- Research article
- Open Access
Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails
© Bock et al; licensee BioMed Central Ltd. 2011
- Received: 14 June 2011
- Accepted: 31 August 2011
- Published: 31 August 2011
Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains.
We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification.
We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.
- Histone Tail
- Modify Peptide
- Peptide Array
- Peptide Spot
- PWWP Domain
Epigenetic signals include the methylation of DNA, the post-translational modification (PTM) of the N-terminal histone tails and non-coding RNAs. In eukaryotes, these epigenetic marks are involved in the regulation of gene expression and chromatin state. The most studied histone tail modifications are acetylation of Lys, methylation of Lys or Arg leading to mono-, di- (symmetric or asymmetric in the case of Arg) or trimethylation in the case of Lys and phosphorylation at Ser or Thr [1–4]. These PTMs are recognized and bound by specific reading domains which mediate most of the biological functions of histone tail PTMs [5, 6]. Up to date more than 100 different PTMs have been discovered in histone tails, with many of them known to have distinct and important roles in the regulation of gene expression, DNA repair and replication, chromatin biology and the cell cycle. While histone lysine acetylation has a general activating role on transcription, histone lysine methylation can function both as an activating or a repressing mark depending on the site of methylation and the number of methyl groups added.
Acetylated histones are recognized by Bromo domains, an about 110 amino acid residues long domain folded into a left-handed four α-helical bundle. The family of Bromo domains has more than 70 identified members which are found in many chromatin-associated factors, including histone acetyltransferases or chromatin-remodeling factors. The Bromo domain containing protein 2 (BRD2) belongs to the Bromo Domain And Extra-Terminal Domain (BET) family, members of which contain two Bromo domains and an additional conserved terminal domain. It was reported that the tandem Bromo domains of BRD2 bind to H4K12ac .
Protein domains belonging to the Royal family include among others Tudor, Chromo and MBT domains. They are known to interact with methylated lysine residues. The Chromatin Organization Modifier (Chromo) domain is about 50 amino acids in size which are folded into a small ß-finger flanked by one α-helix. The Chromo domain family consists of more than 120 identified members. The Chromo domain of the Heterochromatin protein 1 beta (HP1ß) binds specifically to H3K9me3 and with weaker affinity to H3K9me2 and it is involved in the establishment of heterochromatin [8, 9]. Another example of a Chromo domain containing protein, though less characterized, is the M-phase phosphoprotein 8 (MPP8), which has been shown to recognize H3K9me3, but also H3K9me1 and H3K9me2 [10–13].
The Tudor domain folds into a ß-sandwich flanked by one α-helix. Members of this domain family are for example the p53 binding protein 1 (53BP1), which has been shown to interact with H4K20me2 [14, 15] and H3K79me2 , and the Jumonji domain containing protein 2A (JMJD2A) reported to bind to H4K20me3, H4K20me2, H3K4me3, H3K4me2 and H3K9me3 [14, 17, 18].
However, a specific interaction with modified amino acids is possible in other families as well. For example, the PWWP domain (Proline Tryptophan Tryptophan Proline Motif) present in DNA methyltransferase 3a was shown to read H3K36me3  and the Plant Homeodomain (PHD) fingers, which are found in more than 100 proteins, interact with methylated lysine residues. These binding modules are about 50 amino acids long and contain two binding sites for zinc ions. The PHD finger of Rag2, an essential component of the Rag1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly, interacts with H3K4me3 .
The investigation of the PTM specific binding of reading domains to peptides requires testing of binding to as many peptides with different PTMs as possible which is impeded by the high costs of synthetic modified peptides. Recently, we described the application of Celluspots peptide arrays for the quality assessment of commercial antibodies . Peptide synthesis on cellulose membranes by the SPOT method allows the generation of many peptides with variable sequence and modifications at reasonable costs [22, 23]. Peptide SPOT arrays are valuable tools for the analysis of the specificity of peptide modifying enzymes [24–28] or the binding specificity of antibodies and reading domains [19, 21, 22, 26, 28–32]. In the Celluspots technique, peptides are synthesized following the conventional SPOT synthesis on a cellulose matrix, but after the synthesis the cellulose piece together with the peptides is solubilized and spotted on glass slides . Consequently, Celluspots peptide arrays are less expensive, because many arrays can be produced from one synthesis and, due to the fact that they are smaller, the assay can be performed with much less reagent.
In the present study, the binding specificities of seven known reading domains were analyzed using Celluspots peptide arrays comprising 384 peptides from 8 different regions of the N-terminal histone tails, viz. H3 1-19, 7-26, 16-35 and 26-45, H4 1-19 and 11-30, H2A 1-19 and H2B 1-19. The arrays are commercially available from Active Motif and feature 59 post-translational modifications (most of them identified, some of them hypothetical) in many different combinations (Additional file 1). Binding of the GST fused reading domain proteins to peptide arrays was visualized using an anti-GST antibody, followed by a secondary anti-goat-HRP antibody and ECL detection system. The domains were selected to represent the different folds of reading domains and show a wide range of specificities. Control experiments showed that GST alone did not give rise to any signal on the peptide array (data not shown). Each reading domain was tested at least two times on the peptide arrays to ensure that the results are reliable. In case of weak signals, the experiment was repeated with higher protein concentration. In case of an overexposed image, the protein concentration was reduced. For quality control, each glass slide contains two identical copies of the array. The binding intensities for each tested reading domain were analyzed with the Array Analyze program, which calculates the average of the binding intensities to corresponding peptide spots in both copies of the array and prepares a graphical output - one scatter plot illustrating the binding intensities observed at corresponding spots in both copies of the array and a bar diagram showing the distribution of deviations of the binding intensities to the corresponding spots. For all arrays, the main error range of the two internal duplicates was between 0 and 5% indicating that binding of reading domains to the arrays was reproducible.
Peptide binding of the HP1ß Chromo domain
Peptide binding of the MPP8 Chromo domain
Peptide binding of the JMJD2A double Tudor domain
Peptide binding of the 53BP1 tandem Tudor domain
In the past, some histone modifications were reported to interact with the tandem Tudor domain of 53BP1: H3K79me2 , a modification which is not present on the Celluspots peptide array, and H4K20me2, H4K20me1, H3K4me2 and H3K9me2 [14, 15]. Indeed, all H4K20me2 modified peptides (the main target reported for the tandem Tudor domain of 53BP1 ) were specifically recognized by the 53BP1 tandem Tudor domain on the peptide array (Figure 5B). Interestingly, the secondary modifications H4K16ac, H4K12ac and, to a lesser degree, H4R24me2a enhanced the binding affinity of the tandem Tudor domain for H4K20me2, since the peptides carrying H4K20me2 combined with those modifications showed the strongest binding. The other reported interactions with H4K20me1, H3K4me2 and H3K9me2  were not observed on the peptide array.
Peptide binding of the Dnmt3a PWWP domain
Peptide binding of the Rag2 PHD finger
Out of the group of H3K4me3 interacting PHD finger binding modules, we selected the PHD finger of Rag2 for this study which has been reported to interact with H3K4me3 . As expected, the strongest signal was observed for H3K4me3 modified peptides on the Celluspots arrays (Figure 6B). In comparison to H3K4me3, the signal intensity of bound H3K4me2 was greatly reduced and there was almost no binding signal observed for H3K4me1 modified peptides. In concert with literature, we found that the PHD finger of Rag2 is highly specific for H3K4me3, because there were no other modified amino acid residues targeted on the peptide array. The secondary modification H3T3ph completely abolished the binding of Rag2 PHD finger to H3K4me3-modified peptides.
Peptide binding of the BRD2 Bromo domain
The Bromo domain protein BRD2 had been shown to interact with H4K12ac-modified chromatin  and the second Bromo domain of BRD2 was found to recognize H4K5K12-diacetylated peptides [36, 37]. Therefore, we tested the second Bromo domain of BRD2 on Celluspots peptide arrays and found that it bound preferentially to tri- or tetra-acetylated peptides from histone H4 (Figure 6C) with some preference for H4K5acK12ac. The tetraacetylated peptide H4K5ac-K8ac-K12ac-K16ac showed the strongest binding signal. The hypothetical modification H4K20ac is included on the peptide array and the triacetylated H4K12ac-K16ac-K20ac peptide was recognized by the Bromo domain with similar affinity as the other triacetylated H4 peptides. Notably the monoacetylated peptides H4K5ac, H4K8ac, H4K12ac and H4K16ac were not bound and diacetylated peptides containing H4K5ac-K8ac were only weakly bound. This is not surprising, since it was shown in the past that some Bromo domains preferentially bind to multiple acetylated histone tails .
Reading domains mediate PTM specific protein/protein interactions, in the case of epigenetic reading domains, a PTM specific interaction with histone peptides occurs. These protein domains are essential players in epigenetic signaling, because they translate the specific PTM patterns of histones into a biological function. Identification and study of reading domains includes the analysis of their specificity with respect to the primary PTM recognized, the peptide sequence and the influence of additional secondary PTMs nearby. One example for a screening system for the identification of PTM binding proteins is a protein microarray used by Kim et al. in 2006. For that study domains of known chromatin-associated proteins were cloned as GST fusions and spotted onto nitrocellulose-coated glass slides and incubated with fluorophore-labeled N-terminal histone H3 and H4 peptides carrying different modifications . Peptide arrays have been used as an alternative screening tool as well. Bua et al. applied peptide arrays containing biotinylated histone peptides, which were either unmodified or carried a single modification at known PTM sites , later larger peptide arrays also containing combinations of PTMs were used [21, 31, 39, 40]. We applied Celluspots arrays for the screening of antibody binding to modified histone tails , because they allow for a cost effective presentation of many potential targets with different modification patterns.
Recently, we also used Celluspots peptide arrays for the initial screening of the binding specificity of two PHD finger like domains - the ADD domains of ATRX and Dnmt3a [31, 40]. The ADD domain of Dnmt3a was reported to bind to unmodified H3K4 and the structure of this complex had been solved . On the peptide array, the Dnmt3a ADD domain interacted only with peptides where H3K4 is either unmodified or monomethylated, but not when it is di- or trimethylated . While secondary modifications like H3R2me2a/s had no or only a mild effect on the binding affinity, H3T3P, H3S10ph and H3T11ph prevented binding of the Dnmt3a ADD domain. We have shown that the ATRX ADD domain binds to H3K9me3 in the absence of H3K4me2/3 on the peptide array  and confirmed this result using purified peptides. Later, additional experiments confirmed this finding [42, 43].
Here, we tested the binding of several reading domains to Celluspots peptide arrays and show that the binding specificities observed with Celluspots arrays in general agree nicely with literature results. One of the big advantages of this approach is that many different modified peptides are presented on the array such that no initial hypothesis on the binding motif is necessary. In addition, peptides with up to four combined modifications are present, which allows for analysis of combinatorial readout to identify secondary modifications which enhance or reduce the binding affinity to peptides which carry the primary target modification.
An inhibiting effect of some secondary modifications was seen for most of the studied reading domains. For example, HP1ß binding to H3K9me3 was prevented by H3R8Citr, H3S10ph and H3T11ph. All of these modified amino acids are either close or adjacent to the target trimethyl lysine, but an additional modification at an adjacent residue does not necessarily influence binding as seen in the case of the MPP8 Chromo domain. Even though binding was inhibited by H3S10ph and H3T11ph similarly as for HP1ß, H3R8Citr did not have any effect on MPP8 Chromo domain binding to H3K9me3. Trying to understand that difference, we superimposed the structures of HP1ß (pdb entry 1KNE)  and MPP8 Chromo domain (pdb entry 3QO2)  in complex with H3K9me3 peptides and compared the distances of unmodified R8 in the peptides to the nearest side chain atoms of the Chromo domains, which are E23 in HP1ß and E97 in MPP8 (Figure 2). In MPP8, the distance between the E97 side chain atoms and R8 is 2.41 Å indicative of a strong hydrogen bond being formed, that also would be present after citrullination of the arginine. In contrast, in HP1ß the nearest side chain to R8 is E23 with a distance of 5.14 Å, which may provide some electrostatic interaction but does not support a hydrogen bond. The electrostatic contact between E23 and R8 would be lost after citrullination, because citrullination of arginine removes its charge which may explain why citrullination of H3R8 prevents binding of HP1ß but not of MPP8.
We observed with several domains that the presence of one or more additional modifications improved binding to peptides which carried the primary mark. This effect could be due to technical problems like unequal peptide synthesis or surface binding. It could also mean that these combinations of PTMs are biologically important, like in the case of HP1ß only binding to H3K9me3 if S10 is not phosphorylated  or the ATRX ADD domain only binding to H3K9me3 if K4 is unmethylated . Furthermore, one may also speculate that improved binding by the presence of additional PTMs may indicate that the amino acid sequence of the peptides used is not ideal for binding of that reading domain, which would suggest binding to other modified non-histone proteins. Therefore, the biological relevance of enhancing or inhibiting secondary modifications found in an initial screening for specific interactions of a reading domain with modified peptides needs to be further investigated with additional experiments. In the case of HP1ß, for example, it has been shown that phosphorylation of H3S10 during the M-phase of the cell cycle leads to the release of HP1 proteins from H3K9me3 modified chromatin  such that this detail of the array results has a biological meaning.
As described, with the JMJD2A double Tudor domain, we observed combined readout of H3K4me3 and H3K9me3 which is interesting, because both marks have opposing biological effects. Since JMJD2A is known to demethylate H3K9me3 [44, 45], one could speculate that H3K4me3/K9me3 dual modified chromatin is an intermediate in the reactivation of H3K9me3 silenced chromatin, where trimethylation of K4 would recruit the JMJD2A activity that would finalize the switch from H3K9me3 repressed to H3K4me3 active chromatin. Interestingly, the ATRX ADD domain performs a combined readout of H3K4 and K9 as well, but in this case the preferred combination is H3K4me0 and H3K9me3, which is both characteristic of transcriptionally inactive chromatin.
We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post-translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. Since peptide arrays are screening tools, unexpected or novel results need to be confirmed by equilibrium peptide binding experiments using purified peptides. In our experience, such studies often confirmed results from peptide arrays. For example in the case of the Dnmt3a PWWP domain, binding to H3K36me3 on the peptide array could be verified by peptide binding, pull-down of native nucleosomes and functional DNA methylation experiments . Similarly, the initial observation of a combinatorial readout of H3K9me3 when H3K4 is not di- or trimethylated by the ATRX ADD domain on the peptide array was confirmed by chromatin pull-down and peptide binding assays in our laboratory  and later also by others [42, 43]. The same is true for the Dnmt3a ADD domain recognition of unmodified H3K4, which is important for the methylation of DNA by Dnmt3a, where peptide array results  nicely agreed with published equilibrium peptide binding data . Here, we confirmed by peptide binding that MPP8 Chromo domain binding to H3K9me3 is inhibited by S10ph. In all these cases, the initial peptide array results prompted further experiments, which confirmed them and in some cases it was possible to show a biological relevance. We conclude that Celluspots peptide arrays are well suited tools to study the PTM specific interactions of reading domains and reading domain variants with modified histone tails.
Cloning, expression and purification of reading domains
Celluspots peptide arrays spotted on glass slides were provided from Intavis AG (Köln, Germany). They are now commercially available from Active Motif (Cat. No. 13001). The peptide sequences and PTMs are specified in the Additional File 1. For quality control, each glass slide contains two copies of the array. We have shown previously that antibody binding to these internal duplicates was highly reproducible which ensures reproducible peptide spotting . Similar results were observed here with reading domains. By mass spectrometric analysis, we showed previously that the peptide spots contained the full length product and sometimes some shorter by-products as well  which is expected, since unpurified peptides are used. These heterogeneous contaminating peptides did not affect antibody binding in a detectable manner, because the arrays present much more peptides than available surface binding sites for antibodies or reading domain. Antibody binding to arrays prepared from independent peptide synthesis was highly reproducible .
Binding of protein domains to peptide arrays
The array was blocked by incubation in TTBS buffer (10 mM Tris/HCl pH 7.5, 0.05% Tween-20 and 150 mM NaCl) containing 5% non-fat dried milk at 4°C overnight, then washed two times with TTBS buffer, one time with interaction buffer (100 mM KCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 0.1 mM DTT and 10% glycerol), and incubated with purified GST-tagged reading domains: HP1ß full length protein (10 nM), MPP8 Chromo domain (0.5 μM), JMJD2A double Tudor domains (10 nM), 53BP1 tandem Tudor domain (1 μM), Dnmt3a PWWP domain (50 nM), Rag2 PHD finger (2 nM) and BRD2 Bromo domain 2 (10 nM) at room temperature for 2 hours in interaction buffer. After washing with TTBS buffer three times, the array was incubated with goat anti-GST antibody (GE Healthcare #27- 4577-01) 1:5000 dilution in TTBS buffer containing 1% non-fat dried milk for 1 h at room temperature. Then, the membrane was washed three times with TTBS and incubated with horseradish peroxidase conjugated anti-goat antibody (Invitrogen #81-1620) 1:12000 in TTBS containing 1% non-fat dried milk for 1 h at room temperature. Finally, the membrane was washed four times with TTBS and submerged in ECL developing solution (Thermo Fisher Scientific) and image was captured on an X-ray film. Typical exposure times were 0.5-5 min. Analysis was done using the Array Analyze program, which was also used to prepare the graphs shown in Figures 1, 5 and 6. The program runs under MS-Windows and it is available free of charge at http://www.activemotif.com/catalog/667.html or from the authors upon request.
Peptide binding experiments
Peptide binding of the MPP8 Chromo domain was analyzed by fluorescence depolarization using a Varian Carry Eclipse fluorescence spectrophotometer as described . Purified FITC-coupled peptides (H3K9me3 and H3K9me3-S10ph, both amino acids 1-19) were purchased from Intavis AG (Köln, Germany).
This work was supported by the BMBF grant 0315886B. Thanks are due to Intavis AG (Köln, Germany) for providing materials.
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