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Nucleic Acids Res. Feb 2011; 39(3): 874–888.
Published online Oct 4, 2010. doi:  10.1093/nar/gkq870
PMCID: PMC3035464

DNA methyltransferase 3b preferentially associates with condensed chromatin

Abstract

In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a ‘reader’ of higher-order chromatin structure leading to gene silencing through DNA methylation.

INTRODUCTION

Methylation of cytosine C5 of CpG dinucleotides is a characteristic DNA modification in many eukaryotic genomes that plays an important role in gene silencing (1,2). In mammals, DNA methylation patterns change drastically during early development, and the establishment and maintenance of proper genomic DNA methylation is required for normal development (3–5). In somatic cells, 70–80% of all CpG dinucleotides are methylated (6). This epigenetic gene regulation plays an important role in X chromosome inactivation, genomic imprinting and stable silencing of retrotransposons (7–9). Covalent modification at the C5 position is mediated by DNA methyltransferases (Dnmts). Three active Dnmt genes, Dnmt1, Dnmt3a and Dnmt3b, have been identified in mammals (10,11). All three genes are essential for normal mouse development (12,13). However, it is not known how each Dnmt selects target CpG sites in the genome. Gene silencing in eukaryotes is mediated by a variety of epigenetic phenomena in addition to DNA methylation, including histone modification, chromatin remodeling and non-coding RNAs, and there is extensive cross-talk between these mechanisms (14–17). Histones function as structural components for packaging DNA in the nucleus but are also key regulators of gene expression (18). Various post-translational histone modifications, including acetylation, methylation, phosphorylation, ubiquitination and sumoylation, and other histone variants serve as functional codes that dictate the recruitment of protein complexes or change higher-order chromatin structure to achieve gene activation or inactivation (19–21). Histone H3 lysine 9 (H3K9) is trimethylated in heterochromatin, and mutation of the histone H3K9-specific methyltransferase causes a loss of genomic DNA methylation in Neurospora crassa (22,23), Arabidopsis thaliana (24) and mouse cells (25), which suggests that DNA methylation acts downstream from histone H3K9 methylation. In human cells, DNMT3B physically associates with the histone H3K27-specific methyltransferase Enhancer of Zest homolog 2 (EZH2), a member of the Polycomb group of proteins, and targets specific genes depending on its histone methyltransferase activity (26). In addition, recent data indicate that Dnmt3a is recruited to symmetric methylation of histone H4 arginine 3 mediated by the histone arginine methyltransferase PRMT5 (27). Conversely, Dnmt3L, which lacks conserved residues known to be involved in Dnmt activity (28) but interacts with Dnmt3 proteins to stimulate DNA methylation activity (29–31), binds to the N-terminus of histone H3, and this binding is inhibited by methylation of histone H3K4 (32). Crystallographic and biochemical studies revealed the specific interaction of the ATRX-DNMT3-DNMT3L (ADD) domain of Dnmt3L and DNMT3A with unmethylated lysine 4 in the amino-terminal tail of histone H3 (32–34). Accumulating evidence suggests that there is extensive crosstalk between DNA methylation and histone modification. Aside from histone modifications, disruption of histone H1 genes affects DNA methylation in A. thaliana (35) and mouse ES cells (36), suggesting a functional link between DNA methylation and linker histone H1-dependent higher-order chromatin structure. Biochemical studies revealed that Dnmts interact with chromatin (32,37,38), however, it remains to be determined whether Dnmt itself recognizes specific histone modifications or higher-order chromatin structure. The current study characterized the association of two de novo methyltransferase, Dnmt3a2 and Dnmt3b, with nuclear chromatin in ES cells and reconstituted chromatin templates. In the nucleus, Dnmt3b, but not Dnmt3a2, preferentially associated with histone H1-containing chromatin without any significant enrichment of silent chromatin-specific modifications. We demonstrated the preferential interaction of Dnmt3b with nucleosomal DNA rather than naked DNA. In contrast to Dnmt3b, Dnmt3a2 bound weakly to all substrates regardless of DNA structure. The incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b, while histone acetylation reduced Dnmt3b binding in vitro. Consistent with these observations, the NAD + -dependent histone deacetylase SirtT1, the mammalian homolog of yeast Sir2, which specifically deacetylates histone H4 lysine 16 (H4K16) (39,40), was enriched in Dnmt3b-associated chromatin in ES cell nuclei. These results provide molecular evidence that Dnmts differentially interact with higher-order chromatin. We propose that Dnmt3b specifically recognizes higher-order chromatin structures to induce DNA methylation of condensed chromatin.

MATERIALS AND METHODS

Cell culture and in vitro differentiation

Undifferentiated ES cells (ht7) were maintained in DMEM, as described earlier (41). To induce differentiation, ES cells were cultivated in suspension without leukemia inhibitory factor (LIF) to form embryoid body (EB) cells. Five days after induction, EB cells were plated in tissue culture dishes and cultured for an additional 5 days.

Preparation of nuclei

ES cell nuclei were prepared as described earlier, with some modifications (42,43). Briefly, ES cells were resuspended in Nuclei Isolation Buffer (NIB) containing 10 mM Tris–HCl (pH 7.5), 60 mM KCl, 15 mM NaCl, 1.5 mM MgCl2, 1 mM CaCl2, 0.25 M sucrose, 10% (v/v) glycerol, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and EDTA-free protease inhibitor cocktail (Roche). Cells were resuspended in NIB containing 0.1% (v/v) Nonidet P-40 (NP40), allowed to swell for 10 min, and then homogenized with a Dounce homogenizer on ice. The nuclei were pelleted by centrifugation at 500g for 10 min at 4°C to remove the soluble protein (cytoplasmic fraction), washed with NIB, and then resuspended in the same buffer.

Cell fractionation

Cell fractionation was performed as described earlier with some modifications (44). After collecting the cytoplasmic fraction, as described above, nuclei were suspended in NIB, and DNA was measured by UV absorbance at 260 nm in saturated 5 M NaCl, 8 M Urea buffer (20 OD260 units corresponded to 1 mg/ml DNA) (43). The nuclear pellets were diluted to 1.5 mg/ml DNA in NIB. The nuclei were treated with 0.5 U/µl (333 U) of RNase-free DNaseI (Sigma) for 15 min at 37°C. Ammonium sulfate was added to a final concentration of 0.25 M and the samples were incubated for 10 min at 25°C. The solubilized chromatin (chromatin fraction) was collected by centrifugation at 5000g for 10 min at 4°C. The pellets were extracted again with 2 M NaCl in NIB and then incubated for 5 min at 4°C (2 M NaCl wash fraction). To remove the remaining DNA and histones, the samples were centrifuged at 5000g for 5 min at 4°C. The remaining pellets, which contained the nuclear matrix (nuclear matrix fraction), was solubilized in 8 M urea containing 0.1 M NaH2PO4, 10 mM Tris–HCl (pH 8.0), EDTA-free protease inhibitor cocktail and 0.1 mM PMSF.

Chromatin fractionation

Micrococcal nuclease (MNase) digestion was performed as described earlier, with some modifications (42,45,46). Briefly, the nuclei were isolated as described above. The nuclear pellets were suspended in NIB at a concentration of 1.5 mg/ml DNA, pre-incubated for 10 min at 30°C and treated with increasing amounts of MNase (5, 20 or 80 U/mg DNA, Worthington) for 10 min at 30°C. After incubation, the nuclei were rapidly cooled on ice for 10 min and then subjected to centrifugation at 12 800g for 10 min at 4°C. The supernatant (S1 fraction) was collected and the pellets were resuspended in 2 mM EDTA, and then incubated for 10 min on ice. Suspended pellets were subjected to centrifugation at 12 800g for 10 min at 4°C. The supernatant was collected (S2 fraction) and the pellets were resuspended in 2 mM EDTA (P fraction). The amount of DNA in each fraction was measured using Pico Green (Invitrogen), according to the manufacturer’s instructions. Equivalent amounts of DNA (600 ng) were separated by SDS–PAGE and analyzed by immunoblot. For DNA analysis, aliquots from each fraction were treated with 20 µg/ml of RNase A for 1 h at 37°C and then 40 µg/ml of proteinase K overnight at 50°C. The DNA was extracted twice with a solution of phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. The DNA was analyzed by 1.5% agarose gel electrophoresis in 1× TAE and visualized with SYBR-Green I (Invitrogen).

Establishment of a FLAG-Dnmt3b-expressing cell line

The full-length cDNA of Dnmt3b with FLAG sequences fused to the N-terminus was cloned into the pCAGIpuro expression vector (kindly provided by Dr H. Niwa, RIKEN Japan) to construct pCAG-FLAG-Dnmt3b-IRES-puro. To generate ES cells that stably expressed FLAG-tagged Dnmt3b, the expression vector was introduced into parental ES cells (ht7) by electroporation (Bio-Rad).

Purification of Dnmt3b-associated proteins and chromatin

Nuclear extracts (S2 fractions) were prepared from FLAG-Dnmt3b-expressing cells by incubating the cells on ice with nuclei extraction buffer (NEB) [10 mM Tris–HCl (pH 7.5), 2 mM EDTA, 10% (v/v) glycerol, 0.1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche)] containing 400 mM NaCl (NEB400). Extracted nuclear pellets were subjected to centrifugation at 12 800g for 10 min at 4°C. The supernatant was collected (S2 fraction), and a one-third volume of NEB400 containing 0.3% (v/v) NP40 was added. The nuclear extract was incubated with pre-equilibrated FLAG-M2 agarose (Sigma) for 4 h at 4°C with rotation. The immunoprecipitates were washed three times with NEB400 containing 0.1% (v/v) NP40 for 10 min at 4°C with rotation. After washing, FLAG-Dnmt3b immunoprecipitates were eluted with 0.4 mg/ml FLAG-peptide in NEB400 containing 0.1% NP40 for 1 h at 4°C with rotation. The purified proteins were precipitated with acetone and separated on SDS–polyacrylamide gels. Protein bands stained with silver were excised from the gel, in-gel digested with trypsin and analyzed by LC–MS/MS.

To identify the histone modification specificity of Dnmt3b, nuclear extraction and immunoprecipitation were carried out in NEB containing 75 mM NaCl (NEB75) and NEB75 with 0.1% (v/v) NP40, respectively, as described above. For analysis of histone acetylation, nuclear extraction and immunoprecipitation were carried out in NEB containing 65 mM NaCl and 10 mM sodium butyrate (Na butyrate, NEB65B) and NEB65B with 0.1% (v/v) NP40, respectively, as described above. FLAG-M2 agarose was collected and the immunoprecipitates were extracted with Laemmli’s sample buffer (47). SDS–PAGE was performed under standard conditions and the separated proteins were transferred to the nitrocellulose membrane (GE Healthcare). Membranes were probed with primary antibodies as describe below. Blots were then probed with HRP-conjugated secondary antibodies or probed with Alexa 633 anti-rabbit IgG (1:1000; Invitrogen A-21071). Dnmt3b-specific bound histone modification was determined by quantifying the intensity of the bands using a Typhoon PhosphorImager (GE Healthcare) and ImageQuant software (Molecular Dynamics).

Chromatin isolation and sucrose gradient sedimentation

ES cell nuclei were treated with MNase (20 U/mg DNA) and the S2 fraction was extracted at 4°C with nuclei extraction buffer (NEB75), as described above. The S2 fraction was applied to a 10–30% (w/v) sucrose gradient containing NEB75 and centrifuged for 3 h at 50 000 rpm at 4°C in a SW55 rotor (Beckman). After sedimentation, equal volumes of each sample were recovered from the top. Equivalent volumes from each fraction were separated by SDS–PAGE and analyzed by immunoblot. For DNA analysis, fractionated samples were extracted with phenol-chloroform-isoamyl alcohol and precipitated as described above.

Expression and purification of GST-Dnmt3a2 and GST-Dnmt3b

Full-length Dnmt3a2 and Dnmt3b cDNAs were introduced into pGEX-6P1 (GE Healthcare). Escherichia coli BL21 (DE3) were transformed with expression vectors and cultured as previously described with some modifications (48). Harvested bacteria were sonicated in buffer [PBS containing 330 mM NaCl, 0.1% (w/v) Triton X-100, 1 mM DTT and EDTA-free protease inhibitor cocktail (Roche)], and then centrifuged at 12000g for 10 min at 4°C. The supernatant was collected and filtered through a 0.45-µm Millex GV filter (Millipore). The filtered supernatant was loaded onto a GSTrap HP column (GE Healthcare) and eluted with 10 mM glutathione (reduced form) in sonication buffer. The eluted fractions were further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare) pre-equilibrated with gel filtration buffer G [10 mM Tris–HCl (pH 7.5), 1 mM DTT, 1 mg/ml antipain, 1 mg/ml aprotinin, 0.4 mg/ml pepstatin] plus 330 mM KCl. The main fractions were collected and dialyzed against buffer G containing 200 mM KCl, 10% glycerol and 0.5 mM DTT for 2 h, followed by buffer G containing 50 mM KCl, 10% glycerol and 0.5 mM DTT for 2 h. GSTrap HP and HiLoad 16/60 Superdex 200 columns were connected to an ÄKTA Explorer 100 FPLC System (GE Healthcare).

Reconstitution of 12-mer nucleosomal arrays

Hypo- and hyperacetylated core histones were isolated from HeLa cells and treated without or with 10 mM Na butyrate for 24 h, respectively (43). DNA templates containing 1, 4 and 12 tandem repeats of a 208-bp ‘601’ sequence (49) were mixed with HeLa core histone proteins to reconstitute nucleosomes by salt dialysis, as described previously (43). The 208-bp 601 sequence consists of a strong nucleosome positioning sequence and contains 19 CpG sites. Core histone proteins were mixed with DNA in a 1:1 ratio in 2 M NaCl-10 mM Tris–HCl (pH 7.5), 1 mM EDTA (TE), followed by serial dialysis against 1 M NaCl-TE for 4 h, 0.75 M NaCl-TE for another 4 h, and finally TE overnight. The reconstitution quality was examined by Sca I digestion followed by 0.7% nucleoprotein agarose gel electrophoresis in 0.25× TBE, as described earlier (50). The gels were stained with SYBR Green I (Invitrogen) and analyzed using a FluorImager 595 (GE Healthcare).

Linker histone assembly with reconstituted nucleosomal arrays

Nucleosomal arrays were incubated with various amounts of histone H1, purified from HeLa cells, in binding buffer [10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 50 mM KCl and 1 µg/µl BSA] overnight on ice (43). Samples were mixed with an equal volume of loading solution [10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 50 mM KCl and 6% glycerol] and resolved by 0.7% nucleoprotein agarose gel electrophoresis in 0.25× TBE. To determine histone stoichiometry, histone H1-containing nucleosomal arrays were precipitated in binding buffer containing 10 mM MgCl2, as previously described, with some modification (51,52). Briefly, precipitated nucleosomal arrays were washed in binding buffer without BSA five times, and then resuspended in Laemmli’s sample buffer (47). Nucleosomal arrays were separated by SDS–PAGE and visualized by SYPRO-Orange (Invitrogen) staining. Histone stoichiometry was determined by quantifying the intensity of the bands using a FluorImager 595 (GE Healthcare) and ImageQuant software (Molecular Dynamics).

Micrococcal nuclease mapping

Reconstitited nucleosomal arrays (24.26 ng of DNA) in the absence or presence of histone H1 were digested with increasing amounts of MNase (0.141, 0.281, 0.562 and 1.125 U). Products of digestion were labeled with [γ- 32 P]ATP and analyzed by native PAGE as described earlier (53).

Sedimentation velocity analysis of the nucleosomal arrays

Sedimentation velocity experiments were performed using a Beckman XL-I analytical ultracentrifuge equipped with scanner optics, as previously described (54). The initial sample absorbance at 260 nm was between 0.6 and 0.8. Samples (360 µl) were equilibrated in TE (pH 7.5) containing 2.5 mM NaCl at 20°C for 30 min in the analytical ultracentrifuge before sedimentation at 15 000 rpm. Using the UltraScan data analysis program (version 7.4), boundaries were analyzed by the method of van Holde and Weischet (54). Sedimentation coefficients were corrected to S20,w using a partial specific volume of 0.622 ml/g for saturated chromatin arrays (50).

In vitro binding assay

Various lengths of naked 601 DNA or reconstituted chromatin assembled with or without histone H1 were incubated with purified GST-Dnmt3a2 or GST-Dnmt3b for 1 h at room temperature in binding buffer [10 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 5% glycerol, 0.25 mM DTT and EDTA-free protease inhibitor cocktail (Roche)]. After incubation, anti-GST antibody (MBL) was added to a final concentration of 0.025 mg/ml and the samples were incubated overnight at 4°C in 10 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1.25% glycerol, 0.0625 mM DTT and EDTA-free protease inhibitor cocktail. Pre-equilibrated Dynabeads Protein G (Dynal) was added, and the reaction mixture was incubated on ice for 1 h. Protein complexes were washed twice with 10 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM EDTA and EDTA-free protease inhibitor cocktail, and collected by magnetic separation according to the manufacturer’s instructions. Collected protein complexes were eluted in DNA loading buffer (0.1% SDS, 5% glycerol and 0.005% bromophenol blue) by incubation at 50°C for 20 min. The bound DNA was analyzed by 1.5% agarose gel electrophoresis in 1× TAE and visualized with SYBR-Green I (Invitrogen). The amount of bound DNA was measured by quantifying the intensity of the bands using a FluorImager 595 (GE Healthcare) and ImageQuant software (Molecular Dynamics). Immunoprecipitation was performed three or six times. For negative controls, immunoprecipitation was performed without GST-Dnmt proteins.

Immunoblot and dot blot analysis

Protein samples in Laemmli’s buffer (47) were resolved by SDS–PAGE and then transferred onto a PVDF or nitrocellulose membrane. For dot blot analysis, reconstituted chromatin arrays in 10 mM Tris–HCl (pH 7.5), 1 mM EDTA and 1 µg/µl BSA were spotted on a nitrocellulose membrane. Transferred or spotted membranes were probed with the following specific antibodies or anti-serum, as indicated: anti-Dnmt3b and anti-Dnmt3a (1:50 000; provided by Dr E. Li) (55), anti-Dnmt3L (1:1000; provided by Dr S. Yamanaka) (30), anti-Dnmt3a (1:250; Imgenex IMG-268), anti-SirT1 (1:1000; Abcam ab12193), anti-HDAC1 (1:1000; Upstate 06-720), anti-HDAC2 (1:2000; Sigma H3159), anti-HP1α (1:1000; MBL BMP 001), anti-HP1β (1:1000; Euromedex 1MOD-1A9-As), anti-HP1γ (1:1000; Euromedex 2MOD-1G6-As), anti-PCNA (1:1000; Upstate 05-347), anti-Brg-1 (1:500; Santa Cruz sc-10768), anti-Lamin B1 (1:200; MBL JM-3046-3), anti-Oct-3/4 (1:250; Santa Cruz sc-9081), anti-FLAG (1:2000; Sigma F3165), anti-β-actin (1:1000; Sigma A2228), anti-Histone H3 lysine 9 tri-methylation (H3K9me3) (1:500; Abcam ab1186), anti-H3K9me2 (1:1000; Upstate 07-212), anti-H3K9me1 (1:1000; Abcam ab9045), anti-H4K20me3 (1:500; Abcam ab9053), anti-H4K20me1 (1:1000; Abcam ab8895), anti-H3K27me3 (1:1000; Upstate 07-449), anti-H3K4me3 (1:1000; Abcam ab8580), anti-acetyl lysine 9 Histone H3 (H3K9Ac) (1:1000; Upstate 06-942), anti-acetyl-Histone H3 (1:1000; Upstate 06-599), anti-H4K16Ac (1:1000; Upstate 07-329), anti-acetyl-Histone H4 (1:1000; Upstate 06-598), anti-Histone H3 (1:8000; Abcam ab1791), anti-Histone H4 (1:1000; Abcam ab10158) and anti-Histone H1 (1:250; Abcam ab7789). Immunoreactive proteins were visualized using Chemi Lumi One (Nacarai tesque) or ECL Plus (GE Healthcare).

Immunofluorescence

ES cells were cultured on collagen-treated cover slips (Becton, Dickinson and Company). For analysis, EB3 cells were transferred onto collagen-treated cover slips and cultured for 4 h. Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Fixed cells were washed with PBS and then blocked in buffer (PBS containing 5 mg/ml BSA, 50 mM glycine, 2% goat serum and 0.1% sodium azide) for 30 min at room temperature. After blocking, cells were incubated overnight at 4°C with the following primary antibodies in blocking buffer, as indicated: anti-Dnmt3b (1:5000; provided by Dr E. Li) (55), anti-HP1α (1:500; Euromedex 2HP-1H5-As) and anti-Histone H1 (1:250; Abcam ab7789). After washing with PBS, cells were incubated with the following fluorescence-conjugated secondary antibodies in blocking buffer, as indicated: Alexa 546 anti-rabbit IgG (1:500; Invitrogen A-11035) and Alexa 488 anti-mouse IgG (1:500; Invitrogen A-11029). After washing with PBS, cells were counter-stained with 0.5 mg/ml of 4′,6′-diamidino-2-phenylindole (DAPI). After washing in PBS and sterilized ultra pure water, coverslips were mounted with ProLong Gold (Invitrogen). Cells were analyzed using a Radiance 2100 laser scanning confocal microscope system (Bio-Rad) equipped with an inverted Nikon Eclipse TE-2000 microscope (Nikon) or Nikon A1 laser scanning confocal microscope system (Nikon) equipped with an inverted Nikon Eclipse Ti (Nikon).

Statistical analysis

Data represent the means with standard deviation (SD). The Student’s t-test in the Stat View 5.0 software (SAS Institute Inc.) was used to calculate statistical significance.

RESULTS

Dnmt3b localizes to histone H1-enriched heterochromatin

To investigate the targeting mechanisms of de novo methyltransferases in the nucleus, we used ES cells, which express both Dnmt3a and Dnmt3b (55), and examined the expression of Dnmt3 proteins during ES cell differentiation. ES cells were cultured in suspension for 5 days in the absence of LIF to form EB, and then cells were plated in dishes for an additional 5 days to differentiate into primarily mesodermal cells. The expression of Oct3/4, a transcription factor essential for maintaining undifferentiated ES cells (56), decreased by 5 days after induction (Figure 1A) suggesting that differentiation had occurred. Dnmt3b and Dnmt3a2 were upregulated upon induction of ES cell differentiation, and then were gradually downregulated (Figure 1A). Dnmt3b and Dnmt3a2 were reduced in parallel with the reduction of Oct3/4 upon ES cell differentiation. In contrast to Dnmt3b and Dnmt3a2, Dnmt3a was expressed 8 days after induction. The amount of Dnmt3L gradually decreased as the ES cells differentiated. Since both Dnmt3b and Dnmt3a2 were highly expressed in early EB cells, Day 3 embryoid body (EB3) cells were used for further analysis of Dnmt3 proteins.

Figure 1.
Dnmt3b is enriched in histone H1-containing chromatin fractions. (A) Dnmt3b, Dnmt3a2, Dnmt3a, Dnmt3L and Oct3/4 expression during ES cell differentiation. Equal amounts of protein (10 µg) from undifferentiated ES cells (Day 0) and differentiated ...

The subcellular distribution of Dnmt3b in EB3 cells was analyzed using a sequential extraction procedure (44) (Figure 1B). Fractionation efficiency was confirmed by the presence of PCNA in the nucleoplasm fraction that was readily extracted by detergent treatment, core histones in the chromatin fraction, and lamin B1 in the nuclear matrix fraction (Figure 1B). As previously reported, most Dnmt3b, Dnmt3a2 and Dnmt3L were found in the chromatin fraction in EB3 cells (30,37,55,57).

To further investigate the nuclear distribution of Dnmt3b in EB3 cells, we carried out biochemical fractionation using MNase. Isolated nuclei were subjected to MNase treatment in low salt buffer to generate transcriptionally active and inactive chromatin fractions (45,46). Nuclease treatment caused the initial release of mono- and oligonucleosomes, which predominantly contained transcriptionally active chromatin (S1 fraction). After the release of the S1 fraction, the resulting pellets were subjected to hypotonic lysis to release soluble polynucleosomes (S2 fraction). The S2 fraction was enriched in histone H1 and predominantly contained transcriptionally inactive chromatin. The remaining pellets contained the nuclear matrix (P fraction) (Figure 1C). Equivalent amounts of DNA from each fraction were subjected to electrophoresis, and then fluoro staining. As shown in Figure 1D, the S1 fraction was enriched in mononucleosomes, whereas the S2 and P fractions contained polynucleosomes. Nuclear proteins from equivalent amounts of chromatin DNA were separated by SDS–PAGE and then analyzed by immunoblot. Brg1, an ATPase subunit of chromatin-remodeling complexes, and HP1α were enriched in the S1 and P fractions, respectively (42). Dnmt3b and Dnmt3L were enriched in the S2 fraction, whereas Dnmt3a2 was present at equivalent levels in the S1 and S2 fractions (Figure 1E). Neither Dnmt3b, nor Dnmt3a2 accumulated in the P fraction, which is the major HP1α-containing fraction. Thus, biochemical fractionation revealed that Dnmt3b associated predominantly with nuclease-insensitive transcriptionally inactive chromatin, while Dnmt3a2 was distributed throughout the genome, independent of DNA accessibility.

Dnmt3b localizes to chromatin regions with histone H1 but not HP1α

To investigate the mechanism by which Dnmt3b recognizes target genomic sites for methylation, Dnmt3b complexes were purified from ES cells. An ES cell line that stably expressed a FLAG-epitope fused to Dnmt3b (FLAG-tagged Dnmt3b) was established to facilitate purification. Stable ES transformants exhibited <2-fold higher level of Dnmt3b in the undifferentiated and differentiated states (Figure 2A). Endogenous Dnmt3b in EB3 cells localized in the nucleus without forming intensely labeled foci. Results showed that the localization of FLAG-tagged Dnmt3b overlapped with that of endogenous Dnmt3b (Figure 2B and C). Consistent with the biochemical fractionation analysis of Dnmt3b (Figure 1E), nuclear localization of Dnmt3b more closely resembled that of linker histone H1 rather than HP1α, since the latter formed intensely-labeled foci that overlapped with DAPI-stained heterochromatic foci in ES and EB3 cells (Figures 1E, E,2B,2B, B,2C2C and Supplementary Figure S1A and S1B).

Figure 2.
Establishment of a FLAG-Dnmt3b expressing cell line. (A) Expression of endogenous Dnmt3b and FLAG-Dnmt3b in undifferentiated (Undiff) and differentiated (EB3) ES cells. Total protein (4 µg) from parental wild-type ES cells (WT) and FLAG-Dnmt3b-expressing ...

Histone modifications in Dnmt3b associated chromatin

To further investigate the mechanism by which Dnmt3b recognizes target genomic sites for methylation, Dnmt3b-associated proteins were purified from FLAG-tagged Dnmt3b-expressing stable ES cells using immunoaffinity purification in 0.2 M NaCl (FLAG-IP). Nuclear extracts (S2 fraction, Figure 1E) were prepared in 0.4 M NaCl from MNase-treated FLAG-Dnmt3b-expressing ES cells. As a control, a mock purification from wild-type ES cells was performed and no associated proteins were detected by silver staining (Figure 3A). SDS–PAGE analysis of purified Dnmt3b complexes revealed that several polypeptides were specifically associated with FLAG-Dnmt3b. Through analysis of the associated polypeptides by LC/MS/MS, a 75 kDa Dnmt3b-associated protein, Dnmt3a2, was identified (Figure 3A). Six protein bands that corresponded to the sizes of core histones and histone H1 were also detected (Figure 3A). Consistent with a previous report (58), immunoblot analysis confirmed that Dnmt3b associated with Dnmt3a2 and with Dnmt3L even under 0.4 M NaCl extraction conditions (Figure 3B). HP1 proteins, which were not extracted in the S2 fraction under hypotonic conditions (Figure 1E), dissociated from chromatin under high salt conditions (37) and were present in the 0.4 M NaCl extracted S2 fractions. Interactions between Dnmt3b and HP1 proteins in 0.4 M NaCl extracted S2 fractions were not detected (Figure 3B).

Figure 3.
Dnmt3b stably associates with histone H1-containing chromatin in ES cells. (A) Silver staining of Dnmt3b-associated proteins purified from wild-type (Mock) or FLAG-Dnmt3b-expressing EB3 cells (FLAG-Dnmt3b). Dnmt3b-associated proteins were analyzed by ...

In mammals, the silent heterochromatic status is associated with hypoacetylation of histones and specific histone methylation (59–61). To determine whether Dnmt3b recognized specific histone modifications, FLAG-Dnmt3b-associated core histones in the S2 fraction containing 75 mM NaCl were subjected to immunoblot analysis using methylation-specific histone H3 antibodies. Non-specific precipitation of core histones during purification of Dnmt3b-associated chromatin was evaluated by a mock purification with parental wild-type ES cells (Supplementary Figure S2A). Immunoblot analysis using an antibody against the globular domain of histone H3 served as a loading control between bulk chromatin and Dnmt3b-associated chromatin (32,62). As previously observed in Dnmt3L-associated chromatin (32), H3K4me3 was reduced in immunopurified FLAG-Dnmt3b-associated chromatin (Figure 3C and Supplementary Figure S2B). H3K9me1, which is positively correlated with gene expression (59,63), was also slightly reduced in the Dnm3b-bound chromatin (Figure 3C). In contrast, little enrichment of silent chromatin-specific modifications, such as H3K9me3, H3K9me2, H3K27me3, H4K20me3 and H4K20me1 was detected (59,61) (Figure 3C and Supplementary Figure S2B). We further analyzed the effects of histone acetylation on Dnmt3b-binding in the S2 fraction prepared with sodium butyrate. As shown in Figure 3D, the level of H4K16Ac was relatively reduced in Dnmt3b-associated chromatin (Figure 3D). Acetylation of H4K16 induces decondensation of chromatin structures in vitro (64,65). These results suggested that Dnmt3b recognized condensed chromatin structures, rather than specific histone modifications.

Dnmt3b preferentially associates with nuclease-resistant higher-order chromatin in the nucleus

To characterize Dnmt3b-associated chromatin in ES cell nuclei, the interaction between Dnmt3b and chromatin was examined by treating nuclei with different concentrations of MNase. When ES cell nuclei were treated with 5 U/mg MNase, the chromatin was partially digested, whereas most chromatin was digested into mononucleosomes with 80 U/mg MNase (Figure 4A). After MNase treatment, nuclear extracts were subjected to immunoprecipitation using an anti-FLAG antibody to analyze the Dnmt3b–chromatin interaction. The interaction between Dnmt3b and chromatin was disrupted when ES cell nuclei were treated with 80 U/mg MNase (Figure 4B). Sucrose gradient centrifugation was used to further investigate Dnmt3b-associated chromatin. Soluble chromatin in the S2 fraction was subjected to 10–30% (w/v) sucrose gradient sedimentation under physiological salt conditions (75 mM NaCl), which maintains higher-order chromatin structures (66,67). Fractionated nucleosomal DNA was separated by 1.5% agarose gel electrophoresis and then visualized by fluoro staining as well as immunoblot analysis. Dnmt3b was enriched in histone H1-containing long chromatin fractions, and was not detected in shorter chromatin fractions (Figure 4C). These results suggested that Dnmt3b did not simply bind to DNA, but preferentially associated with higher-order chromatin.

Figure 4.
Dnmt3b associates with nuclease-resistant heterochromatin and the association requires higher-order chromatin structure. (A) DNA from nuclei digested with 5 or 80 U MNase/mg DNA. Equivalent amounts of DNA were separated by 1.5% agarose ...

Dnmt3b preferentially associates with higher-order reconstituted chromatin

To investigate the effects of chromatin structure on Dnmt3b binding, the binding affinity of Dnmt3b for differentially reconstituted nucleosomal DNA substrates was determined. Recombinant GST-Dnmt3a2 and GST-Dnmt3b fusion proteins were expressed in E. coli and purified (Figure 5A). We reconstituted mononucleosomes, 4-mer nucleosomes and 12-mer nucleosomal arrays on tandem 208 bp DNA repeats of the CpG rich 601 nucleosome positioning sequence (Figure 5B and Supplementary Figure S3) (49). Reconstituted chromatin or naked DNA was mixed with purified GST-Dnmt3b or GST-Dnmt3a2 at a molar ratio of one recombinant protein molecule per nucleosome core, and then protein-bound DNA was immunoprecipitated using an anti-GST antibody. Bound DNA was analyzed to quantify Dnmt3 binding, and the result was expressed as the percentage of the input DNA (Figure 5C and Supplementary Figures S4 and S5). The binding affinity of GST-Dnmt3b significantly increased with increasing numbers of nucleosomes in the reconstituted chromatin (Figure 5C). Furthermore, GST-Dnmt3b preferentially bound to nucleosomal 12 × 601 DNA as compared to naked 12 × 601 DNA (Figure 5C). In contrast to GST-Dnmt3b, GST-Dnmt3a2 bound weakly to all substrates regardless of DNA size or nucleosomal structure, and there were no significant differences in binding affinity among all substrates (Figure 5C). These results demonstrated that Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA, and suggested that the binding of Dnmt3b is dependent on chromatin structure.

Figure 5.
Dnmt3b preferentially binds to long chromatin in vitro. (A) Purified GST-Dnmt3a2 and GST-Dnmt3b. Purified protein was analyzed by 10–20% SDS–PAGE and then visualized by CBB staining. (B) Mononucleosomes, tetramers (4-mer) and 12-mer nucleosomal ...

Histone H1 binds near linker DNA between nucleosomes, and affects higher-order chromatin structure (68,69). Incorporation of histone H1 into reconstituted nucleosomal arrays induces a compaction of the chromatin fiber (70). To investigate whether Dnmt3b preferentially binds to highly folded chromatin, histone H1 was incorporated into reconstituted 12-mer nucleosomal arrays, and the effect was assessed by nucleoprotein agarose gel electrophoresis. When 12-mer arrays were incubated with increasing amounts of histone H1, a decrease in the migration of nucleoprotein complexes was found (Figure 6A). MNase digestion of histone H1-containing chromatin yielded chromatosomes, which confirmed that histone H1 was stably incorporated into the nucleosomal arrays (53) (Figure 6B). To estimate the relative amount of histone H1 to core histones, the reconstituted 12-mer nucleosomal arrays were precipitated with MgCl2 (51). Precipitated material was separated by SDS–PAGE, and then subjected to SYPRO-Orange staining (Figure 6C and Supplementary Figure S6). The molar ratio of histone H1 to core histones was 0.53 at its maximum in the reconstituted chromatin (Figure 6D). In ES cells, the average ratio of histone H1 to core histones is approximately 0.46 (36). Thus, reconstituted nucleosomal arrays contained physiological amounts of histone H1. We next assessed the binding of GST-Dnmt3b to the reconstituted chromatin substrates (Supplementary Figures S4 and S7). As expected, the affinity of GST-Dnmt3b binding to chromatin increased with the incorporation of increasing amounts of histone H1 into the nucleosomal arrays (Figure 6E). These results suggested that Dnmt3b preferentially binds to higher-order chromatin structures.

Figure 6.
Dnmt3b preferentially binds to histone H1-assembled chromatin. (A) 12-mer nucleosomal arrays (70 nM nucleosome cores) were incubated with various amounts of histone H1 (0, 70, 140 or 210 nM). Binding of histone H1 to reconstituted chromatin ...

Dnmt3b preferentially associates with hypoacetylated nucleosomal arrays in vitro

Histone acetylation neutralizes the net charge of lysine, reduces chromatin–chromatin interactions, and is associated with decondensed transcriptionally active chromatin (71). Recent studies have shown that acetylation of histone H4K16 induces the disruption of condensed chromatin fibers in vitro (64,65). To investigate the effect of histone acetylation on chromatin binding of Dnmt3b, hypo- and hyperacetylated nucleosomal arrays were prepared. When nucleosomal arrays are properly and fully positioned, monomer-sized nucleosomes are observed following the digestion of Sca I restriction sites in the linker DNA of 601 sequences between nucleosomes (50). We detected monomer-sized nucleosomes without naked DNA fragments by gel mobility shift analysis (Figure 7A, lanes 3–5), which indicated that the hypo- and hyperacetylated nucleosomal arrays were properly positioned and fully reconstituted (Figure 7A, lanes 6, 7 and Supplementary Figure S3). The level of histone acetylation in these nucleosomal arrays was analyzed by dot blot analysis. There was an enrichment of histone acetylation in hyperacetylated nucleosomal arrays (Figure 7B). To characterize any structural differences between the nucleosomal arrays, the sedimentation velocity was determined by analytical ultracentrifugation. In 2.5 mM NaCl without MgCl2, hyperacetylated nucleosomal arrays had S20,w values of approximately 33S, while hypoacetylated nucleosomal arrays sedimented at approximately 38S. These results indicated that hypoacetylated nucleosomal arrays had a more compact chromatin structure than hyperacetylated nucleosomal arrays (Figure 7C). Using these two different nucleosomal arrays, a Dnmt3b binding assay was performed (Supplementary Figures S4 and S8). As shown in Figure 7D, Dnmt3b preferentially associated with the more condensed, hypoacetylated nucleosomal arrays. Consistent with the reduced amount of H4K16Ac in Dnmt3b-associated chromatin (Figure 3D), the amount of H4K16Ac was also reduced in GST-Dnmt3b associated chromatin (Supplementary Figure S9). These results prompted investigation into whether Dnmt3b associated with histone deacetylases in the nucleus. Co-immunoprecipitation analysis revealed that both the class I histone deacetylases HDAC 1/2 and the class III histone deacetylase SirT1 interact with Dnmt3b in the nuclease-insensitive S2 fraction under physiological salt conditions (75 mM NaCl) (Figure 7E, left). Specifically, interaction of Dnmt3b with SirT1, but not with HDAC1 or 2, was maintained even under high salt (0.4 M NaCl) conditions, indicating a stable interaction between them (Figure 7E, right). Recently co-localization of SirT1 and Dnmt3b was shown at a specific genomic region (72). Considering the preferential association of Dnmt3b with hypoacetylated nucleosomal arrays (Figure 7D), these results suggest that the deacetylation of histones by HDACs, including SirT1, promotes the recruitment of Dnmt3b into chromatin.

Figure 7.
Dnmt3b preferentially associates with hypoacetylated chromatin in vitro. (A) Gel shift analysis of differentially acetylated 12-mer nucleosomal arrays. 12-mer arrays reconstituted with hypo- (−Ac) and hyper- (+Ac) acetylated core histones were ...

DISCUSSION

The results show that Dnmt3b in differentiated ES cells (EB3 cells) preferentially binds to nuclease-resistant condensed chromatin that contains linker histone H1 and histone deacetylases including SirT1, without a significant enrichment of silent-specific histone methylation. In addition, using reconstituted nucleosomal arrays as DNA substrates, Dnmt3b, but not Dnmt3a2, recognizes higher-order chromatin structures. Although further enzymatic analyses are required, the findings suggest that Dnmt3b is specifically recruited into condensed chromatin regions, recognizing higher-order chromatin structures, to stabilize gene silencing by DNA methylation.

During ES cell differentiation, the levels of Dnmt3b and Dnmt3a2 transiently increased and associated with chromatin (Figure 1A and B). Fractionation of chromatin by MNase treatment of EB3 cell nuclei revealed that Dnmt3b mainly associated with histone H1-containing condensed chromatin in the gene silent S2 fraction (Figure 1E). In contrast to Dnmt3b, Dnmt3a2 was found in both nuclease-sensitive S1 and insensitive S2 fractions equally (Figure 1E). Since the DNA-binding affinity of Dnmt3b increased significantly with increasing numbers of nucleosomes, while the binding of Dnmt3a2 was not affected by nucleosomal DNA structure (Figure 5C), Dnmt3a2 in the S2 fraction might associate with chromatin through interactions with Dnmt3b (Figure 3A and B).

A functional link between DNA methylation and histone H1, which contributes to higher order chromatin structure (69), has been demonstrated across the fungi, plant and animal kingdoms (35,36,73). Mouse ES cells that lack three of six somatic histone H1 genes, in which the ratio of histone H1 to core histones is reduced from approximately 0.46 to 0.25, exhibit altered gene expression patterns and changes in DNA methylation (36). In the current study, the incorporation of histone H1 into reconstituted nucleosomal arrays (to over 0.5 H1 molecules per nucleosome core) significantly promoted chromatin binding of Dnmt3b (Figure 6E). Recent studies using long nucleosomal arrays showed that the incorporation of histone H1 induces a dose-dependent increase in chromatin compaction (64,70). The results suggest that Dnmt3b does not simply bind to DNA, but that it recognizes the condensed nucleosomal structure.

It was demonstrated previously that Dnmt3b associates with HP1α (25), which also contributes to the condensation of chromatin structures (74). HP1α was present predominantly in the nuclear matrix-containing nuclear pellet (P) fraction (Figure 1E) and the nuclear localization of Dnmt3b overlapped with that of histone H1, but not HP1α (Figure 2B and C). Dnmt3b was partially present in the P fraction besides the S2 fraction (Figure 1E), and the Dnmt3b in the P fraction appears to interact with HP1 proteins. The results indicate that most of the stable interactions of Dnmt3b with chromatin are independent of HP1α, as was also recently shown by sedimentation analysis (37).

Recent structural and biochemical studies demonstrated that the ADD domains of Dnmt3 proteins bind to histone H3 N-terminal tails that are unmethylated at lysine 4 (32–34). Consistent with this notion, Dnmt3b-bound chromatin was reduced for tri-methylated histone H3K4 in ES cells. In contrast, no significant accumulation of the inactive chromatin modifications H3K9me2/3, H3K27me3 or H4K20me3 in Dnmt3b-bound chromatin was found (Figure 3C and Supplementary Figure S2B). Furthermore, as shown in Figure 7D, histone deacetylation itself promoted Dnmt3b binding. The results suggest that histone deacetylation, rather than site-specific histone methylation, played an important role in the recruitment of Dnmt3b.

It has been shown that the N-terminus of histone H4 plays an important role in chromatin structure (50). Specifically, H4K16Ac induces the decondensation of reconstituted nucleosomal arrays, as determined by linker histone eviction (64,65). The reduction of histone H4K16Ac from Dnmt3b-bound chromatin (Figure 3D and Supplementary Figure S9) suggested that the physical interaction between Dnmt3b and chromatin was weakened by alterations in higher-order chromatin structures induced by acetylation of histone H4 at K16. In mammals, CpG islands within the 5′-promoter regions of many genes are usually unmethylated (62,75,76). These 5′-CpG islands are nuclease sensitive, reflecting histone hyperacetylation and nucleosome eviction (77–79). Dnmt3b exhibited a low preference for naked DNA (Figure 5C), and was enriched in the nuclease-insensitive S2 fraction (Figure 1E). Characterization of the Dnmt3b–chromatin interaction suggested that the chromatin environment of 5′-CpG islands is not suitable for the stable association of Dnmt3b with chromatin to induce DNA methylation.

The stable association of Dnmt3b with SirT1, which specifically deacetylates H4K16 (40), was found in the high salt S2 fraction (Figure 7E). In contrast, as recently reported (37), the interaction of Dnmt3b with HDAC1/2 (80–82) was disrupted in high-salt buffer (0.4 M NaCl). SirT1 is required for histone H1 recruitment in mammalian cells (39), and contributes to the onset of DNA methylation through the recruitment of DNMT3B to SIRT1-associated silent chromatin (72). It appears that SirT1 functions as a regulator of chromatin structure, creating a platform for the recruitment of Dnmt3b and subsequent DNA methylation (Figure 7F). Although additional studies are needed to elucidate the precise targeting mechanisms and enzymatic activity of Dnmt3b, the results suggest that Dnmt3b functions not only as a writer of DNA methylation but also as a molecular reader of condensed chromatin structure for further gene silencing.

FUNDING

Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Scientific Research (grant numbers 19038015, 21114510, to K.U.); Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO) program Osaka University for female researchers. Funding for open access charge: JST PRESTO program.

Conflict of interest statement. None declared.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

Supplementary Data:

ACKNOWLEDGEMENTS

We thank E. Li for the anti-Dnmt3a and anti-Dnmt3b serum; S. Yamanaka for the anti-Dnmt3L antibody; H. Niwa for the pCAGIpuro DNA; T. Richmond for the 601-208 × 12 DNA; M. Sakai for technical support with the sedimentation velocity analyses; I. Suetake, S. Tajima, C. Masutani and H. Kimura for technical advice; J. Goddi and H. Sasaki for valuable comments on the article and members of our laboratory for support and discussion.

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