Chapter Seven - Rapid Semisynthesis of Acetylated and Sumoylated Histone Analogs
Introduction
Posttranslational modifications (PTMs) of amino acid side chains in eukaryotic histones add epigenetic diversity to the chromatin landscape (Allis et al., 2007, Kouzarides, 2007). Histone PTMs can directly influence chromatin structure and/or mediate protein–protein interactions that drive the transcription, replication, and repair of genetic material (Badeaux & Shi, 2013). Histone PTMs range from small chemical moieties such as methyl, acetyl, phosphoryl, and glycosyl groups to entire proteins such as ubiquitin and the small ubiquitin-like modifier protein (SUMO) (Dhall & Chatterjee, 2011). Given their central role in regulating gene function and repair, the misregulation or misinterpretation of histone PTMs is closely associated with human diseases such as cancers, ataxias, and muscular dystrophy (Portela & Esteller, 2010).
Early genomic approaches with modification-specific antibodies identified distinct sets of histone PTMs at specific chromatin loci and correlated these with transcriptional activity of the associated genes. These observations inspired the histone code hypothesis for gene regulation, which postulates that different histone PTMs may act individually or in combination to regulate chromatin-templated functions (Strahl & Allis, 2000). Advances in mass spectrometric techniques have led to the identification of about 15 chemically unique PTMs in histones, and combinations of these modifications lead to hundreds of uniquely identifiable histone states (Garcia, Pesavento, Mizzen, & Kelleher, 2007). Therefore, synthetic and semisynthetic techniques that yield rapid access to various modified histones are crucial when testing elements of the histone code hypothesis.
Peterson and coworkers first demonstrated the power of protein semisynthesis in interrogating the effects of acetylation at lysine 16 in H4 (H4 K16ac) on chromatin structure (Shogren-Knaak et al., 2006). In their protocol, a 22-amino acid long histone H4 N-terminal peptide containing acetylated lysine 16 and a C-terminal α-thioester were produced by solid-phase peptide synthesis. Full-length acetylated H4 was then generated by native chemical ligation (NCL) (Dawson, Muir, Clark-Lewis, & Kent, 1994) of the acetylated α-thioester peptide with a heterologously expressed C-terminal fragment of histone H4 (residues 23–102) containing an arginine-to-cysteine mutation at the N-terminus. The authors successfully incorporated acetylated H4 protein into nucleosome arrays and observed that H4 K16ac significantly impairs chromatin condensation and higher-order fiber formation. Despite its utility, the synthesis of long peptide thioesters required for NCL continues to be technically challenging and the semisynthesis of acetylated histones by NCL has remained limited to specialized laboratories (Kent, 2009).
In contrast with acetylation, the modification of histone lysines by conjugation with the proteins ubiquitin and SUMO leads to dramatic changes in the overall physical and chemical properties of nucleosomes. However, the fact that ubiquitylation and sumoylation (modification by Ub and SUMO, respectively) are transient modifications that mark a small fraction of core histones in cells (typically < 5%, with ubiquitylated H2A being the exception at ~ 10%) has limited the isolation of quantities of ubiquitylated and sumoylated histones for in vitro mechanistic studies (Davies and Lindsey, 1994, Nathan et al., 2006). Recent progress toward understanding the roles for these modifications has been made through the efforts of several research groups in developing novel chemical handles and ligation auxiliaries that permit NCL at lysine side chains (Long et al., 2014, McGinty et al., 2008). However, the technical challenges in generating wild-type ubiquitylated and sumoylated histones go beyond those encountered in generating semisynthetic acetylated histones.
A key turning point in facile semisynthetic access to posttranslationally modified histones was the discovery that thialysine analogs of methylated lysine side chains in histones are reasonable substrates for chromatin-modifying enzymes (Simon et al., 2007). Thialysine analogs are readily generated by alkylating genetically encoded cysteine residues in full-length histones with N-methylated forms of 2-aminoethyl halides, thereby obviating the need for preparing peptide thioesters for NCL. Several thiol-directed strategies provide rapid access to close functional analogs of wild-type PTMs of lysines and arginines (Dhall & Chatterjee, 2015). In this chapter, we provide detailed methods for chemical strategies that yield analogs of acetylated and sumoylated histones (Dhall et al., 2014). A high-yielding disulfide forming reaction (Rabanal, DeGrado, & Dutton, 1996) is used to generate H4 site-specifically sumoylated at Lys12 (suH4ss) and radical-mediated thiol-ene coupling chemistry is used to introduce acetylated thialysine in histone H3 at positions 14 and 56 (Li et al., 2011). Both strategies provide versatile and straightforward approaches to generate multimilligram quantities of modified histones suitable for a wide range of biophysical and biochemical studies. Furthermore, we describe the incorporation of these modified histones into octamers, which are applied to reconstitute both mononucleosomes (MNs) and chromatin-like nucleosome arrays.
Section snippets
General Materials and Methods
All commonly used chemical reagents and solvents were purchased from either Sigma-Aldrich Chemical Company (Milwaukee, WI) or Fischer Scientific (Pittsburgh, PA). 2xYT medium was reconstituted by mixing 16 g Bacto Tryptone, 10 g Bacto Yeast Extract, and 5 g NaCl per liter of water. A Superdex S-200 10/300 GL size-exclusion column was purchased from GE Healthcare (Waukesha, WI). Chemically competent DH5α and BL21(DE3) cells were purchased from Novagen (Madison, WI). T4 DNA ligase and restriction
Overall Design of the Semisynthesis
Histones that are heterologously produced in Escherichia coli are ideal candidates for installing cysteine-derived PTM analogs. E. coli does not have any known histone-modifying enzymes, which ensures that histones expressed in this organism are devoid of PTMs. Furthermore, of the four core histones, H3 is the only histone in higher eukaryotes with a cysteine at position 110 (Sullivan et al., 2002). This residue can be mutated to an alanine without discernible effects on nucleosome structure or
Overall Design of the Semisynthesis
The incorporation of acetyllysine into histones can be achieved by protein semisynthesis (Shogren-Knaak et al., 2006) or amber suppression (Neumann, Peak-Chew, & Chin, 2008), but these approaches can be low yielding and complex. In contrast, a method reported by Liu and coworkers in 2011 is uniquely well suited for generating functional analogs of acetyllysine in a single step on recombinant histone proteins, which can be purified in large quantities (Li et al., 2011). The acetyllysine analog
Octamer Formation Using Modified Histones
Histone octamers are assembled with minor modification of previous reports with the strict exclusion of reducing agents (Dyer et al., 2004). Briefly, each core histone is dissolved at ~ 4 mg/mL in an unfolding buffer containing 7 M Gn–HCl, 20 mM Tris, pH 7.5. It is important to use Ultrapure Gn–HCl at this step to allow accurate protein quantitation by Abs280. The web-based ExPASy Protein Parameters Tool at http://web.expasy.org/protparam/ is used to compute the extinction coefficient for each
Summary and Conclusions
Synthetic access to uniformly and site-specifically modified histones is key when investigating the molecular details of chromatin-mediated mechanisms that drive gene regulation, replication, and repair. Entirely synthetic (Shimko, North, Bruns, Poirier, & Ottesen, 2011) and combinations of synthetic and recombinant technologies, embodied by the technique of expressed protein ligation (Muir et al., 1998), allow exquisite chemical control of both modification site and type in full-length
Acknowledgments
We would like to thank the Department of Chemistry and the Royalty Research Fund at the University of Washington for generous support. C.C. is supported by NIGMS Grant 1R01M110430. C.E.W. gratefully acknowledges support from the NSF GRFP (Grant No. DGH-1256082) and an ARCS foundation fellowship.
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