Site-specific Incorporation of ε-N-crotonyllysine into Histones

Histones, the major protein components of chromatin, are extensively altered by post-translational modifications (PTMs). Deciphering distinctive patterns of these PTMs (namely the ‘histone code’) is key to understand the epigenetic regulation of diverse biological processes.^[1] For example, lysine acetylation is one of the most common PTMs found in histones, and its regulation has been demonstrated to be closely related to DNA replication/repair, transcriptional regulation of genes, and the status of chromatin assembly.^[2] Misregulation of chromatin PTM is linked to various human diseases such as cancer^[3], central nervous system (CNS) disorders^[4], and autoimmune diseases.^[5] Therefore, the enzymes responsible for lysine acetyl modifications, including histone acetyltransferases (HATs) and histone deacetylases (HDACs), are regarded as important drug targets.^[6]

Besides acetylation, other known lysine modifications in histones include methylation, ribosylation, ubiqutination, and sumoylation.^[7] Recently, Tan and co-workers described the identification of a new type of histone PTM, lysine crotonylation, using a mass spectrometry-based proteomics approach.^[8] This novel PTM is largely associated with transcriptionally active chromatin, and principally interacts with promoter and enhancer regions. Despite their structural similarity, lysine crotonylation substantially differs from lysine acetylation in genomic distribution and regulation. For example, the overexpression of two different histone acetyltransferases (HATs), CPB and p300, had no effect on cellular histone crotonylation levels, and histone lysine deacetylases (HDACs) 1, 2, 3, and 6 showed weak or no lysine decrotonylation activity.^[8] These observations suggest that the two PTMs are likely regulated by separate sets of modifying enzymes and, hence, may possess distinctive cellular functions. The ability to generate histones with well-defined crotonyl modifications at specific lysine residues would facilitate a better understanding of the physiological roles and regulation of this novel PTM.^[9]

A number of different approaches have been developed to prepare histones with defined PTMs.^[10] Attempts to directly modify histones by enzymatic reactions^[11] are limited either by the promiscuous nature of histone modifying enzymes or by incomplete in vitro reactions that result in a heterogeneous mixture.^[12] Semi-synthetic approaches using native chemical ligation (NCL)^[13] and expressed protein ligation (EPL)^[14] have been used successfully to generate site-specifically modified histones.^[15] Additional chemical methods include the introduction of PTM analogues via cysteine-based chemistries^[16], or the genetic incorporation and subsequent modification of reactive unnatural amino acids.^[17] These chemical approaches allow the selective, facile preparation of proteins with chemically defined PTMs in vitro. A complementary approach involves the genetic incorporation of post-translationally modified amino acids into histones directly in living cells using the endogenous translational machinery. Herein, we report the preparation of site-specifically crotonyl-modified histones by means of an orthogonal aminoacyl-tRNA synthetase and tRNA pair, which was evolved to site-specifically incorporate ε-N-crotonyllysine (1) into proteins in response to amber codons in Escherichia coli. Furthermore, the evolved pair was used to overexpress a homogeneously crotonylated protein in mammalian cells.

ε-N-crotonyllysine (1, Kcr) was synthesized from unprotected lysine in two steps in > 80 % overall yield. Alpha-amino and carboxylic acid groups of lysine were transiently protected by chelation with copper (II) ion in basic aqueous solution^[18], and the ε-amino group was subsequently reacted with N-hydroxysuccinimide (NHS) ester of (E)-but-2-enoic (crotonic) acid (2) (Scheme 1). Orthogonal Methanosarcina barkeri tRNA/pyrrollysyl-tRNA synthetase (Mb-PylRS) pairs have been used to encode a variety of lysine analogues, including lysine PTMs such as acetyl lysine^[19] and methyl lysine.^[17d] In order to evolve an Mb-PylRS that can efficiently charge Kcr to Pyl-tRNA, we used an Mb-PylRS library^[20] randomized at residues Leu270, Tyr271, Leu274, and Cys313 (with a fixed Tyr349Phe mutation^[21]) to NNK (N=A, T, G, or C, K= T or G) obtaining a diversity of 1×10⁶ mutants (Supplementary Figure 1). A series of negative and positive selections were then performed in E. coli strain DH10B as previously described.^{[17b, 22]} In brief, in the negative selection, the Mb-PylRS library was introduced into DH10B cells harboring a plasmid containing the toxic barnase gene with amber mutations at two permissive sites (Gln2TAG, Asp44TAG), and cells were grown in the absence of Kcr to remove clones containing mutant synthetases that can recognize endogenous amino acids. The positive selection was carried out by subsequently transforming the library from the negative selection into DH10B cells containing a plasmid encoding chloramphenicol acetyltransferase with a Asp112TAG mutation. Cells were grown in the presence of 2 mM Kcr and 70 μg/mL chloramphenicol to select clones containing mutant synthetases that can recognize the unnatural amino acid. After two alternating negative and positive rounds of selection, single colonies of Mb PylRS mutants were obtained. The sequences of mutant synthetases from 20 colonies all converged to a unique clone: Kcr-RS (Leu270, Tyr271, Leu274Ala, Cys313Phe, Tyr349Phe), and this Kcr-RS was used for further studies.

Scheme 1.

Synthesis of ε-N-corotonyllysine (Kcr, 1)

To determine the fidelity and efficiency of Kcr incorporation into proteins, we expressed a mutant green fluorescent protein (GFP) using the Kcr-RS/Pyl-tRNA pair. In short, codon Asp149 in a C-terminal His-tagged GFP gene was mutated to TAG and was inserted into pLei vector containing a single copy of Pyl-tRNA. pBK-Kcr, which encodes the Kcr-RS gene was cotransformed into E. coli DH10B. Cells were grown in Luria-Bertani (LB) medium supplemented with and without 5 mM Kcr; 20 mM nicotinamide (NAM)^[19] was added after 1 mM IPTG induction at OD₆₀₀ 0.6. The protein was expressed overnight at 37 °C, followed by Ni-NTA affinity purification. SDS-PAGE analysis showed that full length GFP was produced in good yield in the presence of 5 mM Kcr (~10 mg/L), and not observed in the absence of Kcr (Figure 1A). The purified mutant GFP was also characterized by electrospray ionization mass spectrometry (ESI-MS); the observed molecular mass (27786) confirmed that Kcr was site-specifically substituted for Asp149 (Figure 1B).

Figure 1.

A) A mutnat Mb-Pyl Kcr-RS/Pyl-tRNA pair selectively and efficiently incorporates Kcr in response to the TAG codon: GFP-Asp149TAG was expressed with or without 5 mM Kcr (lane 1 and lane 2, respectively) in the presence of an Mb-Pyl Kcr-RS/Pyl-tRNA pair. GFP mutant was purified by Ni-NTA affinity chromatography, analyzed by SDS-PAGE and stained with Coomassie Blue. B) ESI-MS analysis of purified mutant GFP; the peak corresponds to full length GFP-Asp149Kcr-His₆ (expected: 27789, observed: 27786).

Interestingly, when the mutant GFP is expressed in the absence of NAM, ESI-MS analysis shows two peaks: one small peak corresponds to the GFP with Kcr incorporated, and the second large peak has a mass 68 Da lower (Supplementary Figure 2), which corresponds to a GFP mutant bearing a lysine at Asp149. Previous studies have shown that CobB, a sirtuin family nicotinamide adenine dinucleotide (NAD)-dependent enzyme, can deacetylate acetyllysine (Kac) containing proteins in E. coli; the enzyme can be inhibited by high concentration (~20 mM) of NAM.^[19] We speculate that CobB may also possess decrotonylation activity, and when we expressed the mutant GFP in the presence of 20 mM NAM, indeed a single MS peak of mutant GFP with intact Kcr was obtained (Supplementary Figure 2).

We next expressed a site-specifically crotonylated human histone subunit 2B (H2B) in E. coli. Multiple lysine crotonylation sites have been identified in H2B.^[8] We choose to substitute Kcr for K11, since the N-terminal domain of histone is known to play an essential role in modulating nucleosome structures.^[23] Furthermore, Lys11 residue is a unique crotonylation site while other residues (K5, K12, and K15) can be acetylated or crotonylated. The human H2B gene with an N-terminal His-tagged and Lys11TAG mutation (His₆-H2B-K11TAG) was inserted into pCDF vector^[24] containing a single copy of Pyl-tRNA. pBK-Kcr, which encodes the Kcr-RS gene was cotransformed into E. coli BL21(DE3) strains. Cells were grown in LB medium supplemented with and without 5 mM Kcr; 20 mM NAM was added after induction with 1 mM IPTG at OD₆₀₀ 0.8. After overnight expression at 37 °C, cells were harvested and lysed with BugBuster (EMD), and protein was purified with Ni-NTA agarose beads (Qiagen) under denaturing conditions. SDS-PAGE analysis showed that full length H2B was produced in good yield in the presence of 5 mM Kcr (~2 mg/L) while full length His₆-H2B was not observed in the absence of Kcr (Figure 2A). Western blot analysis was carried out to confirm the incorporation of Kcr using an anti-corotonyllysine polyclonal antibody (PTM BioLab); Both His₆-H2B-wt and His₆-H2B-K11Kcr revealed a band with the anti-His₆ antibody, but only the His₆-H2B-K11Kcr mutant was detected with the anti-crotonyllysine antibody (Figure 2A). In addition, the purified mutant H2B was also characterized by electrospray ionization mass spectrometry (ESI-MS); the observed mass (16071) confirmed that Kcr was site-specifically substituted for Lys11 (Figure 2B).

Figure 2.

A) Selective incorporation of Kcr into recombinant human histone H2B: Wild type H2B was expressed (lane 1); the His₆-H2B K11Kcr mutant was expressed with or without 5 mM Kcr (lane 2 and 3, respectively). Proteins were purified by Ni-NTA affinity chromatography under denaturing conditions and analyzed by SDS-PAGE. The gel was stained with Coomassie Blue (upper). Western blot analysis was carried out with anti-crotonyllysine (middle) and anti-His₆ antibodies (lower) to confirm the specific incorporation of Kcr. B) ESI-MS analysis reveals a mass corresponding to His₆-H2B K11Kcr with the N-terminal Met removed (expected: 16020, observed: 16017).

It also has been shown that the Mb-PylRS/Pyl-tRNA pair is orthogonal in mammalian cells.^[20] We therefore next demonstrated the feasibility of shuttling the Kcr-RS/Pyl-tRNA system from E. coli to mammalian cells for expressing homogeneous crotonylated proteins in the latter cells. A plasmid pCMV-Kcr containing Kcr-RS and Pyl-tRNA under the CMV and U6 promoters, respectively, was transiently co-transfected with a plasmid pEGFP-Tyr39TAG containing a C-terminal His-tagged enhanced green fluorescent protein (EGFP) gene under the CMV promoter into HEK293T cells. Cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) media supplemented with 10 % FBS (fetal bovine serum) in the presence or absence of 1 mM Kcr for 48 hours. Analysis of the cells by fluorescence microscopy revealed bright green fluorescence only in the presence of 1 mM Kcr (Figure 3A). After confirming Kcr-dependent TAG suppression, we also purified EGFP proteins and confirmed the site-specific incorporation of Kcr by ESI-MS (Figure 3B, expected: 29713, observed: 29714)

Figure 3.

A) Selective incorporation of Kcr into EGFP-Y39TAG with the Mb-Pyl Kcr-RS/Pyl-tRNA pair in HEK293 cells; green fluorescent (top) and bright field (bottom) images were obtained to confirm the expression of full-length EGFP in the presence of 1 mM Kcr (left). The fluorescent signal was not observed in the absence of Kcr (right). B) ESI-MS analysis of the purified protein revealed a single mass corresponding to EGFP-Y39Kcr (expected: 29713, observed: 29714).

In conclusion, we have evolved an Mb-PylRS to site-specifically incorporate Kcr into proteins with high fidelity and efficiency both in E. coli and mammalian cells. The ability to produce histones with homogenous, site-specific Kcr modifications will be valuable in elucidating the biological role of this recently identified PTM.