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Previous Article | Next Article 
Molecular and Cellular Biology, July 2008, p. 4459-4468, Vol. 28, No. 14
0270-7306/08/$08.00+0 doi:10.1128/MCB.00410-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, California 90033
Received 11 March 2008/ Returned for modification 16 April 2008/ Accepted 1 May 2008
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent reports demonstrate that multiprotein complexes that modify histones and those that bind histone modifications play critical roles in developmental pathways, in part, by regulating the expression of genes involved in lineage specification (3, 20, 33). One well-defined example of this is the Drosophila ESC-E(Z) complex, which contains a nucleosome-specific histone methyltransferase, E(z), which specifically trimethylates histone H3 lysine 27 (H3K27) (5, 17, 23). Trimethylated H3K27 binds the chromodomain of Polycomb, a component of the PRC1 repressive complex (9, 22). This binding event and the subsequent repression of the homeotic (Hox) gene cluster represent a critical step in establishing positional identity within a developing embryo (28).
Similar to E(z), we recently codiscovered the nucleosome-specific PR-Set7 histone H4 lysine 20 (H4K20) methyltransferase, also known as Set8 or KMT5A, which is enriched within transcriptionally repressed chromatin (1, 8, 25). PR-Set7 is the only known enzyme that specifically monomethylates H4K20 in mammals; the Suv4-20 enzymes are responsible for the bulk of H4K20 di- and trimethylation (6, 35, 50). Recent structural findings demonstrated that the tandem malignant brain tumor (MBT) repeats of the L3MBTL1 repressor protein bind several mono- and dimethylated histone lysine residues in vitro (15, 18, 21). These studies also showed that L3MBTL1 preferentially binds monomethylated H4K20, a histone modification associated with repressed chromatin, strongly suggesting that they cooperate to regulate gene expression. Consistent with this, it was found that the binding of L3MBTL1 to monomethylated H4K20 creates a transcriptionally nonpermissive chromatin structure in vitro and that L3MBTL1 negatively regulates the expression of a subset of E2F target genes (44). However, unlike for Polycomb-mediated repression, the biological importance of this gene regulation pathway remains undetermined.
In this study, we extend our previous findings regarding a trans-tail histone code involving monomethylated H4K20 and H3K9 (36). We discovered that both modifications are dependent upon PR-Set7 and that the modifications are targeted to the same genomic regions. Importantly, the recruitment of L3MBTL1 to these regions is dependent upon PR-Set7-mediated H4K20 monomethylation and this event is required to induce gene repression. One of the genes that we discovered that is regulated by this pathway is RUNX1, a key regulator of hematopoietic differentiation (12). We demonstrate that the lack of monomethylated H4K20 in the human K562 multipotent cell line induces the spontaneous differentiation of these cells to megakaryocytes, in part, by activating RUNX1. Our findings indicate that this silencing pathway is required to prevent megakaryopoiesis of K562 cells and suggest that it may function similarly in other multipotent cell types to regulate differentiation.
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MATERIALS AND METHODS |
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Cell culture and transfections. HeLa, HEK293, and K562 cells were cultured as previously described (49). Six million HeLa cells per 10-cm plate were grown for 24 h before transfection using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 1 day posttransfection, cells were selected with 6 µg/ml puromycin for 4 days before harvesting for chromatin immunoprecipitation (ChIP), Western analysis, or quantitative reverse transcription-PCR (qRT-PCR). For siRNA, 105 HeLa cells were plated per well in six-well plates for transfection of RNA duplexes by use of Oligofectamine (Invitrogen) as previously described (14). Two million K562 cells were incubated with 9 µg of the indicated plasmids and 1 µg FLAG-GFP in buffer (100 mM HEPES, pH 7.4, 10 µg DEAE-dextran, Opti-MEM medium [Invitrogen]) prior to electroporation at 1,000 µF and 150 V. Cells were incubated at 37°C for 10 min before adding RPMI 1640-10% fetal bovine serum to 2 ml and equally divided into each well of a six-well plate. Forty-eight hours later, cells were pelleted, resuspended in medium with 3 µg puromycin, and grown for 5 days. Cells were washed and resuspended in RPMI 1640-10% fetal bovine serum supplemented with 0.01% dimethyl sulfoxide containing 0.1 nM, 1 nM, 10 nM, or 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 days before collecting cells for fluorescence-activated cell sorting (FACS) and Western analysis.
Western analysis. Western analysis was performed as previously described (36), using the following antibodies and dilutions: H4K20 methyl-specific antibodies (LP Bio) (36), H3K9 methyl-specific antibodies (Millipore) (31), H4 general (1:60,000; Abcam), FLAG M2 (1:2,500; Sigma), H3 general (1:500,000; Abcam), RUNX1 (1:500; Abcam), G9a (1:1,000; Millipore), PR-Set7 (1:1,000) (32), and L3MBTL1 (1:1,000; LP Biologicals); horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (1:5,000; Jackson Immunologicals) were used prior to the addition of enhanced chemiluminescence plus reagent (GE). The results were visualized by exposure to film for 1 min.
Differentiation and sorting. K562 cells were differentiated using 10 nM TPA (Sigma) for 5 days or 50 µM hemin (Chemika) for 4 days. Megakaryocytic differentiation was analyzed by flow cytometry using anti-CD41-fluorescein isothiocyanate (eBiosciences) or anti-CD41-phycoerythrin (MoFlo; Cytomation) antibody. Erythrocyte differentiation was analyzed by benzidine staining and/or measurement of soluble hemoglobin as previously described (10, 47).
ChIPs. Cells were treated with 1% formaldehyde for 8 min before adding glycine (final concentration, 125 mM) to quench the reaction. Cells were pelleted and nuclei were extracted in NEB buffer {5 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], pH 8, 85 mM KCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml each of aprotinin, leupeptin, and pepstatin}, pelleted, and resuspended in lysis buffer (50 mM Tris, pH 8, 10 mM EDTA, 1% sodium dodecyl sulfate) to give a final concentration of 108 cells/ml. Soluble chromatin was sheared to <1 kb by sonication at 4°C for 20 seconds at 40% output (Misonix) and diluted in ice-cold ChIP dilution buffer (150 mM NaCl, 16.7 mM Tris, pH 7.5, 3.3 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate) to give a final concentration of 25,000 cells/µl (final volume, 200 µl) before incubation overnight at 4°C with the following antibodies: H4K20 mono- and trimethyl (4 µl and 13 µl, respectively), H3K9 monomethyl (9 µl), hemagglutinin (HA) (1 µg; Roche), myc (1 µg; Roche), H3 general (1 µg), and L3MBTL1 (20 µl). Protein A beads (GE) were incubated with the immunoprecipitated material for 1 h at 4°C, followed by a series of washes (two with ChIP dilution buffer, four with radioimmunoprecipitation assay-LiCl buffer [50 mM HEPES, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 0.5 M LiCl], and two with TE [10 mM Tris, 1 mM EDTA]), before elution of the bound DNA by use of the Chelex method as previously described (24). PCRs were performed using increasing amounts of the eluted DNA template (0.15%, 0.5%, and 1.5%) at 30 cycles for all primer sets (see Table S1 in the supplemental material).
Expression analysis.
Total RNA was extracted from HeLa and K562 cells by using an RNeasy miniprep kit (Qiagen). RT reactions were performed using a reverse transcriptase kit (Applied Biosystems). Ten nanograms of the resultant cDNA was used for either semiquantitative or quantitative real-time PCRs. Quantitative PCRs were performed in quadruplicate, and change values were calculated using the 2–
CT method and plotted relative to the values for mock-treated cells, normalized to 1.
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RESULTS |
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PR-Set7 monomethyltransferase activity is required to repress RUNX1. High-throughput genome-wide technologies were employed to identify specific genes regulated by PR-Set7 and monomethylated H4K20 (data not shown). One of the identified target genes was AML1/RUNX1, a master regulator of hematopoietic differentiation and a gene commonly translocated in leukemias (16). qRT-PCR was performed on the PR-Set7 shRNA and control shRNA HeLa cells to confirm these findings (Fig. 2A). As predicted, there was a >2-fold increase in RUNX1 expression in the absence of PR-Set7 and monomethylated H4K20. RUNX1 expression was also analyzed for HeLa cells transfected with the PR-Set7 CD plasmid to determine if the increase in expression was directly correlated with a loss of monomethylated H4K20. As we had previously observed with several other genes enriched in monomethylated H4K20 (data not shown), the absence of this histone modification resulted in a >3-fold increase in RUNX1 expression. Concomitant with an increase in RUNX1 mRNA levels, we also observed a significant increase in RUNX1 protein levels in the PR-Set7 shRNA cells (Fig. 2B). These findings strongly suggest that the monomethylation of H4K20 by PR-Set7 plays a key upstream regulatory role in RUNX1 expression. Furthermore, these findings suggest that the presences of PR-Set7 protein itself and monomethylated H3K9 are not sufficient for RUNX1 repression.
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12-fold enrichment of monomethylated H4K20 at the RUNX1 promoter compared to the levels in the upstream region in the HeLa cells (null). Based on the findings described above, we predicted that monomethylated H3K9 would also be specifically targeted to this region. ChIPs were performed with an H3K9 monomethyl-specific antibody, and visual inspection of the resultant PCR amplifications revealed an enrichment of monomethylated H3K9 at the RUNX1 promoter compared to the levels in the upstream region (Fig. 2C). Quantitative analysis confirmed an 5-fold increase of monomethylated H3K9 at the RUNX1 promoter, providing further evidence of the selective targeting of this trans-tail histone code.
To further verify these findings, ChIPs were performed with HeLa cells transfected with the PR-Set7 shRNA plasmid that depletes cells of global levels of monomethylated H4K20 (Fig. 1B). Visual inspection of the PCR amplifications from the PR-Set7 shRNA cells revealed a significant reduction in monomethylated H4K20 at the RUNX1 promoter compared to the levels in the null cells (Fig. 2C). Quantitative analysis indicated an 10-fold decrease in monomethylated H4K20 at the RUNX1 promoter, similar to the levels observed at the upstream region (Fig. 2D). ChIP analysis performed with the monomethyl-specific H3K9 antibody in the PR-Set7 shRNA cells also revealed an 12-fold reduction of this modification at the RUNX1 promoter compared to the levels in the null cells, similar to the levels observed at the upstream region. These findings indicate that both H4K20 and H3K9 monomethylations at the RUNX1 promoter are dependent on PR-Set7.
Additional ChIP analysis was performed with HeLa cells transfected with the PR-Set7 CD plasmid, which serves as a dominant negative by reducing monomethylated H4K20 in the cells without decreasing PR-Set7 levels (Fig. 1C). As predicted, visualization of the PCRs demonstrates a marked reduction in monomethylated H4K20 at the RUNX1 promoter compared to the levels in the null cells (Fig. 2C). Quantitation of the samples confirmed the reduction; however, the levels of monomethylated H4K20 at the RUNX1 promoter were consistently higher than those in the PR-Set7 shRNA cells (Fig. 2D). This is most likely due to the inherent competition between wild-type and mutant PR-Set7 proteins in the transfected cells, resulting in the observed residual monomethylation of H4K20 at the RUNX1 promoter. Consistent with the findings described above, ChIP analysis demonstrated no observable change in monomethylated H3K9 at the RUNX1 promoter in the PR-Set7 CD cells compared to the levels in the null cells (Fig. 2C and D). These findings provide further evidence that both histone modifications are dependent upon the targeting of PR-Set7 to specific regions in the genome. It is also important to note that the continued presence of monomethylated H3K9 at the RUNX1 promoter in the PR-Set7 CD cells does not inhibit RUNX1 derepression compared to that in the PR-Set7 shRNA cells, providing further evidence that monomethylated H3K9 is not sufficient for RUNX1 repression (Fig. 2A).
Decreased monomethylated H4K20 results in reduced L3MBTL1 at RUNX1. It was previously reported that the MBT repeats of the L3MBTL1 repressor protein selectively bind monomethylated H4K20 in vitro (15, 18, 21). Due to this association, we theorized that L3MBTL1 was directly binding monomethylated H4K20 in vivo to repress RUNX1 expression. Consistent with this theory, depletion of L3MBTL1 by RNA interference resulted in a >4-fold increase in RUNX1 expression, similar to the increase observed in the absence of monomethylated H4K20, indicating a role for L3MBTL1 in regulating RUNX1 (Fig. 2A). Based on these findings, we predicted that L3MBTL1 was targeted specifically to the RUNX1 promoter. To test this, ChIP analysis using an L3MBTL1 antibody was performed with HeLa (null) cells. Visualization and quantitation of the PCR amplifications confirmed a significant enrichment of L3MBTL1 at the RUNX1 promoter compared to the levels in the upstream region (Fig. 2C and D). Importantly, the depletion of PR-Set7 and monomethylated H4K20 in the PR-Set7 shRNA HeLa cells resulted in a dramatic reduction of L3MBTL1 at the RUNX1 promoter compared to the levels in the null cells, similar to the levels observed in the upstream region. The loss of L3MBTL1 from the promoter was coincident with the derepression of RUNX1 (Fig. 2A). ChIP analysis with the PR-Set7 CD cells revealed a marked reduction of L3MBTL1 enrichment at the RUNX1 promoter compared to the levels in the null cells (Fig. 2C and D). However, the decreased levels of L3MBTL1 did not reach those observed in the PR-Set7 shRNA cells, most likely due to persistence of low levels of monomethylated H4K20 caused by the competition between the mutant and endogenous wild-type PR-Set7 proteins. Collectively, these findings indicate that the presence of L3MBTL1 at the RUNX1 promoter is associated with RUNX1 repression and that the recruitment of L3MBTL1 to this region is most likely due to its interaction with monomethylated H4K20.
Monomethylated H4K20 is required for L3MBTL1 recruitment to repress RUNX1. To verify that PR-Set7 was targeted to the RUNX1 promoter, a myc-tagged eukaryotic expression vector encoding full-length PR-Set7 was transfected into HeLa cells for ChIP analysis using myc antibodies, since a reliable ChIP-grade PR-Set7 antibody is not yet available. While we anticipated that overexpression of the PR-Set7 monomethyltransferase would result in global increases in monomethylated H4K20, Western analysis demonstrated a significant reduction of this histone modification compared to the levels in the myc-null cells (Fig. 3A). This reduction was proportional to a dramatic global increase in trimethylated H4K20, but with no observable changes in dimethylated H4K20. ChIP analysis was performed with various antibodies at the RUNX1 promoter in HeLa cells transfected with the myc-PR-Set7 plasmid or the myc-null control plasmid (Fig. 3B). ChIPs performed with the myc antibody demonstrated an enrichment of PR-Set7 at the RUNX1 promoter compared to the levels in the myc-null cells, confirming that PR-Set7 is targeted to the RUNX1 promoter (Fig. 3B). Consistent with the Western analysis, ChIPs with cells overexpressing myc-PR-Set7 demonstrated a significant reduction in monomethylated H4K20 at the RUNX1 promoter and a concomitant increase in trimethylated H4K20. No observable changes in monomethylated H3K9 were detected at the RUNX1 promoter. Since these cells were cotransfected with an HA-tagged eukaryotic expression plasmid encoding full-length L3MBTL1, ChIP analysis was performed using an HA antibody to assess its enrichment at the RUNX1 promoter in these different backgrounds. The results demonstrate that HA-L3MBTL1 was selectively reduced in the myc-PR-Set7 cells containing decreased monomethylated H4K20 and increased trimethylated H4K20, but no change in monomethylated H3K9, at the RUNX1 promoter compared to the levels in the myc-null cells. Importantly, the reductions of monomethylated H4K20 and L3MBTL1 at the RUNX1 promoter in the myc-PR-Set7 cells are coincident with increased RUNX1 expression (Fig. 2A). Collectively, these findings indicate that PR-Set7 and H4K20 monomethylations are targeted to the RUNX1 promoter and strongly suggest that RUNX1 repression is mediated by the specific binding of L3MBTL1 to monomethylated H4K20.
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Loss of monomethylated H4K20 and L3MBTL1 at the RUNX1 promoter is an early event of megakaryopoiesis.
Based on the findings described above, we predicted that the components of this new silencing pathway would be absent at the RUNX1 promoter, specifically in the TPA-treated K562 cells, compared to the levels in vehicle control or hemin-treated cells. ChIP analysis was performed with the different heterogeneous cell backgrounds, where 42% of hemin-treated cells were positively stained for benzidine and 33% of the TPA-treated cells were CD41 positive (see Fig. S3 in the supplemental material). Visualization of the subsequent PCR amplifications confirmed the enrichment of monomethylated H4K20, monomethylated H3K9, and L3MBTL1 at the RUNX1 promoter in both the vehicle control K562 cells and those treated with hemin compared to the levels in the RUNX1 upstream region (Fig. 5A). As predicted, there were marked reductions in monomethylated H4K20 and L3MBTL1 at the RUNX1 promoter in the TPA-treated K562 cell population. In contrast to the global analysis, there was an observable decrease in monomethylated H3K9 at the RUNX1 promoter. Quantitation of the PCR products from three independent biological replicates, as described above, confirmed these observations (Fig. 5B). Therefore, the loss of monomethylated H4K20 and L3MBTL1 at the RUNX1 promoter is associated with increased RUNX1 and the selective differentiation toward the megakaryocytic lineage.
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Catalytically active PR-Set7 is required to prevent megakaryopoiesis.
It was previously reported that the expression of RUNX1 sensitizes K562 cells to megakaryocytic differentiation upon TPA treatment (7). Therefore, our findings predicted that the depletion of monomethylated H4K20 and the associated activation of RUNX1 would have a similar sensitizing effect. To test this hypothesis, K562 cells were first cotransfected with a plasmid that constitutively expresses GFP and either a FLAG-tagged RUNX1 plasmid or an empty FLAG-null plasmid as the negative control prior to treatment with TPA. FACS analysis was performed with these different cells at 5 days posttransfection to determine the number of CD41-positive cells within the GFP-positive cell population (see Fig. S4 in the supplemental material). Consistent with the previous report, we observed an 50% increase in the number of CD41-positive cells in the FLAG-RUNX1 TPA-treated cells compared to the levels in the control (see Fig. S5 in the supplemental material). Similar experiments were performed using the FLAG-null plasmid or the FLAG-PR-Set7 CD plasmid that acts as a dominant negative by depleting cells of monomethylated H4K20 without reducing levels of PR-Set7. Similar to the results in HeLa cells, Western analysis confirmed the specific decrease of monomethylated H4K20 and increased RUNX1 in the FLAG-PR-Set7 CD cells compared to the levels in the control cells (Fig. 6A). These cells were treated with a vehicle control or increasing amounts of TPA, and FACS analysis was performed as described above. As predicted, we observed a dose-dependent increase in the number of CD41 positive cells in the FLAG-null cells with increasing amounts of TPA (Fig. 6B). In contrast, the FLAG-PR-Set7 CD cells displayed a >50% increase in the number of CD41 positive cells compared to the levels in the FLAG-null control cells when treated with only the vehicle. Importantly, the number of CD41-positive cells did not increase with increasing amounts of TPA in the FLAG-PR-Set7 CD cells, strongly suggesting that they had already achieved the maximal differentiation potential. Collectively, these findings indicate that the absence of catalytically active PR-Set7 and monomethylated H4K20 results in the spontaneous megakaryocytic differentiation of K562 cells.
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DISCUSSION |
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10-fold excess of wild-type or CD PR-Set7 compared to endogenous PR-Set7 (data not shown) did not result in visible increases in global levels of monomethylated H3K9. While there are several possible explanations for these observations, they cannot be properly tested until the H3K9 monomethyltransferase has been identified. Besides interacting with an H3K9 methyltransferase, an alternate possibility is that PR-Set7 interacts with an unknown H3K9 di-/tridemethylase to achieve monomethylated H3K9 both on a global scale and at target loci. Both possibilities are currently under investigation. We report here, for the first time, that monomethylated H4K20, monomethylated H3K9, and the L3MBTL1 repressor protein converge at specific genomic regions in vivo and that they function cooperatively to repress transcription. Our findings strongly suggest that the interaction between monomethylated H4K20 and L3MBTL1 is essential for the observed repressive effect as the depletion of either results in the derepression of RUNX1 in different cell lines. It is possible that L3MBTL1 is recruited to these regions by an interaction with PR-Set7; however, we have been unable to detect such an interaction by coimmunoprecipitation (see Fig. S6 in the supplemental material). While the expression of myc-PR-Set7 led to an increase in PR-Set7 at the RUNX1 promoter, we consistently detected decreases in both monomethylated H4K20 and L3MBTL1 at this region, further suggesting that PR-Set7 and L3MBTL1 do not interact in vivo. The residual presence of L3MBTL1 in these experiments could be due to the persistence of low levels of monomethylated H4K20 at the RUNX1 promoter. However, recent in vitro binding studies indicate that the MBT repeats of L3MBTL1 can also bind monomethylated H3K9, and since monomethylated H3K9 remains present at the RUNX1 promoter in these experiments, these observations suggest that this modification may play a role in recruiting or stabilizing L3MBTL1 to this region (18). Consistent with this, in the PR-Set7 CD HeLa cells, where monomethylated H4K20 is dramatically reduced, we observed residual enrichment of monomethylated H3K9 and L3MBTL1 at the RUNX1 promoter, again suggesting a possible in vivo interaction between them. However, the continued presence of monomethylated H3K9 at the RUNX1 promoter was not sufficient to repress RUNX1 transcription, suggesting that this modification does not play a direct role in gene repression. It is possible that monomethylated H3K9 and the unidentified methyltransferase are important in other aspects of this pathway, but this will remain unclear until the methyltransferase is discovered. These findings also indirectly reinforce that the interaction between monomethylated H4K20 and L3MBTL1 appears to be the critical step for repression.
PR-Set7 and monomethylated H4K20 were originally identified as being associated with repressed chromatin, and our findings here and those of another study indicate that PR-Set7 and monomethylated H4K20 clearly participate in a gene repression pathway by recruiting and binding the L3MBTL1 repressor protein in vivo (8, 25, 44). However, several recent reports document an association of monomethylated H4K20 with actively transcribed genes (2, 40, 46). One possible explanation for these apparent differences is that this histone modification could participate in both transcriptional activation and repression pathways, depending on the specific gene, similar to what has been observed for di- and trimethylated H3K9 (45). This could be achieved by the recruitment of distinct H4K20 monomethyl-binding regulatory proteins that could activate or repress transcription (30). However, a more likely possibility is that the underlying function of this pathway is to fine-tune the dosage of the corresponding gene rather than completely ablating its transcription. This theory is consistent with both observations: that monomethylated H4K20 is found within several active genes and that the depletion of this modification consistently results in the increased expression of these genes (data not shown). Further studies are required to validate this hypothesis.
RUNX1 is an important transcription factor that controls the expression of a variety of lineage-specific genes and, therefore, itself must be tightly regulated to prevent premature differentiation (12). In this report, we demonstrated that in the multipotent K562 cells the newly described repressive trans-tail histone code operates at RUNX1 to control its expression and, therefore, megakaryocytic differentiation. We discovered that the loss of PR-Set7 and monomethylated H4K20 was an early event in differentiation, occurring prior to DNA ploidy and cell morphology changes, indicating that this repressive pathway participates in regulating megakaryopoiesis. Contrary to our earlier observations, global levels of monomethylated H3K9 were retained in the TPA-treated K562 cells despite the overall reduction of PR-Set7, although monomethylated H3K9 was reduced at the RUNX1 promoter. It is interesting to note that hemin-treated cells also displayed reduced levels of PR-Set7 but retained both monomethylated H4K20 and H3K9. Taken together, these observations imply that these histone modifications are relatively stable in the two separate differentiation pathways, even in the absence of PR-Set7, with the exception of monomethylated H4K20 in the TPA-treated cells. They also imply that specific reduction of monomethylated H4K20 during megakaryopoiesis is a defining mechanistic feature of this pathway compared to erythropoiesis. It remains unclear how the selective reduction of this histone modification is achieved, although it is tempting to speculate that there is an unknown H4K20 demethylase that operates specifically in the precursor cells to initiate megakaryocytic differentiation, in part, by activating RUNX1. We demonstrated that the ectopic expression of RUNX1 sensitized K562 cells to the differentiation effects of TPA but itself was not sufficient to induce megakaryopoiesis (see Fig. S5 in the supplemental material). Surprisingly, in cells lacking monomethylated H4K20, TPA was not required to induce megakaryopoiesis; the cells tended to spontaneously differentiate. While RUNX1 is likely to be an important component of this effect, we hypothesize that other unidentified genes that contribute to megakaryopoiesis are also regulated by this repressive pathway and that the lack of monomethylated H4K20 results in their activation, culminating in megakaryocytic differentiation. It is important to note that the differentiation-associated changes in monomethylated H4K20 are not restricted to hematopoiesis. A previous report demonstrated that global levels of monomethylated H4K20 were highest in mouse neuroblasts and myoblasts and that these levels were significantly reduced during their differentiation, similar to what we observed during megakaryopoiesis (4). Collectively, these findings strongly suggest that this repressive pathway plays a fundamental role in developmental programs by preserving a multipotent phenotype via repression of certain lineage-specific genes. Consistent with this theory, a recent study demonstrated that homozygous PR-Set7 knockout mice were embryonic lethal, suggesting a role for PR-Set7 and monomethylated H4K20 in mammalian embryogenesis (11). Similarly, Drosophila organisms lacking PR-Set7 fail to progress past the third instar, again demonstrating a critical role for PR-Set7 and monomethylated H4K20 in development (8, 25). The identification of the genes regulated by this repressive pathway will provide critical insights into the complex biological problem of development and differentiation.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Pew Charitable Trusts and from the NIH to J.C.R. (GM075094). J.K.S. was partially supported by a predoctoral traineeship from the NIH (T32 GM067587).
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FOOTNOTES |
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Published ahead of print on 12 May 2008.
Supplemental material for this article may be found at http://mcb.asm.org/.
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-G9a, anti-G9a. (B) HeLa cells were transfected with two different PR-Set7-specific shRNA constructs, and Western analysis was performed to detect global changes in the methylated states of H4K20 and H3K9 compared to the levels in the empty vector (null). (C) Similar Western analysis was performed with HEK293 cells transfected with a FLAG-tagged control plasmid (null) or a dominant-negative PR-Set7 R256G CD point mutant (PR-Set7 CD) that depletes cells of monomethylated H4K20.
