Proteins are subject to numerous post-translational modifications (PTMs) that can alter the chemical structure, and hence function, of the molecule. The astonishing diversity of PTMs possible on proteins is exemplified by histones, nuclear proteins that form the protein core of the nucleosome particle. Histones can be modified in a variety of ways including acetylation, phosphorylation, methylation, ADP-ribosylation and ubiquitylation. Moreover, many, if not all, of these modifications can occur in combination.[4, 5] Indeed, there is growing evidence that functional cross-talk between histone PTMs is essential for the regulation of gene expression and ultimately cell fate and identity. Biochemical studies into the role of histone PTMs are often confounded by the difficulty associated with obtaining large quantities of homogeneously modified proteins. For this reason chemical approaches to obtaining post-translationally modified histones have received considerable attention in recent years.[7–9] Among the available strategies, the protein ligation approach, expressed protein ligation (EPL), offers the most flexibility in terms of the number and type of PTMs that can be incorporated. To date, EPL has been used to generate phosphorylated, acetylated, and methylated forms of histone H3, acetylated H4, and ubiquitylated H2B. Nonetheless, many modified histones have yet to be accessed using semi-synthesis. A notable case in point is the N-terminal region of H2B, which has been described to possess several PTMs, including (poly)lysine acetylation and serine 14 phosphorylation, which have been implicated in transcription and apoptotic chromatin compaction, respectively. Differentially modified semi-synthetic H2B analogs would be useful to assess the affect of acetylation on both antibody recognition as well as on the efficiency of phosphorylation. In this report, we describe a general semi-synthetic route to H2B that allows the installation of PTMs into an otherwise native polypeptide background.
Previous efforts to generate semi-synthetic H2B have focused on C-terminal modifications. Therefore, to obtain N-terminally modified H2B analogs we first needed to develop a suitable ligation strategy. In designing a useful semi-synthetic route, we wanted to employ a traceless-ligation method (i.e. one that would ultimately yield the native H2B amino acid sequence) that would be compatible with the most common PTMs, and allow synthetic access to several known modifications, especially phospho-serine 14 and neighboring Nε-acetyllysines. EPL is a semi-synthetic version of native chemical ligation (NCL) and as such requires the presence of a cysteine residue at the desired ligation junction. However, H2B lacks any native cysteine residues, regardless of the species of origin. Thus, it is necessary to introduce a non-native cysteine for the purpose of ligation and thereafter remove it. We have previously shown that chemical desulfurization is an efficient way to convert a non-native cysteine to a native alanine in the context of H2B. Conveniently, H2B contains an alanine at position 17, suggesting that the native H2B sequence could be generated by desulfurization of the product of a ligation between a synthetic peptide, corresponding to H2B residues 1–16, and a recombinant polypeptide corresponding to residues 18–125 of H2B preceded by a cysteine (Scheme 1).
NCL, and by extension, EPL requires the presence of an α-thioester group on the N-terminal peptide fragment. Typically, these α-thioesters are incorporated into synthetic peptides through Boc-based solid-phase peptide synthesis (SPPS). Problematically, serine/threonine phosphate esters are labile to the HF cleavage conditions used in of Boc-SPPS. To address this incompatibility, we turned to two recently described peptidyl-resin linker systems that allow the synthesis of peptide α-thioesters by Fmoc-based SPPS. To avoid the rapid hydrolysis of thioesters during the basic conditions of Fmoc-deprotection, these new linkers are based on the concept of a latent thioester that can be unmasked following chain elongation. One linker, 2-hydroxy-3-mercaptopropionic acid, was developed by Botti et al. and involves chain-assembly off a stable peptide-(oxy)ester that can later be converted to the corresponding α-thioester under reducing conditions. Previously we employed this methodology to synthesize cyclic thioester peptide inhibitors of transmembrane receptors involved in S. aureus virulence. The second linker, diaminobenzoic acid, was developed by Dawson and colleagues. In this case, the linker is acylated post chain-assembly to give an active N-acylurea moiety, which, following cleavage, can be displace by thiols in a strategy analogous to the safety-catch linker developed by Kenner et al. We successfully applied these two linker strategies to synthesize three differentially modified α-thioester peptides corresponding to residues 1–16 of H2B, 1a, 1b, and 1c (Figure S1). Of note, these linkers also bypass a complication associated with previously described syntheses of N-terminally modified histones,[7, 8, 11] namely the possibility of epimerization of C-terminal residues during solution phase α-thioester formation.
The C-terminal portion of H2B, 2, was obtained through recombinant expression of a fragment comprising residues 18–125, preceded by a methionine-cysteine dipeptide. Overexpression in E. coli resulted in the spontaneous removal of the initiator methionine and exposure of the N-terminal cysteine. Purification and characterization of this protein, however, revealed +28 and +72 mass shifts suggestive of the presence of thiazolidine adducts of the cysteine. This was confirmed by in vitro methoxylamine treatment, which led to the clean conversion to a single species with mass consistent with the expected protein, 2 (Figure S2). With the synthetic and recombinant building blocks in hand, EPL was carried out through incubation of 1.5 equivalents of 1a, 1b or 1c with 1.0 equivalent of 2. The reactions were monitored by HPLC and ESI-MS, and were found to be >80% complete (based on consumption of 2) after 29–44 hours (Figure S3). Full length modified H2B proteins (3a, 3b, and 3c) were purified by RP-HPLC and their identities confirmed by MS. To obtain the native H2B sequence, in each case the purified material was treated with activated Raney-nickel as previously described. This selective desulfurization converts the lone cysteine used for ligation to an alanine, rendering the ligation strategy traceless (Scheme 1). The desulfurization reaction was found to proceed smoothly for each of the H2B analogs, 3a–c, to give the final products, 4a–c (Figure 1). Ligations and desulfurizations typically generated 1.0–4.1 mg of 4a–c, corresponding to 25–45% overall isolated yield. Of note, the modified amino acids used here, namely phosphoserine (pSer) and Nε-acetyllysine (Lys(Ac)) are stable to the desulfurization procedure as no mass shift below that of expected was observed. Thus, we have established a strategy to generate H2B that is amenable to synthetic installation of phosphoryl and acetyl groups. In an effort to improve the yield of the final desulfurization step of our synthesis we compared a radical initiated homogeneous desulfurization procedure, described by Danishefsky and collegues, to the Raney nickel method. Comparative desulfurization of 3a revealed the radical initiated approach was amenable to phospho-proteins (Figure S4) and also resulted in a significant twofold increase in isolated yield of homogenous product (Table 1). The decreased yield with the Raney nickel procedure presumably arises from insoluble protein aggregation, which is lost during removal of nickel. These findings suggest that desulfurization of aggregation prone proteins, including histones, may be better suited to the radical initiated strategy.
Antibodies are a ubiquitous tool in the field of epigenetics and are commonly used to detect the presence of specific modifications from cell extracts. An important consideration when using these tools, however, is the effect of other modifications on antigen recognition. We decided to test a commercial polyclonal antibody (Upstate, 07–191), raised against a synthetic peptide corresponding to residues 6–16 of H2B with a pSer residue 14, against semi-synthetic H2B-pS14 (4a) and H2B-pS14-4xAc (4b). To assess the affect of acetylation on antigen recognition, 4a and 4b were resolved by SDS-PAGE, transferred to PVDF and probed with the α-H2B-pS14 antibody. As expected, semi-synthetic H2B-pS14, as well as endogenous H2B-pS14 obtained from apoptotic cells, was detected at the appropriate molecular weight (Figure 2A and 2B). However, introduction of acetyl groups at neighboring residues completely ablated all recognition of the phosphorylated protein (Figure 2A). This result highlights the limitations of antibodies in broad genome screens, such as ChIP on chip experiments, where histone proteins are heterogenous and often bear multiple PTMs. Recent mass-spectrometry based proteomic efforts have shown that H2B exists in several polyacetylated states.[25, 26] This MS data, together with our findings, complicates antibody dependent efforts to quantify endogenous levels of H2B-pS14.
To assess the effect of these acetyl groups on enzyme-substrate recognition we tested H2B-4xAc (4c) against human mammalian sterile twenty-like (Mst1) kinase. Mst1 has been demonstrated to phosphorylate H2B both in vivo and in vitro. The kinase domain (residues 1–330) of either wild-type or inactive (K59R) Mst1 with an N-terminal FLAG-tag was transfected into HEK-293 cells. Cells were then harvested and Mst1 was efficiently immunoprecipitated from the cytosolic fraction using α-FLAG agarose gel (Figure S5). Enriched Mst1 was then assayed against recombinant wild-type H2B and 4c substrates using 32P labeled ATP. Unlike the antibody, Mst1 is tolerant of the acetyl marks. The enzyme is able to recognize and phosphorylate both polyacetylated and unmodified H2B (Figure 2C and 2D). Phosphorylation is only observed in samples treated with active Mst1 and not the inactive K59R mutant enzyme (Figure 2C) suggesting the activity being detected is from Mst1 and not a low abundance contaminant from our pulldown. Furthermore, because a recombinant H2B-S14E mutant substrate displayed minimal phosphorylation (Figure 2C and 2D), the modification appears to be predominantly at serine 14.
In conclusion, we have developed a novel traceless ligation strategy for histone H2B that provides synthetic access to acetylated and phosphorylated analogs of the protein. To our knowledge, this is the first reported semi-synthetic strategy for N-terminally modified H2B. This strategy could be modified and applied to study other histone phosphorylation events including H2A-pS1, H3-pS10, and H4-pS1. The proteins synthesized here were then used to demonstrate that a commercial antibody raised against H2B-pS14 did not recognize this modification in the presence of acetylated lysine 5, 11, 12, and 15. This differed from in vitro kinase activity assays, where Mst1 was able to phosphorylate serine 14 on both unmodified and polyacetylated H2B. The work herein also highlights the importance of understanding the potential shortcomings inherent to antibody-generated data sets. The use of modification specific antibodies in the area of epigenetic research are of great value but the data generated should be considered in the context of broader studies that are beginning to elucidate the density of histone modifications.[4, 5, 31]