Review article
Various post-translational modifications (PTMs) have been identified on histone proteins, which occur at hundreds of different sites. Histone PTMs influence the chromatin structure and serve as binding sites for reading domains, which further mediate downstream effects. Histone PTM antibodies or recombinant proteins derived from reading domains are unique research reagents essentially required to study histone modifications. To validate their specificity, histone PTM peptide arrays are used, because they allow to investigate the binding of proteins to a large number of different peptides in one experiment. Furthermore, histone PTM peptide arrays can be used to characterize reading domains and study the specificity of histone modifying enzymes. Here, we provide an overview of histone PTM peptide arrays, highlight some of their applications and compare different commercial histone PTM peptide arrays, viz. MODified Histone Peptide Array, AbSurance Pro Histone Peptide Microarrays, EpiTriton Histone Peptide Array and Histone Code Microarrays. These arrays contain histone peptides with several post-translational modifications in many different combinations, but they differ in peptide synthesis and immobilization methods, peptide and PTM coverage, and PTM combinatorial potential. In addition, some special applications of histone PTM peptide arrays like custom arrays or double peptide arrays are described.
The nucleosome is the organizational unit of the eukaryotic chromatin. It consists of 147 base pairs of DNA wrapped around a histone octamer containing two copies of each of the histone proteins H2A, H2B, H3 and H4. The histone proteins expose unstructured N-terminal tails, which are massively decorated with hundreds of post-translational modifications (PTMs). Currently, 25 different PTMs have been identified on histone proteins, including methylation, acetylation, ubiquitylation, and phosphorylation [1], which occur at hundreds of different sites (Table 1).
Histone PTMs can affect the DNA binding of nucleosomes and the interaction of adjacent nucleosomes. Additionally, they can serve as specific binding sites for histone tail interacting domains (so-called “reading domains”). These reading domains are parts of proteins or protein complexes, which specifically bind to modified histones and regulate several downstream chromatin-templated processes such as gene expression, DNA replication or DNA repair [[2], [3], [4]].
The N-terminal histone tails are among the regions in the human proteome with the greatest density of PTMs (Fig. 1). Of note, different PTMs can co-occur next to each other thereby mutually influencing all processes involved in their generation, removal and readout. Each individual post-translational histone modification has specific influences on the chromatin network, but in 2000 the possibility of a “histone code” was introduced, proposing that combinations of different chromatin modifications act synergistically to fulfill specific functions which can differ or go beyond the roles of the individual marks [5]. These cooperative effects refer to histone PTMs adjacent on one tail, histone PTMs within the same nucleosome and even histone PTMs present in neighboring nucleosomes. The histone code hypothesis is strongly supported by the fact that almost all chromatin interacting complexes contain multiple reading domains specific for different individual histone tail modifications, which may result in a specific combinatorial readout.
Two classes of proteins are essential interactors of modified histone tails. One is the natural reader proteins, which interact with modified histones in a modification specific manner and target most of the biological downstream effects [2,3,6]. The second class is histone PTM antibodies, which are central research reagents used in many applications like chromatin immunoprecipitation to enrich histones or nucleosomes carrying particular modifications. The key role of these two types of proteins makes the analysis of their specificity in modified histone tail binding an important technical challenge. Different methods can be used to investigate the binding specificity of reading domains and histone tail antibodies including peptide arrays.
Short synthetic peptides are ideal to mimic parts of the unstructured N-terminal histone tails and are regularly used to investigate the binding specificity of reading domains and antibodies in vitro. Furthermore, the influence of neighboring marks on the binding specificity can be analyzed using short synthetic peptides [[7], [8], [9], [10], [11], [12], [13], [14]]. Peptide arrays represent a simple but powerful method allowing the investigation of binding of an antibody or reading domain to a large number of differently modified peptides in one experiment in a cost efficient and rapid manner. Therefore, peptide arrays have been evolved as an important tool to validate the binding specificity of different batches of commercial histone tail antibodies before their application in elaborate experiments, because pronounced differences in their binding specificity were observed in several studies [7,[15], [16], [17], [18], [19], [20], [21], [22]].
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Peptide arrays were introduced in 1984, when peptides were synthesized on packed polyethylene rods [23]. In 1991, the concept of light-directed, spatially addressable chemical synthesis [24] was introduced, which is the basis of some current peptide array technologies. Additionally, the SPOT array synthesis method was developed in 1992 [25], which generates peptides on cellulose fibers, presenting them in a hydrophilic environment compatible with many biochemical assays. However, peptide SPOT
Because of their great applicative value, the SPOT peptide arrays were further improved. Therefore, the peptides were first synthesized on cellulose by conventional SPOT synthesis and afterwards the individual spots were cut out and the cellulose, together with the peptides, was solubilized and spotted on a glass slide [28,29]. In these CelluSpots™ Arrays, the peptides are still presented in a hydrophilic cellulose matrix, which is favorable for many biochemical applications and represents an
In general, modified histone peptide arrays can be used to study the modification specific binding of many different biological samples, like antibodies, proteins, serum and cell lysate using small amounts (depending on the array dimensions). Generally, the read-out of these arrays is done by fluorescence, luminescence, radioactivity or phosphorescence. Several papers have documented the successful application of peptide arrays for the analysis of histone PTM binding proteins and many companies
Peptide arrays reflect the natural combinatorial diversity of adjacent chromatin modifications, but the combinatorial effect of PTMs on different histone tails or far away from each other on one tail cannot be studied with standard peptide arrays. To approach this question, mixed peptide arrays were developed [60]. In this approach, differently modified and unmodified 19mer-peptides are generated via the standard SPOT synthesis and solubilized (Fig. 7A and B). Equal volumes of two peptide
Peptide arrays are a powerful tool to investigate the relationship between histone tail interacting domains or histone tail antibodies and histone tail PTMs. Short peptides perfectly mimic parts of the unstructured N-terminal tail. Therefore, peptide arrays can be used in a fast and efficient way to study the in vitro binding specificity of histone PTM reading proteins and to validate the specificity of histone tail PTM antibodies. However, this technique has limitations, because the synthesis
This work was supported by DFG grant JE252/26.
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