Semisynthesis of site-specifically succinylated histone reveals that succinylation regulates nucleosome unwrapping rate and DNA accessibility.

Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.

Toolbox for chemically synthesized histone proteins.

Histone post-translational modifications play significant roles in gene regulation processes. Among many approaches, chemical protein synthesis has been a successful and promising method for the preparation of homogeneous products of site-specifically modified histones for elucidation of their biological significance. In this short review, we describe the recent advances in synthetic toolbox for histone proteins such as thioester precursors, chemical ubiquitination, and one-pot peptide ligation.

Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications.

Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable "lnc-tag," we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.

PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome.

The poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

Chemical Synthesis of Cys-Containing Protein via Chemoselective Deprotection with Different Palladium Complexes.

We report selective removals of N-terminal and internal Cys protecting groups using different palladium complexes to facilitate the efficient chemical protein synthesis. Utilizing the orthogonal deprotection pairs, we accomplished chemical synthesis of histone H3 containing trimethylated Lys through the combination of Pd(0)-mediated Alloc deprotection for one-pot multiple peptide ligation and Pd(II)Cl2-mediated Acm deprotection to recover native Cys residues after desulfurization.

Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage.

Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a nonhydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B.

A Modular Ligation Strategy for Asymmetric Bivalent Nucleosomes Trimethylated at K36 and K27.

In nature, individual histones in the same nucleosome can carry identical (symmetric) or different (asymmetric) post-translational modification (PTM) patterns, increasing the combinatorial complexity. Embryonic stem cells exhibit "bivalent" nucleosomes, some of which are marked by an asymmetric arrangement of H3K36me3 (an activating PTM) and H3K27me3 (a repressive PTM). Here we describe a modular synthetic method to access such asymmetrically modified nucleosomes and show that H3K36me3 inhibits the activity of the methyltransferase PRC2 locally while still prolonging its chromatin binding time.

Molecular Mechanism of the Pin1-Histone H1 Interaction.

Pin1 is an essential peptidyl-prolyl isomerase (PPIase) that catalyzes cis-trans prolyl isomerization in proteins containing pSer/Thr-Pro motifs. It has an N-terminal WW domain that targets these motifs and a C-terminal PPIase domain that catalyzes isomerization. Recently, Pin1 was shown to modify the conformation of phosphorylated histone H1 and stabilize the chromatin-H1 interaction by increasing its residence time. This Pin1-histone H1 interaction plays a key role in pathogen response, in infection, and in cell cycle control; therefore, anti-Pin1 therapeutics are an important focus for treating infections as well as cancer. Each of the H1 histones (H1.0-H1.5) contains several potential Pin1 recognition pSer/pThr-Pro motifs. To understand the Pin1-histone H1 interaction fully, we investigated how both the isolated WW domain and full-length Pin1 interact with three H1 histone substrate peptide sequences that were previously identified as important binding partners (H1.1, H1.4, and H1.5). NMR spectroscopy was used to measure the binding affinities and the interdomain dynamics upon binding to these sequences. We observed different KD values depending on the histone binding site, suggesting that energetics play a role in guiding the Pin1-histone interaction. While interdomain interactions vary between the peptides, we find no evidence for allosteric activation for the histone H1 substrates.

Triple Function of 4-Mercaptophenylacetic Acid Promotes One-Pot Multiple Peptide Ligation.

One-pot multiple peptide ligation is a key technology to improve the efficiency of chemical protein synthesis. One-pot repetitive peptide ligation requires a cycle of three steps: peptide ligation, removal of a protecting group, and inactivation of the deprotection reagent. However, previous strategies are not sufficient because of harsh deprotection conditions, slow deprotection rates, and difficulty in quenching the deprotection reagent. To address these issues, we developed a rapid, efficient deprotection and subsequent quenching strategy using an allyloxycarbonyl group to protect the N-terminal cysteine residue. 4-Mercaptophenylacetic acid (MPAA), a thiol additive for native chemical ligation, functioned not only as a scavenger for π-allyl palladium complexes, but also as a quencher of palladium(0) complexes. By utilizing the multifunctionality of MPAA, we carried out a one-pot five-segment ligation to afford histone H2AX (142 amino acids), which was isolated in 59 % yield.

Palladium prompted on-demand cysteine chemistry for the synthesis of challenging and uniquely modified proteins.

Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site. Here, we demonstrate the tuning of palladium chemoselectivity in aqueous medium for on-demand deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. These tools allow the preparation of highly complex analogues as we demonstrate in the synthesis of the copper storage protein and selectively modified peptides with multiple Cys residues. We also report the synthesis of an activity-based probe comprising ubiquitinated histone H2A and its incorporation into nucleosomes and demonstrate its reactivity with deubiquitinating enzyme to generate a covalent nucleosome-enzyme complex.

Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action.

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module's molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Inhibition of Methylglyoxal-Induced Histone H1 Nε-Carboxymethyllysine Formation by (+)-Catechin.

Reactive dicarbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are common intermediates in protein damage, leading to the formation of advanced glycation end products (AGEs) through nonenzymatic glycation. (+)-Catechin, a natural plant extract from tea, has been evaluated for its ability in trapping GO and MGO. However, (+)-catechin is also reported to have both antioxidant ability and pro-oxidant properties. Until now, whether (+)-catechin can inhibit the formation of nonenzymatic glycation and the mechanism of the inhibition in nucleoprotein nonenzymatic glycation is still unclear. In the present study, histone H1 and MGO were used to establish an in vitro (100 mM phosphate buffer solution (PBS), pH 7.4, 37 °C) protein glycation model to study the trapping ability of (+)-catechin. Our data show that MGO caused dose-dependent protein damage, and the content of MGO-induced Schiff base formation was inhibited by (+)-catechin when the molecular ratio of catechin:MGO was 1:6. The formation of Nε-carboxymethyllysine (CML) was reduced significantly when the ratio of (+)-catechin and MGO was 1:1, which was similar to the inhibition effect of aminoguanidine (AG). The formation of CML under in vitro conditions can be inhibited by low concentration (12.5-100 µM) of (+)-catechin but not with high concentration (200-800 µM) of (+)-catechin. The reason is that the high concentration of (+)-catechin did not inhibit CML formations due to H2O2 produced by (+)-catechin. In the presence of catalase, catechin can inhibit MGO-induced CML formation. In conclusion, the trapping ability of (+)-catechin may be more effective at the early stage of nonenzymatic glycation. However, a high concentration (200-800 µM) of (+)-catechin may not inhibit the formation of CML because it induced the increase of H2O2 formation.

Studies of biochemical crosstalk in chromatin with semisynthetic histones.

Reversible post-translational modifications of histone proteins in eukaryotic chromatin are closely tied to gene function and cellular development. Specific combinations of histone modifications, or marks, are implicated in distinct DNA-templated processes mediated by a range of chromatin-associated enzymes that install, erase and interpret the histone code. Mechanistic studies of the precise biochemical relationship between sets of marks and their effects on chromatin function are significantly complicated by the dynamic nature and heterogeneity of marks in cellular chromatin. Protein semisynthesis is a chemical technique that enables the piecewise assembly of uniformly and site-specifically modified histones in quantities sufficient for biophysical and biochemical analyses. Recent pioneering efforts in semisynthesis have yielded access to histones site-specifically modified by entire proteins, such as ubiquitin (Ub) and the small ubiquitin-like modifier (SUMO). Herein, we highlight key studies of biochemical crosstalk involving Ub and SUMO in chromatin that were enabled by histone semisynthesis.

Synthetic post-translational modification of histones.

Chromatin is the physiological template of genetic information in all eukaryotic cells, a highly organised complex of DNA and histone proteins central in regulating gene expression and genome organisation. A multitude of histone post-translational modifications (PTMs) have been discovered, providing a glance into the complex interplay of these epigenetic marks in cellular processes. In the last decade, synthetic and chemical biology techniques have emerged to study these modifications, including genetic code expansion, histone semisynthesis and post-translational chemical mutagenesis. These methods allow for the creation of histones carrying synthetic modifications which can in turn be assembled into designer nucleosomes. Their application in vitro and in vivo is now beginning to have an important impact on chromatin biology. Efforts towards introducing multiple labile modifications in histones as well as expanding their use in cellular biology promise new powerful tools to study epigenetics.

Chemical chromatin ubiquitylation.

Histone modifications dynamically regulate chromatin structure and function, thereby mediating many processes that require access to DNA. Chemical protein synthesis has emerged as a powerful approach for generating homogeneously modified histone analogues in workable amounts for subsequent incorporation into nucleosome arrays for biochemical, functional and structural studies. This short review focuses on the strength of total chemical protein synthesis and semisynthetic approaches to generate ubiquitylated histones in their native or non-native forms and the utility of these analogues to decode the role of ubiquitylation in epigenetics.

Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes.

Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester). This task is challenging, with obstacles relating to the preparation and ligation of hydrophobic peptides, as well as the requirement for multiple purification steps due to protecting-group manipulations during the polypeptide assembly process. To address this, our approach uses an easily removable solubilizing tag for the synthesis and ligation of hydrophobic peptides, as well as a more efficient and better-yielding method to remove Cys-protecting groups that uses palladium chemistry (specifically [Pd(allyl)Cl]2 and PdCl2 complexes). The utility of this approach is demonstrated in the syntheses of ubiquitinated H2B at Lys34, phosphorylated H2A at Tyr57 and unmodified H4. Each of these analogs can be prepared in milligram quantities within ∼20-30 d.

Total chemical synthesis of methylated analogues of histone 3 revealed KDM4D as a potential regulator of H3K79me3.

Histone H3 methylation plays an important role in regulating gene expression. In histones in general, this mark is dynamically regulated via various demethylases, which found to control cell fate decisions as well as linked to several diseases, including neurological and cancer. Despite major progress in studying methylation mark at various positions in H3 histone proteins, less is known about the regulation of methylated H3 at Lys79. Methylation at this site is known to have direct cross-talk with monoubiquitination of histone H2B at positions Lys120 and 34, as well as with acetylated H3 at Lys9. Herein we applied convergent total chemical protein synthesis to prepare trimethylated H3 at Lys79 to perform initial studies related to the regulation of this mark. Our study enabled us to identify KDM4D lysine demethylase as a potential regulator for trimethylated H3 at Lys79.

Development and crystallographic evaluation of histone H3 peptide with N-terminal serine substitution as a potent inhibitor of lysine-specific demethylase 1.

Lysine-specific demethylase 1 (LSD1/KDM1A) is a flavoenzyme demethylase, which removes mono- and dimethyl groups from histone H3 Lys4 (H3K4) or Lys9 (H3K9) in complexes with several nuclear proteins. Since LSD1 is implicated in the tumorigenesis and progression of various cancers, LSD1-specific inhibitors are considered as potential anti-cancer agents. A modified H3 peptide with substitution of Lys4 to Met [H3K4M] is already known to be a potent competitive inhibitor of LSD1. In this study, we synthesized a series of H3K4M peptide derivatives and evaluated their LSD1-inhibitory activities in vitro. We found that substitutions of the N-terminal amino acid with amino acids having a larger side chain were generally not tolerated, but substitution of Ala1 to Ser unexpectedly resulted in more potent inhibitory activity toward LSD1. X-ray crystallographic analysis of H3K4M derivatives bound to the LSD1·CoREST complex revealed the presence of additional hydrogen bonding between the N-terminal Ser residue of the H3 peptide derivative and LSD1. The present structural and biochemical findings will be helpful for obtaining more potent peptidic inhibitors of LSD1.

The convergent chemical synthesis of histone H3 protein for site-specific acetylation at Lys56 and ubiquitination at Lys122.

Deposition of (H3-H4)2 tetramers is believed to be the critical step in nucleosome assembly. Site-specific acetylation and ubiquitination of histone H3 have been speculated to synergistically facilitate the formation and deposition of (H3-H4)2 tetramers. Here we report our endeavors toward the first chemical synthesis of homogenous histone H3, which bears Lys56 acetylation and Lys122 ubiquitination, for in vitro biochemical and biophysical studies.