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| Proc Natl Acad Sci U S A. 2008 November 4; 105(44): 16964–16969. doi: 10.1073/pnas.0809273105. | PMCID: PMC2579361 |
Copyright © 2008 by The National Academy of Sciences of the USA Cell Biology Staged assembly of histone gene expression machinery at subnuclear foci in the abbreviated cell cycle of human embryonic stem cells Prachi N. Ghule,ab Zbigniew Dominski,c Xiao-cui Yang,c William F. Marzluff,c Klaus A. Becker,b J. Wade Harper,d Jane B. Lian,b Janet L. Stein,b Andre J. van Wijnen,b and Gary S. Steinab1 aCenter for Stem Cell Biology and Regenerative Medicine, bDepartment of Cell Biology and Cancer Center, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655; cProgram in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; and dDepartment of Pathology, Harvard Medical School, Boston, MA 02115 Received August 28, 2008. |
Abstract Human embryonic stem (hES) cells have an abbreviated G1 phase of the cell cycle. How cells expedite G1 events that are required for the initiation of S phase has not been resolved. One key regulatory pathway that controls G1/S-phase transition is the cyclin E/CDK2-dependent activation of the coactivator protein nuclear protein, ataxia–telangiectasia locus/histone nuclear factor-P (p220NPAT/HiNF-P) complex that induces histone gene transcription. In this study, we use the subnuclear organization of factors controlling histone gene expression to define mechanistic differences in the G1 phase of hES and somatic cells using in situ immunofluorescence microscopy and fluorescence in situ hybridization (FISH). We show that histone gene expression is supported by the staged assembly and modification of a unique subnuclear structure that coordinates initiation and processing of transcripts originating from histone gene loci. Our results demonstrate that regulatory complexes that mediate transcriptional initiation (e.g., p220NPAT) and 3′-end processing (e.g., Lsm10, Lsm11, and SLBP) of histone gene transcripts colocalize at histone gene loci in dedicated subnuclear foci (histone locus bodies) that are distinct from Cajal bodies. Although appearance of CDK2-phosphorylated p220NPAT in these domains occurs at the time of S-phase entry, histone locus bodies are formed ≈1 to 2 h before S phase in embryonic cells but 6 h before S phase in somatic cells. These temporal differences in the formation of histone locus bodies suggest that the G1 phase of the cell cycle in hES cells is abbreviated in part by contraction of late G1. Keywords: HiNF-P, p220NPAT, Cajal body, coilin, G1/S transition |
The abbreviated cell cycle of human embryonic stem (hES) cells represents a unique cellular adaptation that expedites self-renewal and is reflected by a very brief G 1 phase ( 1, 2). Competency of somatic cells for proliferation is linked to growth factor-dependent passage through the restriction (R) point in G 1 when cells commit toward onset of S phase ( 3, 4). However, hES cells lack a classical R point and have the capacity for continuous cell division. A principal mechanism that is required for the initiation of S phase in hES cells is the induction of histone gene expression, which is essential for the packaging of newly replicated DNA into chromatin by specific transcription factors ( 1, 2, 5– 12). In both somatic and hES cells, key histone gene regulatory factors are organized in a limited number of subnuclear foci. For example, recruitment of the coactivator protein p220 NPAT (nuclear protein, ataxia–telangiectasia locus) by transcription factor HiNF-P (histone nuclear factor-P) to histone H4 gene promoters, as well as cell cycle-dependent phosphorylation of p220 NPAT by cyclin E/CDK2 to induce histone gene transcription occur at these intranuclear sites ( 7, 8, 13– 17). Newly synthesized histone transcripts are not polyadenylated, and their cleavage requires a U7 small nuclear ribonucleoprotein complex (U7 snRNP) that contains U7 snRNA and the protein subunits Lsm10 (U7 snRNP-specific Sm-like protein LSM10) and Lsm11 (U7 snRNP-specific Sm-like protein LSM11), whereas a specific RNA hairpin in histone transcripts interacts with stem loop binding protein (SLBP) ( 18– 22). Studies with somatic cell types have shown that at least some factors mediating 3′-end processing of histone primary transcripts are organized in Cajal body-related foci that contain coilin ( 23). However, Cajal bodies are not evident in all somatic cell types and are distinct from subnuclear foci that contain p220 NPAT ( 24– 27). In this study, we used the subnuclear organization of histone gene transcription and processing factors as a paradigm to define mechanistic differences in the G1 phase of hES and somatic cells. We show first that the Lsm10 and Lsm11 protein subunits of the U7 snRNP, as well as SLBP, are recruited to p220NPAT foci at histone gene loci in both hES and somatic cells. These results establish that cells in G1 phase preassemble regulatory structures analogous to nucleoli to provide a unique microenvironment for the production of histone mRNAs in S phase. Furthermore, we show that these p220NPAT foci are formed at different stages of the G1 phase in embryonic versus somatic cells. Cell type-specific differences in the temporal assembly of p220NPAT foci provide insight into the regulatory organization of G1 and the coordination of transcription and processing of gene transcripts at the G1/S-phase cell cycle transition in hES cells. |
Results Foci of p220NPAT Associate with the Two Major Histone Gene Clusters at 6p22 and 1q21 in hES Cells. Our studies are directed toward understanding the spatial and temporal organization of the regulatory machinery for histone gene expression during the abbreviated cell cycle in hES cells (H9/WA09). We performed immunofluorescence (IF) microscopy for the histone gene regulatory factor p220 NPAT combined with fluorescence in situ hybridization (FISH) using probes spanning genomic segments near histone gene loci on chromosomes 6 and 1. The FISH results show that p220 NPAT foci as well as phospho-T1270-p220 NPAT foci are associated with the histone gene clusters on 6p22 ( Fig. 1) and 1q21 (data not shown) in asynchronous populations of hES cells ( Fig. 1A, left column, top and middle rows) and normal fibroblasts ( Fig. 1A, right column, top and middle rows). Depending on the stage of the cell cycle, hES cells have either 2 or 4 p220 NPAT foci ( Fig. 1B). Two of the four p220 NPAT foci are always associated with the histone clusters on chromosome 6 ( Fig. 1A), and the remaining 2 foci are associated with the histone gene clusters on chromosome 1 (data not shown). The association of p220 NPAT with histone genes indicates that the histone gene transcriptional complexes are architecturally linked with their target genes in hES cells as they are in normal fibroblasts.  | Fig. 1.The p220NPAT foci are associated with histone gene loci in H9 hES cells and normal diploid WI-38 fibroblasts. (A) IF microscopy images were obtained using antibodies against p220NPAT (green), phospho-T1270-p220NPAT (green), or coilin (blue), and a DIG-labeled (more ...) |
The p220 NPAT foci are related to Cajal bodies that have been shown previously by our group ( 26, 28) and others ( 14, 15, 23) to be associated with histone gene loci. We find that 50–60% of hES cells stain for the Cajal body marker coilin, whereas >95% of cells are stained for p220 NPAT ( Fig. 1A). Not all p220 NPAT foci contain coilin and vice versa, consistent with previous findings ( 26). Importantly, p220 NPAT foci but not coilin consistently associate with histone gene clusters on 6p22 ( Fig. 1A, left column, bottom row) and 1q21 (data not shown). These results indicate that Cajal bodies and p220 NPAT foci are 2 distinct nuclear domains and that p220 NPAT foci are the subnuclear sites where histone loci are organized in hES and somatic cells. We investigated how histone gene loci are associated with p220 NPAT foci and Cajal bodies in different stages of the cell cycle using Ki-67 as a marker. Human ES cells in S phase but not G 1 consistently exhibit costaining of p220 NPAT and the histone gene locus at 6p22 ( Fig. 1B, left column), whereas a strict correlation is not evident for coilin (data not shown). In somatic WI-38 cells, p220 NPAT foci are present in mid to late G 1 and S phases and associate with histone gene loci ( Fig. 1B, right column). In contrast, only 15% of WI-38 cells show focal staining for coilin. However, when coilin foci are present, they tend to be in proximity to p220 NPAT and histone gene loci ( Fig. 1A, right column, bottom row). Thus, hES cells and WI-38 cells exhibit fundamental differences in association of coilin with histone gene-containing p220 NPAT foci. The p220NPAT Foci Colocalize with Factors Mediating 3′-End Processing of Histone Pre-mRNA. Because U7 snRNPs that mediate maturation of histone gene primary transcripts are focally organized ( 23), we investigated whether p220 NPAT foci and histone mRNA processing factors reside at the same subnuclear locations. We studied Lsm10 and Lsm11, which are integral to U7 snRNPs, as well as the SLBP and 3′ histone exonuclease (hExo), which each recognize opposite sides of the histone mRNA-specific 3′ hairpin structure. Costaining of hES cells with either p220 NPAT or coilin, along with one of the 3′-end processing factors, reveals that all p220 NPAT foci colocalize with Lsm10 and Lsm11 in all cells ( Fig. 2, left column, rows 1 and 2). These findings indicate that the machinery for both initiation and processing of histone mRNAs resides in the same subnuclear domains. SLBP compartmentalizes in both the cytoplasm and nucleus of 50–60% of asynchronous hES cells, and a subset of nuclear SLBP is adjacent to or partially overlaps with p220 NPAT foci ( Fig. 2, left column, row 3). Similarly, Lsm10 and Lsm11 reside at p220 NPAT foci in most (>90%) somatic WI-38 cells, whereas SLBP only partially colocalizes with p220 NPAT foci ( Fig. 2, right column, rows 1–3). These results indicate that 3 principal 3′-end processing factors that recognize histone transcripts are spatially linked to p220 NPAT foci.  | Fig. 2.Colocalization of factors mediating histone gene transcriptional initiation and 3′-end transcript processing of histone mRNA. IF microscopy images were obtained for H9 hES cells (Left column) and normal diploid WI-38 cells (Right column) using (more ...) |
Although p220 NPAT foci are clearly linked with active synthesis of histone transcripts, the mechanistic role of Cajal bodies in histone gene expression is less evident. Although only a subset of hES cells and somatic WI-38 cells have focal coilin staining (see above), there is partial or complete overlap of Lsm10, Lsm11, or SLBP with one or more coilin foci in these cells ( supporting information (SI) Fig. S1). Thus, some Cajal bodies may have an auxiliary role in maturation of histone mRNAs, whereas others appear to be unrelated to histone gene expression. In addition to the factors supporting synthesis of mature histone mRNAs, we examined in situ localization of the exonuclease 3′ hExo that specifically interacts with the stem-loop in histone mRNA and may degrade histone mRNA at the completion of DNA synthesis. This enzyme is present at neither p220 NPAT nor coilin foci, but 3′ hExo foci show complete colocalization with PML/ND10 (promyelocytic leukemia domain/nuclear domain 10) bodies in both hES cells and somatic WI-38 cells ( Fig. 2, rows 4 and 5, and Fig. S1). Hence, 3′ hExo is spatially concentrated at domains distinct from p220 NPAT foci. Temporal and Spatial Association of p220NPAT with the Factors Mediating Processing of Histone mRNA at Histone Gene Loci. To understand the temporal coordination between p220 NPAT foci, 3′-end processing factors, and histone loci, we synchronized hES cells in G 2/M phase using nocodazole. Cell cycle entry and progression in synchronized hES cells were monitored using Ki-67 as a marker ( Fig. 3, row 1) ( 1). Cells also were examined for localization of Lsm10 or SLBP to either p220 NPAT or coilin foci. Triple labeling by combining double-label IF microscopy with histone gene-specific FISH was performed to determine whether these factors associate with histone chromosomal loci ( Fig. 1B).  | Fig. 3.Association of p220NPAT and 3′-end processing factors with histone gene loci during the hES cell cycle. Mitotically synchronized hES cells at various cell cycle stages were monitored by IF microscopy for association of Lsm10 or SLBP (red) with (more ...) |
At 1 h after release from mitotic block, when cells progress through early G 1, we do not observe focal organization of either p220 NPAT or Lsm10, nor is there evidence of association with histone loci (data not shown). However, in mid to late G 1 (2 h after release), p220 NPAT foci show complete colocalization with both Lsm10 and histone loci, while coilin foci do not ( Fig. 3, rows 2, 4, and 5). This colocalization is maintained throughout S phase and the remainder of the cell cycle until mitosis (data not shown). Because Lsm10 colocalizes with histone genes containing p220 NPAT foci as soon as these foci form, our results demonstrate that 5′-end transcriptional initiation and U7 snRNP required for histone mRNA maturation are spatially, temporally, and functionally linked within the same subnuclear domains. In contrast to Lsm10, SLBP only partially colocalizes with p220 NPAT in ≈40% of the cells in mid to late G 1 (2 h after release), and this association increases as cells enter S phase (4 h after release) ( Fig. 3, row 3). Hence, the timings of recruitment of SLBP to p220 NPAT foci differ from that of Lsm10. It is possible that Lsm10 is a prerequisite for SLBP association with p220 NPAT foci. Alternatively, de novo transcription of histone genes is required for SLBP recruitment. Regardless of the mechanism, the observation that presence of Lsm10 at p220 NPAT foci precedes that of SLBP indicates that the supramolecular assembly of the histone gene expression machinery is a staged process. The Staged Spatial Assembly of p220NPAT Foci Defines Mechanistic Differences Between the Abbreviated Cell Cycle of hES Cells and the Cell Cycle of Human Diploid Fibroblasts. Our analysis of the ordered assembly of histone gene transcription and mRNA processing components in human embryonic stem cells reveals that subnuclear foci containing histone genes, p220 NPAT, and U7 snRNP are rapidly established following mitosis ( Figs. 1– 3). This rapid spatial assembly reflects the abbreviated G 1 phase during the short cell cycle of hES cells that will enter S phase within 2.5 h after completion of mitosis ( 2). However, in somatic cells S phase is typically initiated 8 to 10 h after mitosis, indicating that temporal aspects of the assembly of p220 NPAT foci may differ between the somatic and embryonic cells. Therefore, we investigated whether the subnuclear organization of p220 NPAT, Lsm10, SLBP, and coilin in relation to histone gene loci differs between hEScells and somatic cells ( Figs. 3 and 4).  | Fig. 4.Colocalization of 3′-end processing factors SLBP and Lsm10 with p220NPAT during the cell cycle in somatic cells. Synchronized WI-38 fibroblasts were examined at various cell cycle stages by IF microscopy for spatial interactions between Lsm10 (more ...) |
We rendered WI-38 cells quiescent by serum deprivation as reflected by absence of Ki-67 staining, and these cells retain small rudimentary foci containing both p220 NPAT and Lsm10 at histone genes ( Fig. 4). Robust p220 NPAT/Lsm10 foci are detected within 6 h of serum stimulation when cells have entered the G 1 phase of the cell cycle based on Ki-67 staining. Both p220 NPAT and Lsm10 remain associated with histone genes at the 6p22 locus during S phase (12 to 18 h) and G 2 (24 h), but not in mitosis when p220 NPAT foci are disassembled ( Fig. 4, middle row, and data not shown). Cells exhibiting coilin foci were infrequently observed (<10%) and, if detected, usually were associated with p220 NPAT/Lsm10 foci (data not shown). The similarities in localization between p220 NPAT, Lsm10, and histone gene loci for hES cells and WI-38 fibroblasts indicate that the preassembly in G 1 of a subnuclear domain dedicated to histone gene expression machinery is an integral part of the preparation for the onset of S-phase transition in both cell types. More importantly, the formation of these domains occurs at least 6 h before S phase in somatic cells, but just 1 to 1.5 h before S phase in hES cells. In serum-stimulated WI-38 cells, the percentage of cells in which SLBP associates with p220 NPAT increases as cells progress through the cell cycle. For example, a small subset (25–30%) of the cells contain SLBP in G 1 (6 h), and more than half of the cells (55–60%) in late S (24 h) have SLBP associated with p220 NPAT ( Fig. 4, bottom row). Furthermore, the association of SLBP with p220 NPAT within each cell becomes more frequent as the cell cycle progresses. SLBP did not consistently localize at Cajal bodies in WI-38 cells (data not shown) and SLBP does not colocalize with Cajal bodies in Hela cells ( 30). Apart from focal SLBP localization at p220 NPAT foci, SLBP is distributed throughout the nucleus in G 1-phase (6 h and 12 h after serum stimulation) and S-phase (18 h and 24 h) cells. SLBP is not detected in quiescent WI-38 when histone mRNAs are not present. SLBP may be recruited to p220 NPAT foci as a protein–protein interaction with other components mediating histone gene processing. Alternatively, localization of SLBP at p220 NPAT foci may reflect interaction with the cognate stem-loop structure in histone primary transcripts that begin appearing as cells enter S phase. Importantly, the temporal association of SLBP with p220 NPAT foci differs fundamentally between hES cells and somatic cells. The differences in subnuclear organization of p220 NPAT/Lsm10 foci, Cajal bodies, and SLBP between hES and somatic cells parallel known differences in the duration of their G 1 phases. |
Discussion In this study, we used IF microscopy to show that p220 NPAT foci associate with the 2 major human histone gene clusters at 6p22 and 1q21, as well as the histone transcript processing components Lsm10, Lsm11, and SLBP in both somatic and embryonic stem cells. Our findings indicate that the factors supporting synthesis and processing of histone transcripts are architecturally organized to form unique subnuclear domains. Previous studies in somatic cells have demonstrated that Cajal bodies are enriched for U7 snRNP components ( 23) and are often in proximity to histone gene loci ( 14, 15, 26, 28). Consistent with these data, we found that Cajal bodies are sporadically associated with histone gene loci in hES cells. Foci of p220 NPAT and coilin exhibit limited overlap. In contrast, p220 NPAT foci consistently associate with the 2 major histone gene clusters on 6p22 and 1q21 in hES cells. Because our data show that p220 NPAT foci are distinct from Cajal bodies and PML/ND10 bodies, and they contain histone genes and mRNA processing factors, we designate these domains histone locus bodies (HLBs), a term originally proposed for similar domains in Drosophila ( 24, 25). Our findings support the concept that formation of p220 NPAT foci at histone gene loci, the recruitment of Lsm10 and SLBP, and the phosphorylation of p220 NPAT by CDK2 at HLBs are staged events that mediate histone gene expression. We have applied this staged spatial assembly of HLBs to define mechanistic differences between the cell cycles of hES cells and normal human diploid fibroblasts. In this and previous studies ( 26), we have shown that p220 NPAT, the defining resident protein of HLBs, rapidly forms detectable foci (≈1.5 h) following mitosis in hES cells. However, the assembly of these foci and their associated processing factors still occurs just ≈1–1.5 h before the G 1/S transition, reflecting the abbreviated G 1 phase (≈2.5–3 h) of hES cells ( 2). In contrast, results presented here and in previous studies ( 14, 15) for normal human somatic cells (with a G 1 phase of ≈8–12 h) establish that HLBs assemble at least 6 h before S phase. Thus, the striking differences in cell cycle times between somatic and ES cells are directly reflected by in situ differences in the spatiotemporal assembly of the histone gene expression machinery. HLBs contain CDK-phosphorylated p220NPAT immediately preceding and following the G1/S-phase transition in both somatic and embryonic stem cells. Hence, the shortened interval between formation of p220NPAT foci and their phosphorylation indicates that the mid-to-late G1 cell cycle stage is dramatically contracted in hES cells. In addition, because p220NPAT foci are detected only by mid G1 in somatic cells but appear rapidly following exit from mitosis in hES cells, the truncated G1 phase of hES cells may also eliminate events coupled with early G1. In conclusion, we show that both embryonic stem cells and somatic cells use a novel dedicated subnuclear domain (HLB) to coordinate synthesis and processing of histone gene transcripts. The temporal differences in the assembly of this domain between the 2 cell types provide insight into the abbreviated cell cycle of hES cells. |
Materials and Methods Cell Culture and Cell Synchronization. Human ES cells were cultured on a mitotically inactivated mouse embryonic fibroblast layer as described previously ( 1, 26). The cells were allowed to grow for 3–5 days after passaging before use. WI-38 cells were grown in complete medium containing MEM, penicillin–streptomycin, l-glutamine, sodium pyruvate, nonessential amino acids, and 10% FBS (Atlanta Biologicals). The hES cells were synchronized using Nocodazole (200 ng/mL) (Sigma) for 16 h, and samples were taken at different time points after release. WI-38 cells were synchronized by serum depletion. Cells were allowed to grow for 7 days without changing the media and were released by adding fresh media containing 20% serum. Samples were collected at different times after serum stimulation. Antibodies. The antibodies, their working dilutions, and their supplier are as follows: monoclonal p220 NPAT (mouse monoclonal; 1:1000; BD Biosciences), polyclonal p220 NPAT (rabbit polyclonal; 1:1000) ( 14, 15), coilin (pdelta; mouse monoclonal; 1:500; Santa Cruz Biotechnology), coilin (rabbit polyclonal; 1:500; ref. 29), PML (H-238; rabbit polyclonal; 1:250; Santa Cruz Biotechnology), PML (mouse monoclonal; PG-M3; 1:250; Santa Cruz Biotechnology), Lsm10 (mouse monoclonal; 1:500; Biomatrix Research), Lsm11 (rabbit polyclonal; 1:500), SLBP (rabbit polyclonal; 1:250), 3′ hExo (rabbit polyclonal; 1:200; refs. 22 and 30, and Z.D., X.Y., and W.F.M., unpublished data). Secondary antibodies were goat anti-mouse Alexa 488, goat anti-rabbit Alexa 488, goat anti-mouse Alexa 594, goat anti-rabbit Alexa 594, goat anti-mouse Alexa 350, goat anti-rabbit Alexa 350 (all 1:800 dilution in 1× PBSA) (0.5% bovine serum albumin (BSA) (Sigma) in 1× phosphate buffered saline (PBS)). For FISH secondary antibody we used anti-digoxigenin (anti-DIG) rhodamine [1:500 in 4× standard saline citrate (SSC)/1% BSA; Roche]. IF Microscopy. Human ES cells or WI-38 cells were grown on gelatin-coated coverslips. IF was carried out as described previously ( 26). Briefly, cells were fixed with 3.7% formaldehyde for 10 min, permeabilized by 0.25% Triton X-100 for 20 min, and then treated with primary antibody for 1 h at 37°C, followed by detection using appropriate fluorescent-tagged secondary antibody. The nuclei were counterstained with DAPI. FISH. Probes were made of BAC clones spanning a region at or very near to the histone gene loci on chromosome 6p22 (RP11-2p4) and 1q21 (CTD-2018M10) (Children's Hospital Oakland Research Institute, Oakland, CA). BAC DNA was purified by using Qiagen columns, and probe DNA was labeled using the DIG Nick translation kit (catalogue no. 11745816910; Roche Diagnostics) according to the manufacturer's protocol. The probe mixture was prepared by adding 50–100 ng probe DNA, 70% formamide, 10 μg human Cot-1 DNA, and 10 μg salmon sperm DNA. FISH was performed after hES cells or WI-38 cells were subjected to IF as described above. After IF, cells were fixed by passing through ethanol grades (70%, 85%, and 100%) and were briefly air dried. Cells were then codenatured with the probe mixture at 80°C for 8 min and allowed to hybridize overnight at 37°C in a moist chamber. Cells were washed by using 50% formamide/4× SSC for 15 min at 37°C, followed by washes in 2× SSC for 15 min at 37°C and 1× SSC for 15 min at room temperature. Probes were detected by incubation with appropriate secondary antibody, followed by 3 washes with 4× SSC, 0.1% Triton/4× SSC, and 4× SSC at room temperature with shaking. Some cells then were counterstained with DAPI and mounted in Prolong-Gold (Invitrogen). Cells were viewed under an epifluorescence Zeiss axioplan 2 microscope and images were captured using a Hamamatsu (C4742-95) charged coupled device (CCD) camera and analyzed by Metamorph imaging software (Universal Imaging). All images were captured at 100× magnification unless noted otherwise. |
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Acknowledgments. We thank the members of our laboratories, and specifically Matthew Mandeville, Shirwin Pockwinse, Jacqueline Akech, Hassan Mohammad, and Margaretha van der Deen, for stimulating discussions and/or the sharing of expertise and reagents. We also thank Judy Rask for assistance with manuscript preparation. This work was supported by National Institutes of Health Grant R01 GM032010 and a Human Embryonic Stem Cell Supplement. |
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