De novo reconstitution of chromatin using wheat germ cell‐free protein synthesis

DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell‐free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP‐1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone‐modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research.

In eukaryotic cells, chromatin structure plays various roles in gene regulation, DNA repair, DNA replication and inheritance of epigenetic information from generation to generation. The basic building block of chromatin is the nucleosome, which contains about 147 bp of DNA wrapped 1.7 times around two copies each of the histones H2A, H2B, H3 and H4 [1,2]. The basic structure of chromatin is identical in almost all eukaryotes, but histone variants and chemical modifications of the histones introduce a considerable heterogeneity in chromatin structure [3][4][5][6]. Although a tremendous amount of information has accumulated in the past decades regarding the identity of the posttranslational modifications of histones [2], it is extremely challenging to understand their effects on chromatin structure, dynamics and physiological functions. To fully understand the biochemical role of histone modifications on chromatin, it is necessary to reconstitute chromatin in vitro with defined components as much as possible.
The reconstitution of chromatin in vitro first involves the preparation of the histones in the correct stoichiometry before chromatin assembly. Some of the earliest methods of histone purification involved acid Abbreviations Acf1, ATP-dependent chromatin assembly factor large subunit; AMP-PCP, b,c-Methylene adenosine 5'-triphosphate; ISWI, chromatinremodeling complex ATPase chain Iswi; MNase, micrococcal nuclease; MS, mass spectrometry; NAP-1, nucleosome assembly protein NAP-1; SC-pBS, supercoiled pBluescript; SRM, selected reaction monitoring. or high-salt extraction of the intact histones from eukaryotic nuclei followed by reassembly of the octamer and purification before chromatin reconstitution [7][8][9]. However, the earlier-mentioned extraction conditions might denature the proteins and require substantial manipulation. Alternatively, histones can be prepared by the synthesis and purification of recombinant proteins from Escherichia coli [10][11][12]. In this method, the expressed recombinant histones are needed to be extracted from the inclusion bodies and then refolded.
There are two conventional methods for the reconstitution of chromatin on DNA [13,14]. In the first series of methods, purified histones from living tissues or recombinant histones are mixed together in equal stoichiometry to form octamers, then mixed with DNA at high-salt concentrations, followed by stepwise dialysis resulting in the assembly of nucleosomes [15,16]. This method is usually unable to evenly space nucleosomes unless the DNA template contains arrays of nucleosome positioning sequences and is primarily used to reconstitute chromatin onto short fragments of DNA [17][18][19]. In the second series of methods, chromatin is assembled using chromatin assembly factors, such as histone chaperones and ATP-dependent chromatin remodeling factors [7,[20][21][22]. The methods also require purified histones at equal stoichiometry.
To date, in vitro-reconstituted chromatin has been widely used to investigate fundamental processes of molecular biology, such as transcription [23], gene silencing [24], centromeric chromatin and others [25][26][27]. However, purification of histones and chromatin assembly factors from living tissues is time consuming and also suffers additional technical limitations. For example, core histones purified from eukaryotic cells are highly heterogeneous, containing different subtypes, and possess multiple posttranslational modifications, all of which are not desirable for the controlled chromatin reconstitution in vitro [8,28]. In contrast, although recombinant core histones produced in bacterial cells are chemically homogeneous, refolding is necessary after the expression of proteins. For these reasons, recombinant histones are less efficient at forming nucleosomes compared with native histones [22,29]. Moreover, supplementation of salts and macromolecules, such as polyethylene glycol or bovine serum albumin, during assembly introduces extraneous molecules that could interfere with the downstream processes.
Recently, we developed a novel reconstitution method using a wheat germ cell-free coexpression system and showed both human and Drosophila chromatins were assembled onto a circular DNA in the presence of mRNAs coding four core histones and appropriate chromatin assembly factors [30]. Both supercoiling assay and micrococcal nuclease (MNase) digestion were used to assess the reconstituted chromatins. In this study, we detailed more insights into our reconstitution system by using Drosophila chromatin components with regard to chemical modification profiles of synthesized histones, the kinetics of histone production in the cell-free system and the determination of essential components in the assembly. Furthermore, we showed that the reconstituted chromatin can be easily isolated via the aid of divalent cations with moderate purity, and the isolated chromatin retained the expected stoichiometry of core histones after recovery. We examined histone modifications on the reconstituted chromatin and found that the chromatin was modified with dGCN5 and dSet8 that resulted in acetylation of nucleosomal H3 and methylation of nucleosomal H4, respectively. The described method might be applicable toward the straightforward preparation of defined chromatin and applicable to a wide range of areas in chromatin structure and function research.

Cell-free protein synthesis
The methods for cloning of genes from Drosophila melanogaster into the pEU expression plasmid vector, preparation of a cell-free extract from wheat embryos and the transcription and translation reactions via a membrane dialysis device (Slide-A-Lyzer, Product #8841; Thermo Scientific, Rockford, IL, USA) were described previously [30][31][32]. In vitro transcription was performed as previously described using the pEU vector carrying an individual gene [33]. The transcribed reaction mixture was treated with RNase-free DNase1 (1 UÁ100 µL À1 ; Nippon Gene, Tokyo, Japan) for 30 min at 37˚C and deprotonated by phenolchloroform extraction followed by EtOH precipitation. To eliminate residual nucleotides from the transcription reaction, we extensively washed RNA precipitate with 75% EtOH, and mRNA was dissolved in RNase-free Milli-Q water. In the dialysis cup mode translation, a 100-µL translation mixture contained 4 µg of creatine kinase (Roche Diagnostics, GmbH, Mannheim, Germany) and 17 µL of the stock wheat germ extract (A 260 = 240; thus, final 40 A 260 unitsÁmL À1 ) (WEPRO7240H, Lot: 15AQ02; Cell-free Sciences, Yokohama, Japan). A total of 25 µg of the total amount of mRNA, which was predetermined to be optimum, was used for the individual, or each of 12.5 µg H2A/ H2B or H3/H4 and each of 6.25 µg of H2A/H2B/H3/H4 for the cotranslation. The translation reaction was performed by dipping the dialysis device in 5 mL of substrate solution at 15˚C for 50 h. The translation mixtures and substrate buffer solution were composed of the following: 30 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine, 2.5 mM DTT, 0.3 mM each amino acid, 1.2 mM ATP, 0.25 mM GTP and 16 mM creatine phosphate. For radiolabeling of the proteins, the translation reaction was performed in the presence of 0.18 MBqÁmL À1 14 C-leucine (11.1 GBqÁmmol À1 ; PerkinElmer, Foster, CA, USA) in the translation mixture and substrate buffer. Concentrations of synthesized histones were determined and adjusted to 4.0 pmolÁµL À1 for individually expressed ones and each 2.0 pmolÁµL À1 for coexpressed histones H2A and H2B and also H3 and H4 by diluting with a separately incubated translation mixture in the absence of mRNA. Aliquots of product solutions from the cup device were rapidly frozen in liquid nitrogen and stored at À80˚C until use. Solubility (%) of products was the ratio of hot trichloroacetic acid (TCA)-insoluble radioactivity in the supernatants to that of the total after centrifugation at 10 000 g for 10 min, measured by liquid scintillator (Ultima Gold XR; Packard). The counting efficiency of 14 C in proteins on filters was corrected and normalized by using purified 14 C-labeled red fluorescent protein in solution. The amount of synthesized protein was calculated by moles of leucine incorporated and the number of residues in each protein. Relative intensities among coomassie brilliant blue (CBB)-stained or the bands on the autoradiogram were measured by densitometry (Typhoon FLA 7000BGR; GE Health Care, Pittsburgh, PA, USA).

Wheat germ cell-free in vitro chromatin assembly
Three types of the 30 µL of standard chromatin assembly mixture were prepared by simply mixing translated solutions containing 20 pmol each of individually synthesized four core histones, cotranslated histones, H2A/H2B and H3/H4, or cotranslated all four histones (H2A/H2B/H3/ H4) and 2.0 pmol each of the supernatant fraction of dNAP-1, dISWI and dAcf1 in the standard reaction. The concentration of wheat germ extract in the assembly mixture was adjusted to the final 40 A 260 by adding a separately incubated translation mixture without mRNA, and 5 µg of creatine kinase was supplemented. Unless otherwise stated in the figure legends, an assembly mixture was preincubated with 60 U MNase (Nippon Gene) with 1 mM Ca 2+ for 30 min at 26˚C to digest the endogenous wheat DNA; then the reaction was halted by adding 5 mM EGTA. The assembly reaction was initiated by adding 1 µg supercoiled pBluescript (SC-pBS) (pBluescript SK+ ; Agilent, Santa Clara, CA, USA) and incubated for 3 h at 26˚C. A b,cmethyleneadenosine 5 0 -triphosphate (AMP-PCP) was used as nonhydrolyzable ATP analogue (Wako, Osaka, Japan). Topoisomerase I and II inhibitors, camptothecin (Wako) and ICRF-193 (Sigma-Aldrich, St. Louis, MO, USA), were purchased.

MNase assay
Either fresh or freeze-thawed reconstituted chromatin con-taining~25 ng of each plasmid DNA and core histones was digested in the presence of 120 U MNase (TaKaRa, Shiga, Japan) in 20 mM Tris-HCl (pH 8.0), 5 mM NaCl and 2.5 mM CaCl 2 at 37˚C, and the reaction was halted by adding 5 mM EGTA, followed by phenol-chloroform extraction and EtOH precipitation. Samples were suspended in Milli-Q water with 0.01 µgÁmL À1 RNase (Nippon Gene), then separated on a composite gel (2% soft agarose and 0.66% agarose) in TAE buffer and visualized with ethidium bromide staining [13,14].

Isolation of chromatin
After chromatin assembly reaction, which contained 2.7 mM magnesium acetate in the translation reaction, reconstituted chromatin mixtures were transferred to 1.5-mL tubes and diluted with 3 vol of HD buffer (25 mM HEPES, pH 7.8, 1 mM DTT); then MgCl 2 was added, and the concentration was adjusted as indicated in Fig. S5. In the standard protocol, the assembled chromatin mixture was brought to 10 mM MgCl 2 and incubated for 5 min at room temperature, followed by centrifugation for 10 min at 10 000 g at 20˚C. The pellet was suspended in HD buffer and directly used as nucleosome samples for further experiments or frozen in liquid nitrogen and stored at À80˚C until use.

Results and Discussion
Synthesis of histones, chromatin assembly factors and histone modification enzymes in the wheat germ cell-free translation To produce chromatin in vitro using the wheat germ cell-free protein synthesis, we first attempted to synthesize the following Drosophila proteins: histones H2A, H2B, H3 and H4; histone chaperone, dNAP-1; ATPdependent chromatin remodeling factors, dAcf1 and dISWI; and chromatin-modifying enzymes, dGCN5 and dSet8. pEU plasmids harboring the earlier genes were used as templates for the SP6 RNA polymerase to generate the mRNAs in vitro. Synthesized mRNAs were treated with DNase I to remove residual pEU plasmid and then used for in vitro wheat germ cell-free translation to produce Drosophila proteins for 50 h at 15˚C in dialysis mode. The in vitro-translated products were analyzed by SDS/PAGE, followed by CBB staining or autoradiography (Figs 1 and S1). The four core histones of the correct size can be seen among the wheat germ extract proteins. Autoradiograms show that all products were intact and of the expected molecular weight except for dSet8, which was larger than the expected size of 76 kDa and will be discussed later. The calculated concentration (pmolÁµL À1 ) and solubility (%) from at least three independent experiments of individually synthesized histones H2A, H2B, H3 and H4 were 13.8 AE 2.8, 14.4 AE 1.4, 16.2 AE 0.7 and 15.5 AE 1.4 pmolÁµL À1 and 56% AE 5%, 60% AE 2%, 51% AE 5% and 59% AE 4%, respectively. Similarly, concentration (pmolÁµL À1 ) and solubility (%) of dNAP-1, dAcf1, dISWI and histone-modifying enzymes, dGCN5 and dSet8, were 24.8 AE 6.8, 3.6 AE 0.7, 2.7 AE 0.3, 15.9 AE 1.0 and 11.1 AE 0.8 pmolÁµL À1 and 100% AE 1%, 12% AE 1%, 32% AE 6%, 11% AE 1% and 21% AE 2%, respectively. The observed solubility of four core histones was greater than 50%, which enabled the immediate use of those products for chromatin assembly in vitro.
After in vitro translation, products were analyzed by MS, and it was confirmed that synthesized histones were N-terminal methionine processed but otherwise lacked any posttranslational modification (Fig. 1B,C; Table S1). As shown in Fig. 1B, both Drosophila and wheat histone sequences were aligned, and the two peptide fragments from each of histones H3 (Ala115-Leu127) and H4 (Arg24-Ile47) were determined to be identical. However, other wheat histone derivatives were not identified by MS/MS analysis. It is also important to note that we did not detect any endogenous wheat histone in the translation mixture in the previous MS analysis [30]. These results provide an important advantage to using the wheat cell-free synthesized histones over heterogeneously modified histones derived from cell nuclei. Furthermore, a major fraction of the synthesized histones remained soluble and had not aggregated, which provides a great advantage over conventional methods that use recombinant histones.

Posttranslational nucleosome assembly
Having developed conditions to efficiently synthesize core histones and chromatin assembly factors using the wheat germ cell-free system, we investigated the ability of in vitro chromatin assembly. We first examined whether the wheat germ extract contained endonuclease activity that could degrade plasmid DNA, because this was not tested in a previous study [30]. SC-pBS was incubated in the translation mixture at 26˚C (Fig. S4), and results showed no detectable level of DNase activity in the extract even after prolonged incubation. Interestingly, the SC-pBS was converted to the relaxed form of pBS within 10 min of incubation (Fig. S3). This result indicated that there is endogenous topoisomerase activity in the wheat germ extract. The observed topoisomerase activity was inhibited by 50 µM topoisomerase Ispecific inhibitor (camptothecin), but not by topoisomerase II-specific inhibitor (ICRF-193). Thus, the observed topoisomerase activity was concluded to be mediated by endogenous topoisomerase type I activity in the wheat extract. This result enabled us to measure the efficiency of chromatin assembly using the extent of supercoiling as a readout for nucleosome deposition [39] without supplementing exogenous topoisomerase.
To optimize the chromatin assembly reaction, we first determined the optimum ratio of individually synthesized four core histones to template DNA in the reaction containing histone chaperone and ATPdependent chromatin assembly factors, dNAP-1, dAcf1 and dISWI ( Fig. 2A). It was determined that 1.0-2.0 µg (1.1-2.2 pmol) of pBS plasmid was the most effective when mixed with 1.0 µg (20 pmol of each core histone) of total core histones in the 30 µL assembly reaction, consistent with previous studies [7,40], whereas 4.0 µg (4.4 pmol) of pBS plasmid to 1.0 µg core histones reduced the formation of supercoiling.
The reaction time was also tested, and we found that a 3-h assembly reaction is sufficient for completion (Fig. 2B), similar to the Drosophila chromatin assembly system reported earlier [7]. In the coexpressionmediated chromatin assembly shown in a previous study [30], the translation-coupled reconstitution took 6 h, whereas the current method does not require a translation process, which differs between two reconstitution modes. Determined conditions were used as standard assembly reactions for the following experiments. In Fig. 2C, we evaluated the nucleosome repeat length in chromatin assembled on pBS under optimized reaction conditions. After chromatin assembly, MNase was added to samples, and a time course of digestion was conducted. Digested DNA was analyzed by agarose gel electrophoresis; then nucleosome repeat length was estimated. The average repeat length was around 165 bp, indicating that histones had indeed been successfully incorporated into nucleosomes and formed chromatin on the DNA.
To determine which component is essential for the chromatin assembly, we eliminated each component and assessed by supercoiling assay (Figs 3 and S4). In the absence of either histone H3 or H4, or both H3/ H4, the formation of supercoils was not detected (Figs 3A and S4A). When either histone H2A or H2B was absent, the supercoiling formation was still observed, but to a lesser extent than with the complete reaction. Consistent with the previous reports, histones H3 and H4 are essential, whereas in the absence of H2A or H2B, supercoils could still be stabilized by the H3/H4 tetramer [11]. We also tested the requirements of the dNAP-1 and ACF (dAcf1 and dISWI) by supercoiling assay [7]. In the absence of dNAP-1, the extent of supercoiled DNA was reduced compared with the complete assembly reaction. When lacking ACF (dAcf1 and dISWI), the amount of supercoiled DNA was slightly diminished. These results differ from previous studies, in which both dNAP-1 and ACF have been shown to be essential in the supercoiling formation [21, 41,42]. It is thought that there are possible chromatin assembly factors present in the wheat germ extract, which were not found in the proteomic analysis in the previous study [30]. Because ACF aids chromatin assembly in an ATP-dependent manner, we next assessed the effect of ATP on chromatin assembly by using a nonhydrolyzable ATP analogue, AMP-PCP (Figs 3B and S4B). As a result, the ATP-regenerating system was essential for the formation of chromatin, indicating that there are alternative ATP-dependent chromatin assembly factors that help the formation of chromatin in our system. In contrast, the previously described coexpression reconstitution reaction contained an ATP-regenerating system required for protein synthesis; thus, the requirement of ATP in the chromatin assembly reaction was not assessed [30]. Although we do not know what chromatin assembly factors present in the extract, it is interesting to determine them in the future study.

Isolation of the reconstituted chromatin
Conventionally, in vitro assembled or native chromatin is purified by sucrose density gradient or size exclusion chromatography, both of which require tens of micrograms of the sample caused by losses during these processes [43]. The effect of divalent or multivalent cations on DNA-protein complex condensation had been described in previous studies [44][45][46]. We therefore decided to test the ability of Mg 2+ to condense and isolate assembled chromatin from a crude assembly mixture. Increasing amounts of Mg 2+ were added to the extract, followed by brief centrifugation to fractionate the reaction into supernatant and pellet (Fig. S5A). At 2.7 mM Mg 2+ , which is the concentration included in the standard wheat translation reaction, most pBS was still found in the supernatant. At 6 mM Mg 2+ , nearly equal amounts of DNA were found in both fractions. At 10 mM Mg 2+ , most of pBS was found in the pellet; thus, we used this concentration throughout the Mg 2+ precipitation of chromatin. In addition, in the presence of histones H3 and H4, pBS was found predominantly in the pellet (Fig. S5B), suggesting that H3/H4 and pBS formed a complex. In contrast, we did not find any pBS in the pellet in the absence of histones. As a control, E. coli ribosomal protein S7, which is a similarly small and basic protein as histones, was tested at 10 mM Mg 2+ , and no pBS was found in the precipitant (Fig. S5B, lane S7). Thus, the precipitated DNA is likely a chromatinized form. We also determined whether Mg 2+ -dependent precipitation was specific for DNA-protein complexes, or whether Mg 2+ could also precipitate RNA-protein complexes. The supernatant and pellet from the samples containing wheat ribosome without RNase treatment were analyzed on an agarose gel (Fig. S5B, lane rRNA). This result shows that a mixture of both ribosomes and tRNAs was found exclusively in the supernatant, while the pBS was found only in the pellet, indicating that the effect of Mg 2+ was specific on the histone-DNA complexes (Fig. S5B). The same samples were subjected to SDS/PAGE analysis to confirm the existence of nucleosomal histones in the Mg 2+ -isolated fraction (Fig. S5C). The majority of wheat germ proteins were found in the supernatant, while histones H2A, H2B, H3 and H4 were found predominantly in the pellet at 10 mM Mg 2+ . Thus, the assembled chromatin was isolated by the use of 10 mM Mg 2+ and subsequent centrifugation. In addition, we were able to isolate histones H3 and H4 with pBS, indicative of the formation of histone H3/H4 tetramers in the assembly (Fig. S5C). Moreover, histones were not isolated when the reaction was carried out in the presence of AMP-PCP, proving that ATP hydrolysis is essential for chromatin assembly.
To ask whether the molar ratios of four histones in the pellet retained the stoichiometry in chromatin before and after Mg 2+ isolation, we performed the chromatin assembly with radioactively labeled histones, fractionated using 10 mM Mg 2+ , and separated histones on SDS/PAGE. The amount of radioactivity in each histone was measured and standardized to the number of leucine residues present in each molecule. These results show that isolated complexes were composed of four histones with close to equimolar ratios (Fig. S6).
An additional advantage of the described chromatin assembly and isolation method is that chromatin could be stored stably at À80˚C. In Fig. S7, we examined the stability of an assembled chromatin upon repeated freezing and thawing by MNase digestion. The digestion profiles of this chromatin before and after four cycles of freezing and thawing were indistinguishable (Fig. S7B), although unfractionated chromatin led to a loss of the MNase pattern likely because of aggregation (Fig. S7A, compared with the freshly prepared sample in Fig. 2C). The observed reversible chromatin isolation by Mg 2+ in our method might also be an effect of currently unknown endogenous proteins in the wheat extract, and they will need to be determined in future studies.

A modification of reconstituted chromatin by histone modifiers
We next asked whether assembled chromatin could serve as a substrate for histone-modifying enzymes, for the first time on the basis of nonchemically modified ATP-dependent assembled chromatin. dGCN5 is a histone acetyltransferase that acetylates the N-terminal tail of both free and nucleosomal histone H3. This activity is aided by other factors, such as dADAs [47,48]. Histone methyltransferase, dSet8, is known to methylate the N-terminal tail of histone H4 [49,50]. The Mg 2+ -isolated chromatin in HD buffer was subjected to histone modification reaction by adding in vitro-synthesized dGCN5 or dSet8 in the presence of radiolabeled acetyl-CoA or S-adenosyl methionine, respectively (Fig. 4A). Acetylation of histone H3 by dGCN5 and methylation of histone H4 by dSet8 were monitored by autoradiography, respectively. Results showed that histones were modified in an enzymedependent manner, demonstrating that isolated chromatin can serve as a substrate for these histonemodifying enzymes. The freeze-thawed chromatin was also tested in this assay, and we found that repeated freeze-thawed chromatin was also modified by both enzymes (Fig. 4B).
The sites of modification on the histones H3 and H4 were determined by MS (Figs S8 and S9). This analysis showed that the H3 peptide (Ser10-Lys23) was acetylated at both the K14 and K18 positions by dGCN5, which is consistent with previous studies [50]. Furthermore, monomethylation of histone H4 K20 by dSet8 was confirmed by MALDI-TOF MS, as was reported earlier [49]. Notably, the discrepancy between the observed molecular weight of cell-free synthesized dSet8 and the expected size has been described in an earlier in vivo study [49]. Thus, we provide evidence that both Mg 2+ -isolated and freeze-thawed chromatin maintained a structure that histone-modifying enzymes could recognize as their substrates, and hence it was also concluded that some residual proteins after Mg 2+ isolation (Fig. S5) do not inhibit subsequent chromatin modification reaction.
Comparison of chromatin assembly using wheat germ cell-free synthesis and conventional methods We have described a method to synthesize histones and reconstitute chromatin by using wheat germ cellfree synthesis. Table 1 presents a comparison of different methods available for in vitro chromatin assembly to date. The salt dialysis method requires either recombinantly expressed histones or extracted core histones from living tissues [12,15,43]. Although recombinantly expressed histones do not possess any posttranslational modification, they need to be refolded, and their ability to be incorporated into chromatin is limited [7,12]. The use of native histones from cells and tissues overcomes these drawbacks, although they are a heterogeneous mixture with various histone subtypes and posttranslational modifications that altogether might confound the downstream analyses [7,8,12,28]. Furthermore, extracted histones from living tissues need high-salt or acid treatment during the purification depending on the source of the histones [51]. Finally, the nucleosomes assembled with salt dialysis are not uniformly spaced unless the underlying DNA template has a repeating array of a defined sequence such as the 601 repeat [17][18][19]. The latter problem can be overcome using histone chaperones and ATPdependent chromatin remodeling factors [7]. Finally, chromatin assembled with conventional methods needs to be stored at 4°C and is stable for a few months, whereas Mg 2+ -isolated chromatin from our method was shown to be stable over several repeats of freezethawing and can be stored stably over several months at À80°C.
In our wheat cell-free chromatin assembly method, the source of histones might be any organism of interest, and histones can be synthesized in a native state. Moreover, these histones are free of any detectable posttranslational modification; thus, chromatin reconstitution can be performed on any circular DNA molecule.  Fig. S1. Proteins synthesized in cell-free translation. Autoradiograms of synthesized ATP-dependent chromatin remodeling factors, dAcf1, dISWI, and histone chaperone, dNap-1 (A), and histone modifiers dGCN5 and dSet8 (B) are shown. Star denotes each product with expected molecular weight. Fig. S2. Cotranslation of histones in the batch and bilayer translation reactions. (A) An equal amount of each of mRNA for cotranslation of two histones H2A/H2B (filled square) and H3/H4 (filled circle) were performed in batch-wise translation. Translation reaction was initiated without pBS, and 2 h after when translation appeared to be ceased, pBS was added at final concentration of 40 lgÁmL À1 marked with arrows. Open square and circle indicate histone H2A/ H2B and H3/H4 after addition of pBS, respectively. Synthesis of histones was monitored by 14 C-leucine incorporation into a hot-TCA insoluble fraction. (B) Autoradiograms of the synthesized histones H3 and H4 products. The bilayer translation containing 5 lg of mRNAs for histones H3 and H4 in the absence or presence of 1.0 lg of pBS in 20 lL of the bottom layer are shown. Fig. S3. Topoisomerase activity was determined in the wheat germ extract. The mixture (60 lL) contained 10 lL of stock wheat germ extract (final 40 A 260 /mL), creatine kinase (40 UÁmL À1 ) and 6 lg of SC-pBS in the absence of histones or chaperones incubated at 26˚C for the indicated time.  The final concentrations at 2.7, 6.0 and 10.0 mM MgCl2 were used after completion of chromatin assembly reaction to isolate histone-DNA complexes followed by the centrifugation. Phenol-extracted plasmid from the total reaction (T), supernatant (S) and pellet (P) was run on agarose gel. Nicked relaxed, linear and supercoiled pBS are indicated as RC, L and SC, respectively. (B) The cotranslated H3/H4 or ribosomal protein S7 was incubated in the presence of pBS. After the addition of 10 mM MgCl2, samples were separated into S and P fractions, and pBSs were separated. In the rRNA lanes, both S and P loading samples were prepared from RNase-untreated complete assembled mixture as in (A) with 10 mM MgCl2. (C) Assembled mixtures with four core histones or with H3/H4 were separated into two fractions, S and P, with 10 mM MgCl2 and then run on SDS/PAGE. Also, a similar experiment in complete assembly reaction was performed in the presence of 1 mM AMPPCP. Amounts of DNA or proteins loaded among lanes were adjusted to an equivalent volume of assembly reaction mixtures. Recovery of proteins in Mg2+-precipitants and purity of histones were calculated by comparison of total intensities of CBB-stained bands to ones of Mg2+-precipitates, or core histones in Mg2+-precipitates by densitometry. Arrows indicate the positions of wheat endogenous nonhistone proteins near histones H3 and H4. Gel images show representative examples of at least three replicates. Fig. S6. Stoichiometry of histones. Autoradiograms of core histones in the mixture (A, total) and in the Mg2+-isolated sample (B) of PNAP assembled chromatin. The intensity of each histone band was measured by the densitometry, and the relative molar ratios of histones H2A, H2B and H4 to H3 were calculated as described in the Materials and Methods and shown on the right. Fig. S7. Effect of freezing and thawing on the purified chromatin. Samples of the assembled reaction mixture or Mg2+-isolated chromatin in the HD buffer were flash frozen by the liquid nitrogen and thawed at room temperature. MNase assay was performed for once freeze-dried unpurified chromatin (A), Mg2+-isolated chromatin (B, left) and four times freezing and thawing Mg2+-isolated chromatin (B, right). The number of nucleosome arrays is indicated with the arrow. Fig. S8. Selected reaction monitoring (SRM) analysis of in vitro histone acetylation by dGCN5. The presence of acetylated lysine residues in the dGCN5-treated histone sample was verified using the LC-SRM analysis, which enables selective detection of the targeted peptides from crude samples. Based on the MS/ MS information obtained from the tryptic peptides of acetylated histone H3, we selected a set of the fragment ions (Q3) for each precursor ion (Q1) and finally established the SRM assays. The SRM analysis coupled with LC separation followed by confirmation of the chromatographic profiles of the targeted peptides was performed. Two acetylated peptides, STGGKAPRKQLATK [10-23 amino acids (a.a.)] and APRKQLATK [15-23 a.a.], were determined only in the dGCN5-treated sample. For the peptide STEL-LIRK [57-64 a.a.] with no reported modification by dGCN5, no difference was observed with or without dGCN5 treatment. K* denotes the acetylated lysine; Q* denotes the deaminated glutamine. Fig. S9. MALDI-TOF MS analysis of in vitro histone methylation by dSET8. (A) After digestion with chymotrypsin, derived digests were subjected to peptide mass fingerprinting (PMF) analysis using the MALDI-TOF mass spectrometer. Asterisk indicates the peptide ions assigned chymotrypsin autolysis products. (B) PMF change by the dSET8 treatment was observed in the range of m/z 1250-1350. The ion peaks at 1306.8 and 1320.8 exhibit a mass difference of 14 Da, a predicted mass shift given by the monomethylation, indicating the presence of lysine methylation by dSET8 in histone peptide GKGGAKRHRKVL. Table S1. Peptide mapping of Drosophila histones by LC-MS/MS analysis.