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Published in final edited form as: Curr Opin Chem Biol. 2018 Feb 27;45:27–34. doi: 10.1016/j.cbpa.2018.02.005

Studies of biochemical crosstalk in chromatin with semisynthetic histones

Calvin Jon Antolin Leonen 1, Esha Upadhyay 1, Champak Chatterjee 1,✉
PMCID: PMC6076846  NIHMSID: NIHMS948023  PMID: 29494828

Abstract

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.

Introduction

The past decade has witnessed an unprecedented surge in chemical biology efforts directed toward studying the regulation of eukaryotic chromatin by reversible chemical modifications [1]. Chromatin is a massive and dynamic nucleoprotein complex that stores vast amounts of genetic material, about 3 billion base pairs of DNA in humans, within the minute cell nucleus. The fundamental building block of chromatin is the nucleosome core particle (NCP), which consists of ~147 bp of double-stranded DNA wrapped about 1.6 turns around a globular protein spool consisting of the core histones H2A, H2B, H3, and H4 [2,3]. Extending outwards from the NCP, and hence exposed to the nucleosol, are the functional group rich histone N-termini or tails. Histone tails are the sites of a wide range of chemically distinct and reversible post-translational modifications (PTMs). These include varying degrees of arginine and lysine side-chain methylation (mono-, di-, and trimethylation), serine, threonine, or tyrosine phosphorylation, and lysine acylation. The last ranges from modification by smaller acetyl to longer crotonyl and butyryl groups and even entire proteins such as ubiquitin (Ub) and SUMO (small ubiquitin-like modifier) (Figure 1) [4–6]. Histone PTMs or marks seldom exist in isolation and the diversity of modified histones revealed in numerous proteomic studies is staggering [7]. Despite the complex landscape of chromatin modifications, specific sets of marks may be correlated with distinct transcriptional states of their associated genes. This led to the hypothesis that combinatorial patterns of marks may constitute a histone code that regulates key DNA-templated processes such as transcription, replication and damage repair [8]. This review will highlight important developments in protein chemistry that have enabled studies of the biochemical relationship, or crosstalk, between the Ub family of histone marks and histone methylation.

Figure 1. Histone tail marks.

Figure 1

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Scheme showing the diversity of marks that have been accessed by chemical protein synthesis and semisynthesis. Chemical groups are indicated as ac= acetyl, m= methyl, p= phosphoryl, sumo= small ubiquitin-like modifier, ub= ubiquitin. The globular core of the nucleosome core particle is shown with histones colored as H2A (gold), H2B (red), H3 (green) and H4 (blue) and double-stranded DNA (gray). PDB code 1KX5.

Mechanisms of chromatin regulation by histone marks

Histone marks influence chromatin structure and function by two mutually non-exclusive mechanisms. In some instances, a mark may directly change local chromatin structure, facilitating or denying access to numerous chromatin-modifying enzymes. Secondly, a mark may serve to recruit chromatin-associated proteins that deposit (writers), remove (erasers), or bind (readers) specific sets of marks [9]. Indeed, a body of literature exists for certain privileged histone marks, such as H4 Lys16 acetylation (H4K16ac), which is associated with actively transcribed genes [10] and an open euchromatin structure [11]. The absence of H4K16ac in chromatin is strongly associated with transcriptional repression, heterochromatin formation, and the appearance of repressive marks such as H3K9me3 [12,13] and H3K27me3 [14,15]. Further complexity in the writing and execution of the histone code arises from the crosstalk between marks, whereby one mark may lead to the addition or removal of others on the same histone or on a different histone [16]. Beyond histone-centric crosstalk alone are the relationships between marks and DNA modifying enzymes [17] or long non-coding RNAs [18], which are also important regulators of cellular outcomes.

Histone semisynthesis to unravel a complex code

The empirical association between histone marks and gene function at different loci is a critical and important step toward understanding chromatin regulation, but it provides little clarity at the molecular level. Cellular signaling is inherently complex and immediate changes in chromatin prior to or following the installation of specific marks are not readily captured in cell-based experiments. Elucidating the precise biochemical crosstalk between sets of marks in chromatin is particularly challenging due to their complex pattern, varying abundance and dynamic nature in cells. Indeed, a molecular understanding of the roles for specific marks requires uniformly and site-specifically modified NCPs, in order to establish a direct line of causality between a mark and its functions. Therefore, the last decade has seen the development of a large number of chemical biology techniques to access homogeneously and site-specifically modified histones [1]. Protein semisynthesis is one early and key technique that has enabled detailed mechanistic studies of crosstalk in well-defined NCP substrates [19].

The enzymatic modification of recombinant histones is a valuable strategy for generating substrates used in mechanistic studies, but it requires knowledge of the site-specific writer and its isolation in an active form. Semisynthesis has several advantages including (1) the ease of scalability, (2) chemospecificity, and (3) the ability to install multiple different marks in close proximity to each other. The latter may be particularly challenging when the natural order of histone modification is unknown, leading to the inhibition of a desired enzymatic activity by partially modified histones.

Native chemical ligation (NCL) is a widely utilized semisynthetic strategy that overcomes inherently limited yields from the solid-phase peptide synthesis of proteins longer than ~50 amino acids, such as the histones [20]. NCL permits chemoselective amide bond formation between two polypeptide fragments, by the incorporation of a C-terminal α-thioester in one fragment and N-terminal Cys residue in the other (Figure 2). An initial reaction between the fragments links them by a thioester, which spontaneously rearranges to the native backbone amide bond at neutral-to-alkaline pH. The fragment with N-terminal Cys may be synthetic or recombinant in nature depending upon its desired PTM state. Expressed protein ligation (EPL) further extends the scope of NCL by employing an intein, a single-turnover enzyme that undertakes protein splicing in bacteria, to generate the C-terminal α-thioester [21]. Thus, either N- or C-terminally modified histone tails are readily accessible by protein semisynthesis employing two asymmetrical fragments, with the smaller synthetically accessible fragment containing any desired marks (Figure 2). The total chemical synthesis of histones by successive NCL steps has also rendered marks in the interior of histones, such as acetylation at H3K56 [22] and phosphorylation at H2AY57, H3Y41 and T45, accessible in good quantities for mechanistic studies [23].

Figure 2. Native chemical ligation and strategies for histone semisynthesis.

Figure 2

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Native chemical ligation leads to the formation of a native amide bond between two polypeptide fragments (top). Different retrosynthetic strategies for the semisynthesis of a modified histone based on the location of the mark near the termini or histone core domain (bottom).

Understanding ubiquitin-signaling in chromatin

Ubiquitin (Ub) is best known as a molecular zip-code that destines proteins for degradation by the 26S proteasome [24]. However, when conjugated with histones it is associated with several non-proteolytic roles including gene silencing, DNA-repair and transcription elongation [25]. All four core histones as well as histone variants and the linker histone H1 are ubiquitylated, but the precise role of Ub in these varying contexts is unclear. This presents an excellent opportunity for the application of chemical techniques to obtain site-specifically ubiquitylated histones for mechanistic studies. Unlike smaller histone marks such as acetyl and methyl groups, however, the installation of Ub on a Lys side-chain during SPPS is extremely challenging. This has inspired the development of several NCL auxiliaries- Cys mimetic amino acid derivatives that permit NCL of Ub-α-thioesters to the target Lys side-chain and are then removed by strong acids, photolysis or reduction to yield the native isopeptide-linked ubiquitylated histone (Figure 3) [26–31]. More recently, chemical analogs of the isopeptide linkage including disulfide [32], thioether [33] or triazole [34] linkages have proven to be useful mimics in biochemical studies of Ub signaling. The obvious limitation when using such analogs is that they must first be compared with the native ubiquitylated histone to ensure that no significant differences arise from the non-natural linkage. Collectively, these semisynthetic efforts have enabled studies of biochemical crosstalk involving histones modified by Ub and its family-members, and shed light on key elements of chromatin regulation.

Figure 3. Cysteine and cysteine-mimetic amino acid derivatives used in histone semisynthesis.

Figure 3

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Cysteine and its mimetics employed in native chemical ligation are related by the 1,2- or 1,3-position of the thiol and amine groups, shown by red bonds. Each derivative is converted to the indicated amino acid in a single step to yield the wild-type modified histone. TCEP= tris(2-carboxyethyl)phosphine, tBuSH= tert-butanethiol, VA-044= 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, TFA= trifluoroacetic acid, TIS= triisopropylsilane, MPAA= 4-mercaptophenylacetic acid, Ub= ubiquitin.

Biochemical crosstalk between H2Bub and H3 K79me2

One of the earliest known targets of modification by Ub are H2B [35] and H2A [36]. Unlike the multimeric K48-linked Ub chains associated with proteasomal targeting of protein, Ub conjugated with histones exists mostly in the monomeric form. Interestingly, ubiquitylation is associated with at least two opposing roles in chromatin. In the form of H2Bub, it marks active genes as the RNA polymerase II transcription complex passes through [37], and as H2Aub (at K119) it marks inactive gene promoters [38]. In the first example of biochemical in-trans crosstalk between two modifications, Allis and Sun demonstrated that ubiquitylation of K123 in yeast H2B (K120 in humans) is a prerequisite for the installation of trimethylation at H3K4 by the Set1 (SET domain containing protein 1) methyltransferase [39]. Later, H2Bub was also demonstrated to be required and sufficient for efficient dimethylation at H3K79 by the non-SET domain histone methyltransferase Dot1L (Disruptor of telomeric silencing 1-like) [40]. However, the precise mechanistic role of H2Bub in Dot1L function remained unknown, due to the low abundance of H2Bub in cells (< 5%) and the inability to purify nucleosomes uniformly modified by this mark. Therefore, the first report of an NCL strategy to site-specifically ubiquitylate peptides at the Lys side-chain ε-amine enabled mechanistic studies of the crosstalk between H2Bub and H3K79me2 [26]. By employing semisynthetic H2Bub reconstituted in NCPs, McGinty et al. demonstrated that the presence of Ub attached to the nucleosome at H2BK120 was necessary and sufficient to stimulate Dot1L activity (Figure 4) [41]. Moreover, Dot1L stimulation was strictly intranucleosomal in nature and the extent of H3K79me2 corresponded 1:1 with the amount of H2Bub in a nucleosome. Subsequent studies with a disulfide-linked analog of H2Bub (H2Bubss) revealed a degree of plasticity in the precise site of Ub attachment on H2B while retaining Dot1L stimulation, and established that spatial positioning of Ub rather than the precise histone target is key to its role [32]. Ala-mutagenesis studies of Ub surface residues revealed the dependence of Dot1L stimulation on an unexpected epitope centered on Leu71/Leu73 near the C-terminus of Ub [42]. This epitope differs significantly from the canonical hydrophobic patch consisting of Leu8/Ile44/Val70 associated with most known functions of Ub. Further studies by Zhou et al. have suggested that Ub binding to the H2A tail may corral Dot1L to a more productive binding mode on nucleosomes [43]. This was demonstrated by the incorporation of the photocrosslinking amino acid photoleucine in place of Leu71 in Ub and its subsequent crosslinking to H2A. The mechanistic implications of the Ub-H2A interaction and precisely how it accomplishes the proposed corralling effect await further interrogation.

Figure 4. Biochemical crosstalk between histone marks in the nucleosome core particle.

Figure 4

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Arrows indicate the known direction of biochemical crosstalk between Ubiquitin (Ub), or the small ubiquitin-like modifier protein (SUMO) and methyl groups (me) at the indicated histone residues. Proteins mediating crosstalk are the Disruptor of telomeric silencing 1-like (Dot1L), the Lysine-Specific Demethylase 1-Corepressor of REST (LSD1-CoREST) complex and core subunits of the Polycomb Repressor Complex 2 (PRC2). These are the Suppressor of Zeste 12 protein homolog (Suz12), Enhancer of Zeste Homolog 2 protein (EZH2), Retinoblastoma-Binding Protein 4 (RBBP4) and Embryonic Ectoderm Development protein (EED). Jumonji And AT-Rich Interaction Domain Containing 2 (Jarid2) and Adipocyte Enhancer-Binding Protein 2 (Aebp2) are two key proteins that associate with PRC2 to mediate positive crosstalk between H2Aub and H3K27me3. SAM= S-Adenosylmethionine.

Biochemical crosstalk between H2Aub and H3K27me3

Ubiquitylation at H2AK119 (H2Aub) is found in ~10% of all eukaryotic H2A [44], and it is a much more abundant modification than H2Bub [39]. The higher abundance of H2Aub led to its successful isolation in early efforts. Consistent with its occurrence in silenced genes, biophysical assays with linear nucleosome arrays reconstituted with cellular H2Aub revealed the formation of compact structures associated with heterochromatin [45]. This contrasts with H2Bub, which inhibits the compaction and higher order oligomerization of 12-mer nucleosome arrays [46].

H2A is ubiquitylated by the E3 ligases Ring1 and Ring1B (or RNF2) that are components of the Polycomb repressive complex 1 (PRC1) [38]. PRC1 plays critical roles in Polycomb silencing of developmentally critical HOX genes and X-chromosome inactivation. It is also implicated in crosstalk with the H3K27 methylating Polycomb repressive complex 2 (PRC2) whereby the two PRCs reinforce each other’s activities [47,48]. With the established crosstalk between H2Bub and H3K79me2 in mind, Whitcomb et al. employed semisynthetic wild-type H2Aub to test crosstalk with H3K27me3 mediated by core components of the PRC2 complex [49]. Surprisingly, they discovered a small but significant inhibitory effect of H2Aub on H3K27 trimethylation by PRC2 in vitro, which could not be readily explained. In a subsequent study, two additional protein components Jarid2–Aebp2 (Jumonji And AT-Rich Interaction Domain Containing 2-Adipocyte Enhancer-Binding Protein 2) were found to bind PRC2 and stimulate its activity ~25-fold on nucleosomes containing H2Aub (Figure 4) [50]. This confirmed the initially proposed crosstalk between PRC1 and PRC2, and indicated that its associated proteins modulate PRC2 function.

Recently, Liu and coworkers also prepared semisynthetic H2Aub by the selective conjugation of a truncated Ub(1-75)-α-thioester to a Gly with N(α)-auxiliary conjugated at K119 in H2A [31]. In a stepwise ligation strategy similar to the genetically encoded orthogonal protection and activated ligation approach pioneered by Virdee et al. [51], the authors prepared a fully amine-protected form of H2A and then generated Lys119 by the reduction of azidonorleucine incorporated at position 119 in response to a Met codon. The high yield of azidonorleucine incorporation in H2A with a mutant methionyl tRNA synthetase was a highlight of this approach. The H2Aub was subsequently incorporated in nucleosomes and tested for crosstalk with various methylated states of H3K36, which mark active genes. Consistent with its role as a repressive mark, H2Aub strongly inhibited the H3K36 di- and trimethylases, NSD2 (Nuclear SET Domain-Containing Protein 2) and SETD2 (SET Domain Containing 2). This confirmed the negative crosstalk between H2Aub and H3K36me2/3 that was first proposed by Zhu and coworkers using oligonucleosomes assembled with histones isolated from HeLa cells [52].

Chasing the high-hanging fruit: biochemical effects of histone sumoylation

Several decades after the discovery of histone ubiquitylation, Eisenman and Shiio reported the modification of the histone H4 tail by two distinct isoforms of the small ubiquitin-related modifier protein, SUMO-1 and SUMO-3 [53]. In the absence of a known histone-specific SUMO ligase, they demonstrated that recruitment of the ΨKXE target-motif-specific E2 ligase, Ubc9, to the promoter region of a luciferase reporter plasmid sufficed to repress transcription and correlated with reduced histone acetylation. In a subsequent study, Berger and coworkers demonstrated similar gene-repressive effects of Smt3, the SUMO homolog in Baker’s yeast, on gene transcription [54]. Despite these pioneering inroads, little remained known regarding the role of SUMO in chromatin or, indeed, its crosstalk with marks of active transcription, such as histone acetylation and methylation. Challenges toward investigating histone sumoylation parallel those of ubiquitylation, and are additionally complicated by the presence of multiple SUMO isoforms, multiple sites of modification, and the lack of sumoylated H4 (H4su) specific antibodies.

Based on mass spectrometric evidence of K12 in histone H4 being a site of modification by SUMO-3 in human cells [55,56], Dhall et al. interrogated the direct effect of histone sumoylation on chromatin structure. By employing a disulfide-linked analog of H4su (H4suss) incorporated in 12-mer nucleosome arrays, they observed that sumoylation inhibits the formation of compact structures associated with heterochromatin [57]. The biophysical effect of H4su paralleled what is known for H2Bub and H4K16ac, which are associated with active genes, and was opposite to the effect of H2Aub. Furthermore, single molecule FRET experiments with fluorophore-labeled mononucleosomes revealed that the mechanism of inhibition by H4su differs significantly from that of H4K16ac, in that H4su reduces the rate at which two nucleosomes associate to form a compact dinucleosome. This surprising effect suggested that H4su might engage in biochemical crosstalk with other histone modifications to repress transcription, rather than directly favoring heterochromatin formation.

Many chromatin-modifying enzymes and their protein interaction partners are either modified by SUMO, or have SUMO-interacting motifs (SIMs) through which they bind SUMO. The current understanding of a SIM is a sequence with less than 10 amino acids and a core of 3–4 hydrophobic amino acids such as Val or Ile, surrounded by acidic residue such as Glu or Asp, or phosphorylated Ser/Thr residues [58]. One critical gene repressive complex that contains a SIM is the LSD1-CoREST-HDAC1 (LCH) complex [59]. LSD1 or KDM1A, is a lysine specific histone demethylase that undertakes the demethylation of H3K4me1/2 and regulates multiple cellular pathways implicated in cellular development, differentiation, and embryonic pluripotency [60]. As a part of the LCH complex, LSD1 represses a subset of neuronal genes in non-neuronal cells in association with the RE1-silencing transcription factor (REST). Recently, it was proposed that LCH also represses REST-independent genes in a SUMO-2/3 dependent manner that requires the SIM in CoREST (the Corepressor of REST) [59]. As both LCH and H4su are associated with transcriptional repression, Dhall et al. tested their potential crosstalk by employing semisynthetic nucleosomes containing both wild-type H4su and H3K4me2 [61]. They discovered an ~2-fold stimulation of LSD1 activity by H4su, which was dependent upon the SIM in CoREST (Figure 4). This led them to suggest that H4su-mediated recruitment of the LCH complex near the H3 tail underlies enhanced demethylation. Consistent with the recruitment model, asymmetric dinucleosomes where H4su and H3K4me2 were on adjacent nucleosomes, were not better substrates for LSD1 than methylated mononucleosomes alone. Thus, the semisynthesis of H4su led to the discovery of the first histone modification that stimulates LSD1 activity. Further in vivo studies are needed to confirm the details of this exciting discovery, which highlights the power of semisynthesis when applied toward elucidating the functional roles for histone marks that have long eluded investigation.

Conclusions

The last decade witnessed an explosion in the number of biochemical studies with well-defined nucleosomal substrates that were aimed at testing proposed mechanisms of crosstalk between marks. Two key aspects of histone proteins- their facile reconstitution in nucleosomes and the presence of only a single dispensable Cys residue (H3C110) between the four core histones- enabled numerous applications of NCL/EPL to study otherwise challenging histone marks. In conjunction with additional techniques such as amber codon suppression [62], methyllysine analogs [63], and sortase-mediated ligation [64], semisynthesis has in principle rendered every atom in the ~210 kDa NCP accessible to chemical modification. The concurrent development of efficient strategies to tracelessly and site-specifically modify proteins by members of the Ub family [26] has led to detailed mechanistic elucidation of the roles for these large protein modifiers in various chromatin contexts. In some cases, such as for histone sumoylation, a semisynthetic approach enabled both the generation and testing of novel hypotheses in the absence of effective molecular biological tools. We envision that as semisynthesis no longer remains the rate-limiting step toward obtaining modified histones, hypothesis-driven investigations of crosstalk between marks and unbiased screens for novel mediators of crosstalk will become routine. Instead of serving as a tool for the mechanistic elucidation of proposed crosstalk, designer semisynthetic nucleosomes with complex patterns of marks will be the launching pad for discovering new paradigms of biochemical crosstalk in chromatin.

Acknowledgments

We thank the Department of Chemistry and the Royalty Research Fund at the University of Washington, Seattle, for generous support. Research in our labs is supported by grants from the NIH R01GM110430 and NSF 1715123. C.J.A.L. was supported by an NIH Molecular Biophysics Training Grant T32GM008268. We apologize to those authors whose excellent work we could not discuss due to space constraints.

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