cycle impartial. Finally, we show that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. MATERIALS AND METHODS hESC culture and differentiation. The H9 hESC collection from WiCell Research Institute (Madison, WI) was managed on hESC-qualified Matrigel (BD Bioscience; catalog no. 354277) in mTeSR-1 medium (Stemcell Technologies; catalog no. 05850) or essential E8 medium (Life Technologies; catalog no. A1517001), as recommended by the supplier. Cells were expanded every 5 to 6 days, using nonenzymatic passaging according to WiCell Research Institute standard protocols. To generate PAX6 cells, undifferentiated ESCs were incubated in mTeSR-1 medium supplemented with 10 M retinoic acid (RA) (Sigma-Aldrich; catalog no. R2625-50MG) for 5 days. The treatment started 1 day after plating of the cells, and medium was changed every day. hESC research was approved by the Institutional Embryonic Stem Cell Research Oversight Committee at the University or college of Vermont. Cell sorting. Pure populations of cells at the G2, mitosis, or G1 phase of the cell cycle were isolated by fluorescence-activated cell sorting (FACS), taking advantage of differences in DNA content to distinguish cells in G2/M from cells in G1 and the unique presence of histone H3 serine 28 phosphorylation (H3S28p) in mitosis to discriminate cells in G2 from those in M phase (Fig. 1A and ?andC).C). As indicated in the physique legends, both nocodazole-synchronized and untreated cells were sorted using the procedure described here. After fixation, cells were permeabilized for 10 min using a moderate permeabilization/wash buffer HOE 33187 made up of saponin (BD Bioscience; catalog no. 51-2091KZ). For ESC isolation, cells were incubated with labeled antibodies to OCT4 (phycoerythrin [PE] conjugated; BD Bioscience; catalog no. 561556) and H3S28p (Alexa Fluor 647 conjugated; BD Bioscience; catalog no. 558609) for 30 min. Labeled antibodies to PAX6, instead of OCT4 (PE conjugated; BD.