Correspondence to: Rudolf Jaenisch, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139. Tel:(617) 258-5186 Fax:(617) 258-6505 E-mail:jaenisch{at}wi.mit.edu.
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Abstract |
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Histone variant macroH2A1 (macroH2A1) contains an NH2-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins -tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.
Key Words: Xist, stem cells, chromatin, microtubule, dosage compensation
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Introduction |
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Recent interest has focused on the histone variant macro H2A1 because it is specifically enriched on inactive X chromosomes (Xi)1 of female mammalian cells. MacroH2A1 forms macrochromatin bodies (MCBs), which are discrete macroH2A1-containing accumulations that colocalize with inactive, but not active, X chromosomes (
Histone macroH2A1 has an unusual domain structure that may account for its nonuniform distribution. The NH2-terminal domain contains a region that is colinear with and 64% identical to a normal core H2A histone (
In the differentiated cells of female mammals, one of two X chromosomes is transcriptionally silenced so that the dosage of X-linked gene expression is similar to that in males. Dosage compensation occurs through an ordered process that insures that only a single X chromosome remains active for each diploid set of autosomes. Proper X inactivation requires the action of the Xist gene, which is expressed from the X-inactivation center (XIC) of the Xi (
Female ES cells provide a useful cell culture model for the X inactivation process because they undergo the X inactivation process upon differentiation (
Here, we report the unexpected finding that macro H2A1 exists in undifferentiated ES cells as prominent focal accumulations centered on centrosomes. This association is labile in the presence of the microtubule-disrupting drug nocodazole in a dose-dependent fashion. Upon differentiation, Xist RNA is first upregulated on the future Xi, and soon thereafter macroH2A1 is recruited to form MCBs. This process occurs in conjunction with a diminution of centrosomal-associated macroH2A1. Therefore, macroH2A1 localization exhibits a sequential shift from a centrosomal to an inactive X chromosomal location during the differentiation and X inactivation processes.
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Materials and Methods |
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Cell Culture
ES cells were grown without feeder cells in standard ES cell medium containing 1,000 U/ml leukemia inhibitory factor. Differentiation was induced by plating ES cells at low density in DME with 15% FCS without leukemia inhibitory factor in the presence of all-trans retinoic acid at a final concentration of 10-7 M as described previously (
Centrosome Preparation
Centrosomes were prepared by the method of
Immunofluorescence, Quantitative Centrosome Immunofluorescence, and Fluorescent In Situ Hybridization Methods
Immunofluorescence on whole ES cells was performed using standard methods. Antibodies specific for macroH2A1 and Skp1 were described previously (- and
-tubulins were purchased from Sigma-Aldrich (catalog nos T-6557 and T9026). Quantitative centrosome immunofluorescence (QCIF) was performed as described (
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Results |
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MacroH2A1 Accumulation Is Centered on Centrosomes of Undifferentiated ES Cells
The intracellular localization of macroH2A1 in undifferentiated ES cells was investigated with indirect immunofluorescence using an affinity-purified polyclonal antibody (-tubulin (an established marker for centrosomes (
-tubulin immunofluorescence) showed substantial concentrations of macroH2A1. The affinity-purified antibody used in these studies (
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Because centrosomes are the nucleation centers for microtubule formation, undifferentiated ES cells were treated with a high concentration of nocodazole (100 µg/ml for 1 h) to see if focal centers of macroH2A1 accumulation require intact microtubules. This treatment resulted in a dramatic disaggregation of centrosomal macroH2A1 (Fig 2), which caused macroH2A1 to assume a dispersed particulate distribution. This result indicated that intact microtubules are necessary for the retention of macro H2A1 at centrosomes in undifferentiated ES cells.
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Established methods for the purification of centrosomes (-tubulin immunofluorescence) that results from the presence of two centrioles within each centrosome (
-tubulin immunofluorescence were confirmed to be centrosomes by the colocalization of
-tubulin with Skp1 immunostaining (Fig 3 b). Skp1, like
-tubulin, is a confirmed component of centrosomes (
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The relative quantities of centrosomes in each sucrose fraction were determined with QCIF, (-tubulin and antimacroH2A1 antibodies. The relative numbers of centrosomes present in each fraction was determined by counting centrosomes on 10 randomly chosen 100x oil immersion fields (Fig 3 c). More centrosomes were isolated from cells pretreated with 10 µg/ml nocodazole, but a lower proportion of these centrosomes retained associated macroH2A1. Though the absolute yield of centrosomes from cells pretreated with 2 µg/ml nocodazole was slightly reduced, virtually all of these centrosomes contained associated macroH2A1. Samples of each sucrose fraction were subjected to immunoblotting using the macroH2A1 antibody (Fig 3 d). The distribution of macroH2A1 detected by immunoblotting of sucrose fractions corresponded well with the relative quantities of centrosomes as judged by QCIF. The absolute yield of macroH2A1 detected by Western analysis was greatly enhanced by the reduced pretreatment with nocodazole (Fig 3 d).
Recruitment of MacroH2A1 to the Inactive X Chromosome
We were surprised to find macroH2A1, a protein previously thought to associate only with chromatin, concentrated in a focal center around the centrosome, an organelle devoid of chromatin. We therefore carried out an analysis of macroH2A1 localization in female ES cells using retinoic acid-induced differentiation (-tubulin immunofluorescence), Xist RNA expression (detected by FISH) and macroH2A1 location (detected by immunofluorescence). We subjected such differentiating cultures to three independent tests:
-tubulin immunofluorescence combined with Xist FISH; macroH2A1 immunofluorescence combined with Xist FISH; and
-tubulin and macroH2A1 double immunofluorescence. These experiments revealed the timing of association of macro H2A1 with the Xi during the X inactivation process.
As expected, biallelic pinpoints of expression of Xist RNA were detected from both X chromosomes in undifferentiated female ES cells, a pattern of Xist expression indicative of a preinactivation state for X chromosomes (-tubulin staining) were spatially separate in these cells (Fig 4 a). MacroH2A1 accumulation was also spatially separate from the location of Xist RNA signals, but macroH2A1 immunostaining invariably overlapped the positions of centrosomes as visualized by
-tubulin immunofluorescence (Fig 4 a). We termed such undifferentiated cells class 1, characterized by centrosomal macro H2A1 and biallelic double pinpoint Xist signals.
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After three days of differentiation, the cells acquired a fibroblast-like morphology and no longer formed colonies like undifferentiated ES cells (data not shown). No class 1 cells were detected and most cells contained intense monoallelic Xist FISH signals indicative of X chromosomes undergoing the X inactivation process (Fig 4 b). Most cells contained robust Xist FISH signals that were distinct from centrosomes and lacked associated macro H2A1concentrations (Fig 4 b). Centrosomal macroH2A1 became increasingly difficult to detect, but when observed, centrosomal macroH2A1 immunostaining was markedly less intense as compared with that observed in undifferentiated ES cells and often occurred in close proximity to Xist signals. We termed differentiating cells that contain intense monoallelic Xist FISH signals that are devoid of macroH2A1 concentrations class 2.
At day 3 and later, we began to observe a significant proportion of cells that contained well defined Xist signals that had acquired concentrations of macroH2A1. These cells, therefore, contained bona fide MCBs as evidenced by a confirmed association of macroH2A1 with X chromosomes. We termed these cells class 3 (Fig 4 c).
Though all combinations of immunofluorescence and FISH using two antibodies (directed against macroH2A1 and -tubulin), and one FISH probe (directed against Xist RNA) were performed, class 1, 2, and 3 cells could be easily distinguished from one another by Xist FISH combined with macroH2A1 immunofluorescence. This allowed us to quantitate the relative proportions of cells in each of the three classes during the course of retinoic acid-induced differentiation (Fig 5). 200 cells were typed as class 1, 2, or 3 from female ES cells undergoing retinoic acid induced differentiation for 0, 3, 6, 9, and 12 d. Cells were ascertained by first looking for Xist expression (Cy3 channel), and then analyzed for their macroH2A1 distribution (FITC channel). The relative proportions of class 1, 2, and 3 cells were determined for each time point on slides whose identity was blinded, and after counting was complete, the results were plotted versus time in differentiation medium (Fig 5). The proportion of class 3 cells increased at the expense of class 2 cells during the differentiation process.
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We wished to investigate whether or not class 3 cells resembled differentiated female somatic cells. Mouse embryonic fibroblasts (MEFs) from female E13.5 mouse embryos were analyzed for their content of macroH2A1, Xist RNA, and -tubulin. Female MEFs resembled class 3 differentiated female ES cells as judged by the presence of MCBs characterized by colocalized Xist RNA and macro H2A1 staining (Fig 6). The positions of MCBs were independent of the positions of centrosomes, similar to late class 3 cells. MCBs also occurred in fibroblasts derived from adult female mouse ears and in a mouse mammary carcinoma cell line called X3 with a stable XXX karyotype (
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Discussion |
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Centrosomal Centers of MacroH2A1 Accumulation
Our results detail the unexpected finding that macroH2A1 accumulates around centrosomes in undifferentiated ES cells. This is surprising since chromatin, the usual site of histone accumulation, is not thought to be directly associated with centrosomes. MacroH2A1 is an unusual histone, however, and the outstanding feature of macroH2A1 is the presence of a large COOH-terminal extension: the nonhistone domain. This domain may be responsible for the unusual distribution of macroH2A1. The nonhistone domain could include a site for centrosome docking. Alternatively, the nonhistone domain might contain sites for microtubule attachment. If so, microtubule-associated motor proteins might be responsible for accumulation and/or retention of macroH2A1 at the centrosome.
Several lines of evidence support the conclusion that macroH2A1 is truly associated with centrosomes in undifferentiated ES cells. The affinity-purified antibody used in these studies to detect macroH2A1 is highly specific for macroH2A1 as judged by Western analysis (Fig 1 c). We detected macroH2A1 (42 kD) by Western blotting in fractions highly enriched for purified centrosomes (Fig 3 d). The distribution of macroH2A1 in these fractions agreed well with the distribution of centrosomes in these fractions. Western blotting showed that the yield of macro H2A1 in fractions containing centrosomes was greatly enhanced at a lower level of nocodazole pretreatment (Fig 3 d). This finding parallels the nocodazole lability of the macroH2A1 centrosomal signal observed by immunofluorescence (Fig 2). MacroH2A1 cosedimented with Skp1 and -tubulin, both established components of centrosomes (Fig 3, a, b, c). Centrosomal staining of macroH2A1 diminished and eventually disappeared upon differentiation, whereas the signal of macroH2A1 at Xi increased.
Numerous studies have shown that centrosomes are the major cellular centers for nucleation of microtubules. The effect of nocodazole on the retention of macroH2A1 suggests a mechanism for the retention of macroH2A1 at centrosomes. Nocodazole is a specific inhibitor of microtubule polymerization. Since the centrosomal accumulation of macroH2A1 is labile in the presence of nocodazole, it seems reasonable that it is retained at centrosomes by virtue of microtubular associations. The amount of macro H2A1 present at centrosomes responds to nocodazole in a dose-dependent fashion and the degree of nocodazole pretreatment had a profound effect on the amount of macroH2A1 detected around purified centrosomes by immunofluorescence (Fig 2 and Fig 3). A reduced treatment with nocodazole allowed us to purify centrosomes that retained an extensive network of associated macroH2A1 (Fig 3 a). Centrosomes with extensive arrays of macro H2A1 exhibited reduced mobility during velocity gradient centrifugation in sucrose. Under these pretreatment conditions, virtually 100% of purified centrosomes retained associated macroH2A1. In contrast, centrosomes isolated from cells pretreated with 10 µg/ml nocodazole had reduced levels of associated macroH2A1 (Fig 3), and many compact centrosomes in fractions 3 and 4 were stripped of detectable macroH2A1 (Fig 3 c). These results strongly suggest that intact microtubules are necessary for the retention of macroH2A1 at centrosomes.
Possible Involvement of Centrosomes in the X Inactivation Process
We can envision two possible explanations for the finding that macroH2A1 resides at centrosomes in undifferentiated ES cells. It is possible that macroH2A1 has some as yet unidentified function at the centrosome, per se. Alternatively, the centrosome may represent a storage site for macroH2A1 in undifferentiated ES cells. We favor the second possibility for a number of reasons. Our results show that macroH2A1 accumulated as MCBs that are associated with Xist RNA signals upon differentiation and concomitant X inactivation. This process occurred at the expense of centrosomal macroH2A1. Therefore, if macro H2A1 has a direct role in centrosome function, this role must be restricted to the undifferentiated state. It seems more likely that macroH2A1 is stored at centrosomes before its incorporation into core nucleosomes of the inactive X. We can envision two potential mechanisms for transfer of macroH2A1 from centrosomes to Xi: macro H2A1 might be transported to the X chromosome during interphase by a novel mechanism; or macroH2A1 might transfer to X chromosomes via microtubules during mitosis when the nuclear membrane is disassembled and microtubules directly connect centrosomes to chromosomes. This second mechanism would not preclude a role for cytoplasmic microtubules in the transport or concentration of macroH2A1 at centrosomes. Future investigations of such potential mechanisms may yield insights into the processes by which chromatin components are targeted to the nucleus. In addition, our results suggest a novel role for centrosomes in nuclear organization.
The timing of acquisition of macroH2A1 by inactivating X chromosomes gives clues as to the possible function of macroH2A1 in the X inactivation process. X inactivation is complete in somatic female cells such as fibroblasts. In such cells macroH2A1 is present as MCBs that colocalize with the inactive X, but not the active X chromosome (
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Footnotes |
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1 Abbreviations used in this paper: ES cell, embryonic stem cell; FISH, fluorescent in situ hybridization; MCB, macrochromatin body; MEF, mouse embryonic fibroblast; QCIF, quantitative centrosome immunofluorescence; Xi, inactive X chromosomes.
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Acknowledgements |
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We wish to thank Peter Jackson for providing Skp1 antibodies and Cathrin Brisken for X3 cells. We thank Anton Wutz, Gyorgyi Csankovzski, and Joost Gribnau for critical readings of this work. This work was conducted using the W.M. Keck Foundation Biological Imaging Facility at the Whitehead Institute.
T. Rasmussen was supported by the National Institutes of Health fellowship GM19510. A. Eden was supported by the European Molecular Biology Organization fellowship ALTF 43-1999. R. Jaenisch was supported by National Institutes of Health/National Cancer Institute grant 5-R35-CA44339.
Submitted: 9 February 2000
Revised: 18 July 2000
Accepted: 18 July 2000
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References |
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