Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells
© Sharma et al; licensee BioMed Central Ltd. 2012
Received: 25 August 2011
Accepted: 27 January 2012
Published: 27 January 2012
DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear.
Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation.
Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.
DNA methylation is an essential epigenetic gene silencing mechanism which interplays with histone modifications and nucleosome occupancy for regulation of tissue-specific gene expression patterns and chromatin architecture in mammalian cells . DNA methylation patterns are established during embryogenesis and then faithfully maintained in differentiated tissues, enabling preservation of cellular identity through multiple somatic divisions. Failure in proper maintenance of methylation patterns can result in development of disease states such as cancer [2, 3]. Thus, it is essential to faithfully maintain DNA methylation patterns in differentiated cells through somatic divisions.
In mammals, the de novo DNA methytransferases, DNMT3A/3B, primarily establish the methylation patterns during embryonic development  and later maintain them in differentiated tissues through cooperation with the maintenenace DNA methyltransferase, DNMT1 [5–7]. Recent studies by our group and others have revealed distinct maintenance mechanisms used by these enzymes for preservation of methylation patterns in somatic cells [8, 9]. While DNMT1 transiently interacts with the chromatin and primarily performs its maintenance activity in association with the replication fork, DNMT3A/3B remain preferentially bound to nucleosomes in chromatin regions containing methylated repeats and CpG islands, which stabilizes these proteins and assists in faithful propagation of DNA methylation within the methylated domains through cooperative activity of DNMT3A/3B and DNMT1 enzymes [9–11]. However, the mechanisms responsible for preferential targeting of DNMT3A/3B to such methylated regions are still poorly understood.
In embryonic stem (ES) cells, targeting of de novo DNMT3A/3B enzymes to specific chromatin regions involves interactions with auxiliary factors in addition to their direct interactions with the nucleosomes [12, 13]. DNMT3A/3B interact with various chromatin-associated proteins including heterochromatin protein 1 (HP1), histone deacetylase 1 (HDAC1), UHRF1 and histone methyltransferases such as EZH2, suggested to play a role in recruitment of DNMT3A/3B to specific chromatin regions for de novo methylation in ES cells [9, 13, 14]. However, recent studies suggest that these auxiliary proteins are not required for interaction of DNMT3A/3B with the nucleosomes in somatic cells suggesting existence of other mechanisms involved in association of DNMT3A/3B with chromatin .
H3K9 methylation has also been established to interplay with DNA methylation for gene silencing in cells. In neurospora crassa, H3K9 methylation directs DNA methylation to transposable elements [15, 16]. A similar link between H3K9 methylation and DNA methylation is present in mammals where heterochromatic H3K9 methyltransferase, Suv39h1, has been shown to direct DNA methylation to major satellite repeats at pericentric heterochromatin . Recently, G9a, a euchromatic H3K9 methyltransferase, has also been found to direct DNA methylation to H3K9 methylated regions in ES cells . G9a, along with its partner protein GLP, is crucial for H3K9 (mainly H3K9me and H3K9me2) methylation of euchromatin and is involved in transcriptional silencing [19, 20]. G9a binds to its own product, H3K9me and H3K9me2 residues, through its ankyrin domain, a mechanism suggested to play a role in propagation of H3K9 methylation through cell divisions [21, 22]. Recent studies have shown that G9a physically interacts with Dnmt3a/3b and recruits them to G9a-target gene promoters, retrotransposons and major satellite repeats for de novo methylation in ES cells , independent of its histone methyltransferase activity . While an essential role of G9a in directing de novo DNA methylation in ES cells has been established, whether it plays a similar role in maintenance of DNA methylation patterns in somatic cells remains unclear.
Here we show that G9a is not required for anchoring of DNMT3A/3B to nucleosomes in methylated chromatin regions and for maintenance of DNA methylation in somatic cells. Using sucrose gradient chromatin fractionation analysis, we show that G9a strongly binds to both mononucleosomes and polynucleosomes, similar to DNMT3A/3B. However, knockdown of G9a in somatic cells does not decrease binding of DNMT3A/3B to nucleosomes and no discernible reduction in DNA methylation levels of G9a-associated target genomic regions occurs even in the absence of G9a, suggesting no role of G9a in maintenance of DNA methylation. However, G9a-knockdown renders cancer cells more sensitive to 5-Aza-CdR (5-aza-2'-deoxycytidine) treatment resulting in increased DNA hypomethylation and cell growth inhibition, indicating that G9a might be involved in re-initiation of de novo methylation and thus could serve as a promising target for combinatorial cancer treatment strategies involving DNA hypomethylating drugs.
G9a strongly associates with polynucleosomes similar to DNMT3A/3B
G9a tightly binds to intact mononucleosomes
To further explore whether these enzymes require intact nucleosomal structures for their association with chromatin, we performed the sucrose gradient analysis on the mononucleosomes treated with ethidium bromide (EtBr) which disrupts the nucleosomal structure by intercalating into DNA but does not interfere with the protein-protein interactions [17, 28–30]. Mononucleosomes were incubated with EtBr prior to loading onto sucrose gradients containing 300 mM NaCl (Figure 2B). The distribution of G9a changed dramatically, similar to DNMT3A/3B, upon disruption of nucleosomal structure by EtBr with the enzymes now sedimenting mainly in factions 3 to 5, which do not contain measureable histone components of the nucleosome. These data show that G9a and DNMT3A/3B enzymes require intact nucleosomal structures for their association with chromatin.
G9a is not essential for maintenance of DNA methylation in somatic cells
DNMT3A/3B do not require G9a for anchoring to nucleosomes
G9a knockdown synergizes with 5-Aza-CdR treatment resulting in increased DNA hypomethylation and inhibition of cell growth
We have previously shown that the majority of DNMT3A/3B strongly anchor to nucleosomes containing methylated DNA in somatic cells. Such binding, which requires the presence of DNA methylation, further stabilizes these proteins [10, 11]. Based on these data, we proposed a revised inheritance model for DNA methylation where DNMT3A/3B remain associated with methylated chromatin domains i.e. in association with their product 5-methylcytosine enabling proper maintenance of DNA methylated states through somatic divisions in cooperation with DNMT1 . This model is similar to proposed inheritance models for histone marks where the histone methyltransferases remain associated with the chromatin domains containing their own mark enabling faithful maintenance [21, 26, 27]. The strong binding of DNMT3A/3B and H3K9 methyltransferases, G9a and SUV39h1, to nucleosomes observed in our sucrose density gradient experiments strongly supports the existence of such proposed inheritance models. Whether a common link exists between these two similar inheritance models for H3K9 methylation and DNA methylation is still unclear. H3K9 methylation and DNA methylation patterns are found to be highly coincident in mammalian cells [14, 43, 44] suggesting the possibility of interplay between these two epigenetic machineries. Indeed recent studies revealed such a link between these two mechanisms in ES cells where G9a was shown to direct DNA methylation to certain loci in ES cells . However, the presence of a similar role of G9a in directing maintenance of DNA methylation in somatic cells is still inconclusive.
Here we have shown that unlike in ES cells, G9a is not essential for propagation of DNA methylation in somatic cells since depletion of G9a in somatic cells did not impair maintenance of DNA methylation at G9a-target loci. Our data are in agreement with some other recent studies indicating that while G9a is essential for de novo methylation, it is dispensable for maintenance of DNA methylation [31, 36]. In addition, our data provide insights into possible mechanisms responsible for such G9a-independent maintenance of DNA methylation in somatic cells. We have shown that DNMT3A/3B remain bound to methylated chromatin regions even in the absence of G9a indicating that these enzymes do not require G9a for their presence at silent methylated domains and they can faithfully maintain DNA methylation in somatic cells independent of G9a.
Based on previous studies in ES cells and our current data, we propose that G9a primarily plays a role in establishment of DNA methylation at target loci in ES cells by recruiting DNMT3A/3B for de novo methylation at such regions. G9a may act in concert with DNMT3L, a regulatory factor expressed only in ES cells which stimulates DNMT3A/3B activities , in the de novo methylation process. However, once the methylation patterns have been established, DNMT3A/3B remain stably associated with nucleosomes in methylated chromatin regions in somatic cells, independent of G9a, and faithfully propagate DNA methylation in such domains in cooperation with DNMT1 through multiple somatic divisions. One possible reason for such differential effects of G9a on DNA methylation observed in ES cells compared to somatic cells might be the greater level of 'epigenetic plasticity' in the ES cells. ES cells require a plastic epigenome since they need to reprogram themselves along specific lineages . In contrast, differentiated somatic cells possess a more restricted chromatin structure which they need to faithfully maintain to preserve their cellular identity through somatic divisions . Therefore, in somatic cells, DNMT3A/3B enzymes remain stably associated with the previously methylated chromatin domains ensuring faithful maintenance of methylated states without causing any aberrant de novo methylation . Interestingly, we also found that the presence/absence of DNA methylation does not affect association of G9a, which binds to H3K9me and H3K9me2 residues, with nucleosomes. While DNMT3A/3B require DNA methylation for their binding to nucleosomes , our current data suggest that impaired DNA methylation does not affect G9a's association with H3K9 methylated chromatin regions and distinct mechanisms are involved in localization of these enzymes in somatic cells.
Our data also show that while G9a is not required for maintenance of DNA methylation in differentiated cells, simultaneous targeting of G9a and DNMTs results in increased inhibition of cell proliferation and greater DNA hypomethylation. The striking increase in inhibition of cell proliferation observed in our experiments highlights the strong potential of combinatorial cancer treatment strategies targeting repressive histone methylation and DNA methylation machineries together. The pronounced cell growth inhibition observed upon simultaneous knockdown of G9a along with treatment with 5-Aza-CdR might be a result of widespread changes in DNA methylation and accompanying changes in histone modifications . Our data also show that such combinatorial targeting approach further increases the efficacy of the DNA hypomethylating drugs in reducing levels of DNA methylation. The increase in DNA hypomethylation might arise from a possible role of G9a in re-initiating DNA methylation at hypomethylated sites after 5-Aza-CdR treatment. G9a might direct DNA methylation at such hypomethylated sites through recruitment of de novo DNMT3A/3B enzymes, similar to its role in ES cells . Inhibition of G9a protein observed during 5-Aza-CdR treatment further supports a role of simultaneous inhibition of DNMTs and G9a in drug-induced DNA hypomethylation . It is also possible that the increased DNA hypomethylation observed upon 5-Aza-CdR treatment of G9a knockdown cells might have arisen from an alteration in the cell growth rate leading to differential drug incorporation in DNA. 5-Aza-CdR has been previously shown to result in severe hypomethylation of rapidly dividing cells . However, 5-Aza-CdR treated G9a-kd cells displayed a reduced growth rate compared to treated G9a WT cells ruling out such a possibility.
Taken together, our data suggest that G9a is not required for preferential targeting of DNMT3A/3B to silent methylated domains and faithful maintenance of DNA methylation in differentiated somatic cells. However, G9a might serve as a potential target for combinatorial cancer treatment strategies involving DNMTs inhibitors to achieve greater drug-induced DNA hypomethylation and anti-proliferation effects.
HCT116 and 293T cells were maintained in McCoy's 5A and DMEM, respectively, containing 10% inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Puromycin was included in the culture medium at 3 μg/ml to maintain infected cells. 293T cells expressing different ΔDNMT3B isoforms were prepared as described previously . For 5-Aza-CdR treatment experiments, cells were cultured in the presence of 0.3 μM 5-Aza-CdR for 24 h. After 24 h, the drug containing media was replaced with fresh media and cells were kept in culture till the mentioned time points. Cell counts were made using Coulter Counter (Beckman Coulter, Brea, CA).
Nuclei were prepared according to the procedure described previously . Briefly, cells were trypsinized and washed once with PBS. The cells were then resuspended in ice-cold RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing protease inhibitors and kept on ice for 10 min before Dounce homogenization in the presence of 0.5% to 1% NP-40 to break up cell membranes. Nuclei were washed twice with RSB plus the protease inhibitors (Roche) without the detergent. Nuclear extracts were prepared by resuspending nuclei in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS) followed by sonication.
MNase digestion and sucrose density gradient centrifugation
Sucrose density gradient experiments were performed as described previously . Purified nuclei (1x108) resuspended in 1 ml of RSB containing 0.25 M sucrose, 3 mM CaCl2, and 100 μM PMSF, were digested with 36 units of MNase (Worthington) for partial and 500 units of MNase (Worthington) for extensive digestion for 15 min at 37°C, and then the reaction was stopped with EDTA/EGTA (up to 10 mM). After microcentrifugation at 5,000 rpm for 5 min, the nuclear pellet was resuspended in 0.65 ml of the elution buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl) containing 5 mM EDTA/EGTA, gently rocked for 1 hr at 4°C followed by microcentrifugation to obtain soluble nucleosomes. A quantity of 0.55 ml of soluble nucleosome containing buffer was fractionated through a sucrose density gradient solution (5% to 25% sucrose, 10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA) containing NaCl of indicated concentrations, by centrifuging at 30,000 rpm for 16 h at 4°C. Fractions were taken from the top of the centrifuge tube to 16 aliquots. Proteins from same volume of each fraction (200 to 250 μl) were concentrated by TCA precipitation and subjected to western blot analysis. The EtBr treatment of the mononucleosome samples was done by adding 20 mg/ml EtBr to the samples (300 μg/ml at final), followed by incubation at room temperature for 10 min before loading onto the gradient.
The lentivirus particles containing N/S, shG9a5 and shG9a7 shRNA sequences were prepared using standard protocols. For lentivirus production, the vesicular stomatitis virus envelope protein G expression construct pMD.G1, the packaging vector pCMV ΔR8.91 and the transfer vector pHRCMVpuroSin8 were used as described previously . Short hairpin RNA sequences encoding non-specific and sequences targeting G9a were as follows: N/S 5'- AAAACTGCAGAAAAAGGGTAGGTTCGACTAGCAGGACTCTTCTCTTGAA
AGAGTCTTGCTAGTTGAACCTACCCGGTGTTTCGTCCTTTCCACAAG-3'; shG9a5 5'- AAAACTGCAGAAAAAGACAGCAAGTCTGAAGTTGAAGCTCTCTCT
TGAAGAGCTTTAACTTCATACTTGCTGTCGGTGTTTCGTCCTTTCCACAAG -3' and shG9a7 5'-AAAACTGCAGAAAAAGGATGAATCTGAGAATCTTGAGGG
CAAG -3'. Infected HCT116 cells were selected in the presence of 3 μg/ml puromycin for two weeks.
Western blot analysis
Proteins from the same volume of each fraction (200 to 250 μl) were concentrated by TCA precipitation, dissolved in SDS/β-mercaptoethanol loading buffer, and resolved on a 4% to 15% gradient SDS/PAGE gel (Bio-Rad, Hercules, CA). Antibodies against H3 (ab1791), DNMT3A (ab2850), and SUV39h1 (ab12405) were purchased from Abcam Inc. (Cambridge, UK); DNMT1 (sc-20701) and DNMT3B (sc-10235) from Santa Cruz Biotech (Santa Cruz, CA); Myc epitope tag (05-724) from Upstate (now Millipore, Billerica, MA) and G9a (G 6919); β-Actin (A 5316) from Sigma (Saint Louis, MO). The image of individual proteins was visualized using ECL detection system (Thermo Scientific, Waltham, MA and Millipore, Billerica, MA).
DNA methylation analysis
Ms-SNuPE assay was performed as described previously [52, 53]. Genomic DNA was prepared from HCT116 cells infected with either N/S, shG9a5, or shG9a7 constructs 14 days after lentiviral infection. To analyze the methylation status of individual DNA molecules, we cloned bisulfite PCR fragments of the loci of interest into the pCR2.1 vector using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA). Primer sequences are available on request. Individual colonies were screened for the insert and the region of interest was sequenced using M13 primers. DNA methylation levels were estimated by calculating the percentage of CpGs remaining methylated from the total number of CpGs assayed among all individual cells.
SS, Ph.D; DSG, graduate student; HFH, graduate student, SJ, Ph.D (Research Assistant Professor); MRS, Ph.D (Professor and Department Chairman); PAJ, Ph.D (Professor and Director of Norris Comprehensive Cancer Center); GL, Ph.D and MD (Associate Professor of Research).
This work was supported by National Institute of Health Grants (RO1 CA124518 to GL, R37 CA082422 to PAJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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