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. 2012 Oct;32(19):3860-71.
doi: 10.1128/MCB.00736-12. Epub 2012 Jul 23.

Epigenetic control of cell cycle-dependent histone gene expression is a principal component of the abbreviated pluripotent cell cycle

Ricardo Medina  1 , Prachi N Ghule, Fernando Cruzat, A Rasim Barutcu, Martin Montecino, Janet L Stein, Andre J van Wijnen, Gary S Stein
Affiliations

Epigenetic control of cell cycle-dependent histone gene expression is a principal component of the abbreviated pluripotent cell cycle

Ricardo Medina et al. Mol Cell Biol. 2012 Oct.

Abstract

Self-renewal of human pluripotent embryonic stem cells proceeds via an abbreviated cell cycle with a shortened G(1) phase. We examined which genes are modulated in this abbreviated period and the epigenetic mechanisms that control their expression. Accelerated upregulation of genes encoding histone proteins that support DNA replication is the most prominent gene regulatory program at the G(1)/S-phase transition in pluripotent cells. Expedited expression of histone genes is mediated by a unique chromatin architecture reflected by major nuclease hypersensitive sites, atypical distribution of epigenetic histone marks, and a region devoid of histone octamers. We observed remarkable differences in chromatin structure--hypersensitivity and histone protein modifications--between human embryonic stem (hES) and normal diploid cells. Cell cycle-dependent transcription factor binding permits dynamic three-dimensional interactions between transcript initiating and processing factors at 5' and 3' regions of the gene. Thus, progression through the abbreviated G(1) phase involves cell cycle stage-specific chromatin-remodeling events and rapid assembly of subnuclear microenvironments that activate histone gene transcription to promote nucleosomal packaging of newly replicated DNA during stem cell renewal.

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Figures

Fig 1
Fig 1
Gene expression profiling during the G1/S-phase cell cycle transition. (A) Comparative microarray analysis of genes exhibiting >1.2-fold differences in expression in human embryonic stem (hES) cells or normal human fibroblasts (TIG-1). Expression profiling was performed using GeneChip human gene 1.0 ST arrays from Affymetrix. (B) Venn diagrams showing the numbers of genes with increased and decreased expression during the G1/S-phase transition in hES versus TIG-1 cells.
Fig 2
Fig 2
Chromatin architecture of the human histone H4 gene in hES cells. (A) DNase I hypersensitivity at the human histone H4 gene (HIST2H4). Nuclei were isolated from hES cells (lanes 4 to 6) and normal diploid fibroblasts (TIG-1 and IMR-90; lanes 7 to 9 and 10 to 12, respectively), and DNA was prepared as described in Materials and Methods. Naked genomic DNA from hES cells digested with increasing amounts of DNase I was used as a control (lanes 1 to 3). The positions of the DNase I hypersensitive sites (HSSs) are shown on the right. A schematic representation of the human histone H4 gene, restriction sites, transcription start site, probe used in Southern blots, and HSSs (arrowheads) is shown on the left. Human ES cells show increased nuclease sensitivity at lower DNase I concentrations than normal diploid fibroblasts (compare lane 6 with lanes 9 and 12); the asterisk (nt −130, lane 5) designates an HSS in hES cells that is observed only at short digestion times. (B) Chromatin landscape of the histone H4 gene in human pluripotent cells. The top panel shows a schematic of the genomic organization of the human histone H4 gene. Transcriptional binding sites I and II, the set of primers (lines 1 to 8) used for analyses and their location, the HSSs (arrowheads), and the sequence representing the typical stem-loop structure found in histone mRNAs are shown. The bottom panel shows chromatin immunoprecipitation analysis for RNA pol II and HINFP at the human histone H4 gene in human embryonic stem cells.
Fig 3
Fig 3
Active chromatin marks at the human histone H4 and H3 gene loci in human pluripotent and normal diploid cells. (A) Interaction of RNA pol II and HINFP with the human histone H4 gene (HIST2H4) in human pluripotent cells (hES H9, iPS A6, iPS D1) and normal fibroblasts (WI-38). The top panel shows a schematic of the genomic organization of the human histone H4 gene as described in Fig. 2. Maximal interaction of HINFP (gray arrowheads) with the histone H4 gene is distal to that of RNA pol II (black arrowheads) in all three pluripotent cells and in normal fibroblasts (WI-38). The values obtained for ChIP antibodies and control IgGs are represented, respectively, by continuous and dotted lines. (B) Chromatin immunoprecipitation analysis of epigenetic marks of the histone H4 gene in human pluripotent cells and normal fibroblasts (WI-38). Solid lines represent the epigenetic marks H3K4me3 (black) and H3K9ac (gray), and dotted lines represent mouse or rabbit ChIP control IgGs for H3K4me3 (black) and H3K9ac (gray), respectively. (C) The human histone H4 gene lacks the epigenetic repressive mark H3K27me3 in all three pluripotent cell lines (hES H9, iPS A6, and iPS D1). Solid lines represent the epigenetic mark (black) and dotted lines represent rabbit ChIP control IgG (black) for H3K27me3. (D) Interactions of RNA pol II and HINFP with the human histone H3 gene (HIST1H3I) in hES H9 cells. The top panel shows a schematic of the genomic organization of the human histone H3 gene. The locations of primers (lines 1 to 8) used for ChIP analyses and the typical stem-loop structure found in histone mRNAs are shown. Values obtained for ChIP antibodies and control IgGs are represented, respectively, by continuous and dotted lines. (E) Chromatin immunoprecipitation analysis of epigenetic marks at the histone H3 gene in hES H9 cells. Solid lines represent the epigenetic marks H3K4me3 (black) and H3K9ac (gray), and dotted lines represent mouse or rabbit ChIP control IgGs for H3K4me3 (black) and H3K9ac (gray), respectively. (F) The human histone H3 gene lacks the epigenetic repressive mark H3K27me3 in hES cells. Solid lines represent the epigenetic mark (black) and dotted lines represent rabbit ChIP control IgG (black) for H3K27me3.
Fig 4
Fig 4
Selective occupancy of human histone genes during the pluripotent cell cycle. Human embryonic stem cells were synchronized in the G2/M phase of the cell cycle by a nocodazole block and then released into the cell cycle. The top portion shows a schematic of ChIP primers relative to the genomic organization of the human histone H4 and H3 genes as described in Fig. 2. (A) Relative mRNA levels of cell cycle markers in synchronized hES H9 cells. An increase in the levels of histone H4 (solid line) and H3 (dashed line) RNAs and a decrease in cyclin E2 RNA (dotted line) confirm transition of cells from G1 to early S phase. (B) Chromatin immunoprecipitation analysis of the human histone H4 (left panel) and H3 (right panel) genes in hES H9 cells at the G1/S-phase boundary. (C) ChIP analysis of the histone H4 (left panel) and H3 (right panel) promoters during the cell cycle in hES H9 cells. Association of HINFP (top panels), the histone cofactor p220NPAT (middle panels), and RNA pol II (bottom panels) with the histone H4 and H3 genes is shown. (D) Temporal changes in binding of proteins at the site of maximal occupancy in the histone H4 locus during the hES cell cycle (around the TSS for RNA pol II and p220NPAT and around site II for HINFP). (E) ChIP analysis of HINFP association with the histone H4 gene during the cell cycle in normal fibroblasts (left panel). Human WI-38 cells were synchronized by serum deprivation and then released into the cell cycle by serum stimulation. Histone H4 transcript profile (right panel) confirms synchrony of WI-38 cells.
Fig 5
Fig 5
Epigenetic landscape of human histone genes. Chromatin immunoprecipitation analysis was performed with hES and WI-38 cells that were synchronized in the G2/M phase by a nocodazole block and in G0 by serum deprivation, respectively (details in Fig. 4 legend). Covalent modifications of histone proteins H3K4me3, H3K9ac, and total histone H3 (A and C) and H4K12ac, H4K16ac, and H3K27me3 (B and D) in hES cells at the human histone H4 and H3 genes are shown. Covalent modifications of histone proteins H3K4me3 and histone H3 (E) and H4K12ac and H3K27me3 (F) in WI-38 cells at the histone H4 locus are shown. Epigenetic marks normalized to total histone signal in hES cells (G) and fibroblasts (H) during the maximal occupancy of histone genes at mid-S phase are also shown. The top panels show the location of ChIP primers for histone H4 and H3 genes as described in Fig. 2.
Fig 6
Fig 6
Molecular events associated with the activation and processing of human histone H4 transcripts. (A) Colocalization of 5′ and 3′ regulatory factors in hES cells. p220NPAT foci (green) are associated with the transcriptional machinery (RNA pol II, top and middle panels, red) and 3′-end processing machinery (LSM11, lower panel, red) at histone gene clusters in human H9 cells. DAPI staining (blue) is used to visualize the nucleus. Panels on the right represent magnified images for each antibody and merged channels from the left panel (white squares). Merged images show that p220NPAT foci, which denote HLBs, colocalize (yellow) with factors mediating histone pre-mRNA processing at sites of histone gene transcription. (B) Chromatin immunoprecipitation analysis of 3′-end-processing factors at H4 and H3 histone genes in human ES cells. Solid lines represent specific interactions above background IgG signal (dotted lines) of the 3′-end-processing factors LSM10 (black, top), FLASH (black, bottom), LSM11 (gray, top), and SLBP (gray, bottom) with the histone H4 and H3 genes. (C) Schematic representations of binding of histone H4-specific gene regulatory factors to sites I and II and binding of RNA pol II are shown (37, 38, 40, 57). Modifications of histone proteins upstream and downstream of the TSS and hypersensitivity to nucleases (HSSs; arrowheads) are also shown; nucleosomes located 1 kb upstream of the TSS are devoid of active histone modifications (unpublished observations). Several 3′-end-processing factors and proteins involved in transcription of the replication-dependent histones colocalize to specific subnuclear foci known as histone locus bodies (HLBs) (21, 22). FLASH protein also localizes to HLBs (7, 23), is required for transcription and 3′-end processing of histone mRNAs (2, 14, 28, 58), and interacts with histone gene loci (Fig. 6B and data not shown). The schematic representation of components involved in 3′-end processing of histone pre-mRNAs was adapted from that of Dominski with permission (15). (D) Proximity ligation assay of the HIST2H4 gene. The top panel shows a schematic of the genomic organization of the human histone H4 gene and a gene desert region (GDR) near the histone H4 locus. The set of primers (open arrowheads) used for analyses and the FatI restriction sites (lines) are shown. The histone H4 locus (bottom panel) shows a higher ligation frequency than a gene desert region, indicating a looping interaction between the 5′ and 3′ ends of the histone H4 gene (**, P < 0.01).
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References

    1. Ahmed K, et al. 2010. Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo. PLoS One 5:e10531 doi:10.1371/journal.pone.0010531 - DOI - PMC - PubMed
    1. Barcaroli D, et al. 2006. FLASH is required for histone transcription and S-phase progression. Proc. Natl. Acad. Sci. U. S. A. 103:14808–14812 - PMC - PubMed
    1. Becker KA, et al. 2006. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J. Cell. Physiol. 209:883–893 - PubMed
    1. Becker KA, Stein JL, Lian JB, van Wijnen AJ, Stein GS. 2007. Establishment of histone gene regulation and cell cycle checkpoint control in human embryonic stem cells. J. Cell. Physiol. 210:517–526 - PubMed
    1. Bilodeau S, Kagey MH, Frampton GM, Rahl PB, Young RA. 2009. SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state. Genes Dev. 23:2484–2489 - PMC - PubMed

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