Methylation of Histone H4 at Arginine 3 Facilitating Transcriptional Activation by Nuclear Hormone Receptor Hengbin Wang, Zhi-Qing Huang, Li Xia, Qin Feng, Hediye Erdjument-Bromage, Brian D. Strahl, Scott D. Briggs, C. David Allis, Jiemin Wong, Paul Tempst, and Yi Zhang

Supplementary Material

Supplemental Figure 1. PRMT1 forms a 330-kD homo-oligomer complex. About 100 g of GST-PRMT1 was digested with thrombin and loaded onto a gel-filtration Superose 200 column. Aliquots of elution fractions were analyzed in a 6 to 12% gradient SDS-PAGE followed by Coomassie staining (top panel) and Western blotting (bottom two panels). The elution profile of protein size markers in this column is indicated on the top. The migration positions of the size markers in the SDS-PAGE are indicated on the left.

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Supplemental Figure 2. Arg 3 of H4 is the major methylation site by PRMT1. In vitro PRMT1 methylated H4 was gel-purified and subjected to NH₂-terminal automated sequencing, and ³H radioactivity eluted from each cycle was counted. The amino acids identified at each cycle of microsequencing are listed.

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Methods

Purification and identification of PRMT1 as an H4-specific HMT. HeLa nuclear proteins were separated into nuclear extract and nuclear pellet. Nuclear extracts (6 g) were further fractionated as 0.1 M, 0.3 M, 0.6 M, and 1.0 M fractions on a 700-ml phosphocellulose P11 column equilibrated with buffer C [20 mM Tris-HCl (pH 7.9), 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF, and 20% glycerol] containing 0.1 M KCl and sequentially eluted with buffer C containing 0.3 M, 0.6 M and 1.0 M KCl. Nuclear pellets (4.8 g) were solubilized in buffer B [50 mM Tris-HCl (pH 7.9), 5 mM MgCl₂, 0.5 mM EDTA, 5 mM DTT, 0.2 mM PMSF, and 25% glycerol] by sonication at 4°C. The suspension was cleared of debris by centrifugation at 35,000 rpm for 1 hour. Ammonium sulfate was then added to a final concentration of 40%, and the suspension was centrifuged at 35,000 rpm for 1 hour. The ammonium sulfate precipitated proteins were resuspended in 1 liter of buffer D [50 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 2 mM DTT, 0.2 mM PMSF, and 25% glycerol] and the ammonium sulfate concentration was adjusted to 0.04 M before loading onto a 700-ml DEAE-52 column. The DEAE-52-bound proteins were eluted with buffer D containing 0.6 M ammonium sulfate and further fractionated on a phosphocellulose P11 column as described above. The 0.1 M fraction derived from the nuclear pellet was loaded onto a high-performance liquid chromatography HPLC-DEAE-5PW column (TosoHaas, 45 ml) that had been equilibrated with BD40, and the bound proteins were eluted with a 10-column volume (10 cv) linear gradient from BD40 to BD400. Fractions containing the enzymatic activity were pooled, adjusted to 500 mM ammonium sulfate before loading to a 22-ml fast protein liquid chromatography (FPLC) Phenyl Sepharose column (Pharmacia). Bound proteins were eluted with a 20-cv linear gradient from BD500 to BD0. Fractions containing the enzymatic activity were pooled, and a portion of the pool was loaded onto a hydroxyapatite (BioRad, 1 ml) column equilibrated with buffer P [5 mM Hepes-KOH (pH 7.5), 10 mM potassium phosphate (pH 7.5), 10% glycerol, 40 mM KCl, 0.01% Triton X-100, 0.01 mM CaCl₂, 0.5 mM PMSF, 1 mM DTT]. The column was eluted with a 20-cv linear gradient from BP10 to BP600. To determine the native size of the enzyme, part of the Phenyl Sepharose pool was concentrated on a 0.2 ml DEAE-52 column and applied to a Superose 200 column (Pharmacia). The enzymatic activity elutes around 330 kD. To identify the 42-kD protein that coelutes with the H4 HMT activity, the 42-kD protein was excised and subjected to in-gel tryptic digestion. The resulting peptides were analyzed by mass spectrometry. All masses obtained have a perfect match with a previously identified protein, the human protein arginine N-methyltransferase 1, PRMT1 (2). However, all the recombinant PRMT1 used in this report is the rat PRMT1 (3), which is highly similar to its human counterpart.

MNase assay, oocytes injection, and primer extension. The MMTV-LTR-CAT reporter construct was generated by inserting a fragment containing the MMTV LTR plus 0.3 kb of CAT sequence into pBluescript II (SK⁺) and the single-stranded DNA (ssDNA) was prepared as described (4). To produce mRNAs encoding AR, PRMT1, and PRMT1(G80R) mutant for transcription analysis, their corresponding cDNAs were cloned into pSP64poly(A) vector. The resulting constructs were linearized with Bgl II, and in vitro synthesis of their corresponding mRNAs were performed using an SP6 Message Machine kit (Ambion) as described by the manufacturer. The preparation of Xenopus stage VI oocytes and the microinjection procedure were performed essentially as described (4). The assembly of the reporter DNA into chromatin via a replication-coupled chromatin assembly pathway (5) in Xenopus oocytes was achieved through injection of the MMTV-LTR reporter as ssDNA (50 ng/l, 18.4 nl/oocyte) into the nuclei of the oocytes. The micrococcal nuclease (MNase) assay of chromatin assembly in Xenopus oocytes was performed as described previously (4). Briefly, about 20 oocytes injected with ssDNA of the MMTV-LTR-CAT were collected after overnight incubation and homogenized in 200 l of MNase buffer (10 mM Hepes, pH 8.0, 50 mM KCl, 3 mM CaCl₂, 1 mM DTT, 0.1% NP-40, and 5% glycerol). The extract was then divided into three fractions (60 l) and digested with 10, 5, or 2.5 units of MNase (Worthington) at room temperature for 20 min. MNase digestions were stopped by addition of 200 l of 20 mM EGTA, 1% SDS. The reactions were treated with RNase A (100 g/ml) for 2 hours at 37°C. Proteinase K was then added to a final concentration of 100 g/ml and incubated at 55°C for at least 2 hours. The DNA was recovered from each sample by phenol/chloroform extraction and ethanol precipitation, resolved by a 1.5% agarose gel, blotted to a nylon membrane, and probed with the random-primer labeled 0.3-kb CAT probe.

For transcriptional analysis, the mRNAs for AR (100 ng/l, 18.4 nl per oocyte), PRMT1 (33 ng/l or 100 ng/l) or the PRMT1(G80R) mutant (33 ng/l or 100 ng/l) were injected into the oocyte cytoplasm 2 to 3 hours before the injection of the reporter DNA (50 ng/l, 18.4 nl per oocyte). Approximately 20 oocytes were injected for each sample. The injected oocytes were incubated with or without 100 nM of the agonist at 18°C overnight. The oocytes from each group were collected, rinsed with 400 ml of MBSH buffer (4), and homogenized in 100 l of buffer containing 0.1 M Tris (pH 8.0) and 10 mM EDTA. To isolate RNA, TRIzol reagent (500 l, GIBCO-BRL) was added to the sample, vortexed, and incubated on ice for 15 min before centrifugation. The clear supernatant was extracted with an equal volume of chloroform. The RNA was then precipitated with 0.7 volume of isopropanol, rinsed with 70% ethanol, and dissolved in diethyl pyrocarbonate---treated water. The levels of transcription from the MMTV-LTR reporter were determined by primer extension using a CAT specific primer as described (4). The primer extension product of the endogenous histone H4 mRNA serves as an internal control (6). Briefly, the total RNA recovered from one or two oocytes was annealed with -³²P end-labeled H4 primer and CAT primer in 10 l of annealing buffer (0.02 M Tris, pH 8.3, 0.4 M KCl) at 65°C for 10 min, 55°C for 10 min, and then 42°C for 25 min. Reverse transcription mixture (30 ml: 67 mM Tris-HCl at pH 8.3, 8 mM MgCl₂, 5 mM DTT, 1 mM dNTP mix, 1 unit of RNasin, and 10 units of Superscript II) was then added and incubated for 1 hour at 42°C. The reaction was stopped by ethanol precipitation. The products were separated on a 6% sequencing gel and visualized by autoradiography.

References

1. Y. Webb et al., J. Biol. Chem. 274, 14280 (1999).
2. H. S. Scott et al., Genomics 48, 330 (1998).
3. W.-J. Lin, J. D. Gary, M. C. Yang, S. Clarke, H. R. Herschman, J. Biol. Chem. 271, 15034 (1996).
4. J. Wong, Y. B. Shi, A. P. Wolffe, Genes Dev. 9, 2696 (1995).
5. G. Almouzni, A. P. Wolffe, Genes Dev. 7, 2033 (1993).
6. J. Wong et al., EMBO J. 17, 520 (1998).