Genetically Encoding Lysine Modifications on Histone H4 - ACS

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Genetically Encoding Lysine Modifications on Histone H4 Bryan J. Wilkins,†,∥ Liljan E. Hahn,†,∥ Svenja Heitmüller,† Holm Frauendorf,‡ Oliver Valerius,§ Gerhard H. Braus,§ and Heinz Neumann*,† †

Free Floater (Junior) Research Group “Applied Synthetic Biology”, Institute for Microbiology and Genetics, Georg-August University Göttingen, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany ‡ Institute for Organic and Biomolecular Chemistry, Georg-August University Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany § Institute for Microbiology and Genetics, Georg-August University Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany S Supporting Information *

ABSTRACT: Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous production of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the production of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The production of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 containing up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.

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K12) are extensively acetylated directly after synthesis and play a role in chromatin assembly.11,12 Acetylations of core residues of newly synthesized histones, for example, H3 K56ac and H4 K91ac, affect the stability of nucleosomes and contribute to the assembly process.13,14 A prerequisite for the analysis of the effect of PTMs on protein function is the ability to produce cleanly modified protein. Synthetic peptides carrying the modification can be fused to the remainder of the protein by native chemical ligation.4 This approach is technically limited to modifications close to one of the termini of the protein. Enzymatic modification usually suffers from the rather low specificity of the enzymes in vitro and requires the knowledge and availability of the enzymes involved. Genetic code expansion facilitates the direct encoding of PTMs in the protein of interest.15 This is achieved by the addition of an evolved aminoacyl-tRNA synthetase/tRNA pair to the protein biosynthesis machinery of the host organism. The tRNA is designed to decode nonsense or frameshift codons, while the synthetase is evolved to specifically recognize an unnatural amino acid with the result that the modified organism decodes the respective codons with

rotein function is regulated by a plethora of posttranslational modifications (PTMs). Histone proteins, despite their small size, are especially rich in PTMs, hosting examples for almost every type discovered so far. This has led to the postulation of the histone code hypothesis, which assumes that specific combinations of PTMs, especially on the flexible amino-terminal tails, regulate downstream events, for example, by recruiting effector proteins to chromatin.1,2 Lysine residues, which are abundant in histones, experience a wide variety of different modification states, such as methylation, ubiquitination, or acetylation. The latter type neutralizes the positive charge of the side chain, often resulting in weakening histone−DNA contacts, and also acts as a recognition motif for specialized protein domains. Several important acetylation sites in histones have been characterized. H4 K16ac has been shown to control the interaction of the H4 amino-terminal tail with an acidic surface of neighboring nucleosomes. This interaction is important for nucleosome fiber compaction in vitro3,4 and contributes an important driving force to the condensation of chromosomes in mitosis.5 Unsurprisingly, imbalanced levels of H4 K16ac are associated with severe consequences, such as cancer, defective DNA repair, and premature senescence.6−8 H4 K20ac has been identified in A. thaliana9 and H. sapiens10 and is assumed to modulate chromatin condensation similar to H4 K16ac. Other lysine residues in the H4 tail (H4 K5 and © XXXX American Chemical Society

Received: October 7, 2014 Accepted: January 15, 2015

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DOI: 10.1021/cb501011v ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Design and characterization of an expression system for recombinant acetylated histone H4. (A) His6-H3(Δ93−98)-TEV-H4 fusion protein is expressed in E. coli. The purified protein is cleaved with TEV protease. During histone octamer reconstitution the H3 fragment precipitates, while H4 is incorporated. (B) Expression test. Two different fusion constructs with an amber codon replacing K16 of H4 were expressed in C321.ΔA.exp cells23 in the presence or absence of 10 mM AcK and 20 mM NAM. Whole cell lysates were analyzed by SDS-PAGE and stained with Instant Blue. (C) Purification and TEV cleavage of His6-H3(Δ93−98)-TEV-H4 K16ac. The fully cleaved products were analyzed by SDSPAGE and stained with Instant Blue. (D) Reconstitution of histone octamers. The cleavage products from C were used to reconstitute octamers by salt gradient dialysis. The precipitate formed during dialysis (1) and octamer fractions from either unmodified H4 (2) or H4 K16ac (3) were analyzed by SDS-PAGE and stained with Instant Blue or detected in Western blots with anti-H4 K16ac antibodies.

the unnatural amino acid. This approach has been adapted to introduce several PTMs genetically, such as tyrosine sulfation and nitration or lysine methylation and acetylation.16−19 Acetylated versions of three of the four core histones, H2A, H2B, and H3, have been produced efficiently in this way.14 However, the same strategy has failed so far to produce significant amounts of histone H4. Acylation of lysine residues is not limited to acetyl groups. In fact, numerous other acylations have been observed, such as propionylation, butyrylation, crotonylation, myristoylation, malonylation and succinylation.20−22 In many cases the enzymes involved in the addition or removal of the modifications (as far as they have been studied) are the same or similar to the ones that acetylate the proteins. Seemingly, lysine acetyltransferases (KATs) lack, to varying degrees, the specificity to discriminate between differently acylated CoA molecules. Few detailed studies addressing the differential biochemical consequences of these newly discovered acylation types have been performed so far and even less is known about how they affect physiological processes. Here we report a method to produce milligram quantities of recombinant acetylated histone H4 and use it to reconstitute nucleosomes with defined modifications of the H4 tail. We combine this with the incorporation of alternative acyl modifications on the same residue to study the ability of commercially available antibodies to discriminate between different acylation states. Our initial attempts to produce acetylated histone H4 by genetic code expansion in the same way as other core histones did not result in detectable amounts of protein. We speculated that this might be due to H4 being less insoluble than the other core histones and, when expressed in reduced amounts, being lost to degradation. Therefore, we constructed a gene fusion

coding for H3 connected to H4 by a linker containing a TEV protease cleavage site under the control of an arabinose inducible promoter in the backbone of pCDF-PylT (Figure 1A). We reasoned that this protein would form inclusion bodies as efficiently as H3 alone. To prevent the H3 part of the molecule from incorporating into histone octamers during refolding, we introduced different deletions into the histone fold domain of H3. All fusion proteins expressed efficiently in E. coli (Supplementary Figure 1). Using MbAcKRS3 AcK was efficiently incorporated into His6-H3(Δ93−98)-TEV-H4 containing an amber codon in place of the codon for K16 of H4 (Figure 1B). The incorporation was dependent on the presence of AcK in the growth medium. The protein was purified under denaturing conditions and cleaved with TEV protease (Figure 1C). Typical yields were approximately 1 mg of H4 per liter of growth medium. The use of an amber codon free RF1 deletion strain (C321.ΔA.exp23) improved yields by a factor of 4 over conventional strains (Supplementary Figure 2). Histone octamers were refolded by salt gradient dialysis using equal molar amounts of all four core histones (Figure 1D). No further purification of the H3−H4 TEV cleavage reaction was necessary, as the H3 fragment precipitated during the dialysis step and was removed by ultrafiltration. Specific incorporation of AcK at the desired site was confirmed by MS/MS of tryptic peptides (Supplementary Figure 3). Recently, production of H4 K16ac by genetic code expansion has been described using an E. coli strain lacking RNase E to stabilize H4 mRNA.24 Instead of the traditionally used Xenopus H4, this report produced Drosophila H4, which is more stably expressed in E. coli (personal unpublished observations). We therefore tested the expression of our fusion construct in the background of an RNase E deletion strain but did not obtain increased amounts of protein (Supplementary Figure 4). B


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active site residues Ala267, Tyr271, Leu274, Cys313, and Met315 to NNK (N = A, T, G, C and K = T or G). We performed three rounds of positive and negative selection (pos./neg./pos.) using reporter constructs with chloramphenicol acetyltransferase or barnase, interrupted by amber codons to identify MbPylRS variants accepting PrK or CrK. Individual mutants were tested for their ability to survive increasing concentrations of chloramphenicol (Supplementary Figure 6). Mutants showing amino acid-dependent growth were used to suppress an amber codon at position 4 in the sperm whale myoglobin gene of pMyo4TAG. From these experiments we identified MbPylRS variants that efficiently incorporated PrK or CrK (Figure 3B). We analyzed the cross-specificity of the evolved synthetase variants and compared them to a previously evolved AcK specific variant (MbAcKRS3,14) and the wild-type enzyme (Table 1). In the presence of 2 mM PrK the evolved MbPrKRS produced similar amounts of myoglobin as the wildtype enzyme in the presence of 2 mM N(ε)-boc-lysine, which is an efficient substrate analogue. Both, MbPylRS and MbAcKRS3, were able to incorporate PrK in myoglobin although less efficiently. The evolved MbCrKRS incorporated CrK in myoglobin with the same efficiency as the wild-type enzyme, while MbAcKRS3 was not able to use CrK. Subsequently we tested the MbPylRS variants for their ability to incorporate butyryllysine (BuK) in myoglobin. The wildtype enzyme performed best in this case with MbCrKRS showing similar activity. None of the newly evolved enzymes showed improved incorporation of AcK. All myoglobin proteins showed specific incorporation of the encoded amino acids by high-resolution ESI-FTICR mass spectrometry (Supplementary Figure 7). In further studies we therefore used MbPrKRS for the incorporation of PrK and MbPylRS to incorporate CrK or BuK. We produced histone H4 with different modifications at lysine-16 and used them to test the specificity of commercial antibodies in Western blots (Figure 3C−E). We found that anti-Kcr antibodies efficiently recognize H4 K16cr. At high concentration (1:4000) the same antibodies also recognize H4 K16bu, while K16ac and K16pr are not, or very inefficiently, detected. Anti-H4 K16ac antibodies recognized H4 K16ac and less efficiently K16pr and K16bu, while K16cr was hardly detected. To rule out the possibility that the histones produced by our method contained a mixture of BuK and CrK (e.g., by enzymatic interconversion), we produced chemically butyrylated histone H4 (Supplementary Figure 8). Again, anti-Kcr and anti-H4 K16ac antibodies recognized butyrylated H4. The degree of specificity of these antibodies should be carefully considered when used in immunofluorescence and ChIP experiments. To demonstrate the efficiency of the system, we produced histone octamers containing H4 modified with acetyl, propionyl, butyryl, or crotonyl at K16 (Figure 3F). Analysis by SDS-PAGE and Western blot demonstrate the formation of stoichiometric octamers containing the desired modifications. We expect that the technical developments described in this study will facilitate the analysis of post-translational modifications on histone H4 by enabling the production of milligram quantities of modified histone octamers for biochemical experiments.

To test the efficiency of our system we created plasmids in which up to four lysine codons were replaced with amber codons and used them to express His6-H3(Δ93−98)-TEV-H4 containing multiple acetylations. We obtained 0.2−0.4 mg of multiply acetylated H4 per liter of medium containing 10 mM AcK (Supplementary Figure 2). A recent report prepared tetraacetylated H4 by in vitro translation using extracts from RF1 depleted BL21 DE3 cells.25 The yields of our approach and that of Mukai et al. are difficult to compare because of the differences in the production strategy. The advantage of the strategy reported here, however, is the simple scalability of cellbased expression. We prepared histone octamers homogeneously acetylated at H4 K16 or K20 to reconstitute nucleosome arrays (Widom 601-200-1226). The arrays reconstituted with H4 acetylated octamers formed with equal efficiency as unmodified arrays as measured by gel mobility shift and MNase digest (Supplementary Figure 5). Next, we analyzed their ability to compact in the presence of increasing concentrations of Mg2+ (Figure 2).

Figure 2. Acetylation of H4 K20 has little influence on nucleosome array condensation. Nucleosome arrays were incubated with increasing concentrations of MgCl2 and precipitated arrays removed by centrifugation. The fraction of nucleosome arrays still in solution was quantified photometrically (OD260) and plotted against MgCl2 concentration, normalized to a control without MgCl2.

As expected, H4 K16ac arrays required significantly higher Mg2+ concentrations than unmodified arrays, consistent with previous observations.4 Surprisingly, the Mg2+ concentration needed to precipitate H4 K20ac arrays was only marginally higher than for unmodified arrays. Apparently, acetylation of H4 K20, which forms water mediated contacts to the acidic patch of a neighboring nucleosome in the crystal structure27 and in an MD simulation,28 has little influence on array condensation, indicating that the major driving force for the interaction with the acidic patch is contributed by H4 K16 and the surrounding residues. Instead, H4 K20ac may act by interfering with methylation of the same site, which functions in multiple processes, such as regulation of gene expression, chromatin condensation, and DNA damage repair.29 Alternatively to acetylation several other lysine acylations have been observed on histone proteins20−22 (Figure 3A). Genetic code expansion offers the possibility to produce the modified histones by encoding the corresponding lysine derivatives. Carell and co-workers used wild-type PylRS from Methanosarcina mazei to produce histone H3 K9 modified with propionyl, butyryl, or crotonyl groups,30 while Schultz and coworkers evolved M. barkeri PylRS to incorporate crotonyllysine (CrK) to produce H2B K11cr.31 We created a library of approximately 30 million MbPylRS mutants by randomizing the

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METHODS

General Methods. Information on cloning, creation, and screening of MbPylRS library, mass spectrometric analyses, and production C


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Figure 3. Installation of PrK, BuK, and CrK on recombinant proteins by genetic code expansion and recognition of H4 K16 modifications by antibodies. (A) Structures of acetyllysine (AcK), propionyllysine (PrK), butyrylysine (BuK), and crotonyllysine (CrK). (B) Sperm whale myoglobin was expressed from pMyo4TAG in E. coli BL21 DE3 transformed with the indicated MbPylRS variant (Wt, MbPylRS; Ac, MbAcKRS3; Pr, MbPrKRS; Cr, MbCrKRS) in the presence of the indicated concentration of unnatural amino acid (in mM). Total proteins were analyzed by SDSPAGE and Western blot using anti-His antibodies. (C) Production of recombinant histone H4 with different acylations at Lys-16. Proteins were expressed and purified as described in the Methods section, cleaved with TEV protease, analyzed by SDS-PAGE, and stained with Instant Blue. (D) Anti-Kcr antibodies recognize H4 K16cr and H4 K16bu. Equal amounts of recombinant H4 with the indicated modification at K16 were analyzed by SDS-PAGE and Western blot using anti-Kcr antibodies. (E) Anti-H4 K16ac antibodies recognize H4 K16ac, K16pr, and K16bu. Recombinant H4 K16 with the indicated modifications was analyzed as in panel D using anti-H4 K16ac antibodies. (F) Histone octamers reconstituted with H4 variants containing the indicated modifications are analyzed by SDS-PAGE and Western blot using anti-Kcr and anti-H4 K16ac antibodies.

Table 1. Sequence and Amino Acid Selectivity of MbPylRS Variants amino acid residue

selectivity

enzyme

266

270

271

274

313

315

AcK

PrK

BuK

CrK

MbPylRS MbAcKRS3 MbPrKRS MbCrKRS

Leu Met Leu Leu

Leu Ile Leu Leu

Tyr Phe Phe Tyr

Leu Ala Leu Leu

Cys Phe Thr Val

Met Met Met Tyr

− + − −

+ + ++ +

++ − − +

++ − + +

allowed to grow at 37 °C with shaking, until an OD600 > 1.0. The culture was then supplemented with 10 mM acetyllysine (AcK) and 20 mM nicotinamide (NAM) and incubated for another 30 min before induction with 0.2% arabinose. Protein expression was carried out at 37 °C with shaking for 16 h. Cells were washed with PBS containing 20 mM NAM. Other acylated versions of histone H4 were produced by the same protocol using the appropriate MbPylRS variant and 2 mM acylated lysine. Cells were lysed in 30 mL of PBS containing 20 mM NAM, 1 mM DTT, 1 mM PMSF, 1xPIC protease inhibitor cocktail, and 0.2 mg

of recombinant proteins and nucleosomes can be found in the Supporting Information as well as a list of E. coli strains (Supplementary Table 1), primers (Supplementary Table 2), and antibodies (Supplementary Table 3). Production and Purification of Recombinant Histones. Recombinant expression of acetylated H4 fusion proteins was performed in C321.ΔA.exp cells (Addgene, #49018),23 transformed with pBK-AcKRS314 and the appropriate amber mutant vectors, in LB broth supplemented with 50 μg mL−1 kanamycin and 75 μg mL−1 spectinomycin. Cells were inoculated from an overnight culture and D


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ACS Chemical Biology mL−1 lysozyme. Cells were incubated at 37 °C for 20 min with shaking and then lysed by high-pressure homogenization on ice. Lysates were centrifuged (15 min, 18000 rpm, Beckmann JA-20) and washed in PBS supplemented with 20 mM NAM, 1 mM DTT, and 1% (w/v) Triton X-100. Inclusion bodies were collected as above, washed (PBS with 20 mM NAM and 1 mM DTT), macerated in 1 mL of DMSO, and incubated at RT for 30 min. Proteins were extracted from inclusion bodies by shaking for 1 h at 37 °C in 30 mL of a mixture of 6 M guanidinium chloride, 20 mM Tris (pH 8.0), and 2 mM DTT, centrifuged as above, and loaded onto a Ni2+-NTA column. The column was washed with 100 mL of 8 M urea, 100 mM NaH2PO4, and 1 mM DTT (pH 6.2). Proteins were eluted with 7 M urea, 20 mM sodium acetate, 200 mM NaCl, and 1 mM DTT (pH 4.5). The eluates were dialyzed against 5 mM β-mercaptoethanol, supplemented with 50 mM Tris-HCl (pH 7.4) and 20 μg mL−1 TEV protease, and incubated until cleavage was complete (monitored by SDS-PAGE). Salts were removed as above and the protein lyophilized.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

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S Supporting Information *

Additional methods, tables, and figures. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J. Chin for plasmids, G. Church for E. coli strain C321.ΔA.exp, and K. Hiragami-Hamada for help in reconstituting nucleosome arrays. B.J.W. is grateful for a postdoctoral fellowship of the Alexander von Humboldt Foundation. Research in the laboratory of H.N. is funded by the German Research Foundation (Emmy-Noether Programme) and the Freefloater-Programme of the University of Göttingen, the Cluster of Excellence and DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain, and the Fonds der Chemischen Industrie.

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Genetically Encoding Lysine Modifications on Histone H4 - ACS

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