Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.</p

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies

Study of protein-protein interactions in molecular epigenetics

Molecular epigenetics describe the dynamic changes of chromatin structure, consisting of histones, associated proteins and DNA, which ensure a coordinated expression of genes during cellular differentiation. In the present PhD work, we have developed tools for the analysis of protein-protein interaction and the activity of protein (histone) lysine methyltransferases. We have also studied the substrate specificities of histone lysine methyltransferases (G9a and SET7/9) and the binding specificities of reading domains (PWWP domain of Dnmt3a and ADD domain of ATRX). DNA methylation patterns are set during embryogenesis by Dnmt3a and Dnmt3b enzymes. However, the mechanisms guiding these enzymes to their target regions are not well understood. In the present study, we have shown that PWWP domain of Dnmt3a recognizes the histone 3 lysine 36 trimethylation marks and we propose that this guides the enzyme to its target regions. ATRX is a chromatin remodeling protein, which is often found to be mutated in patients having alpha-thalassemia and mental retardation X-linked syndrome (ATR-X) syndrome. Nearly half of the disease causing mutations lies in the ADD domain of ATRX. However the function of this domain is unknown. We have shown that the ADD domain of ATRX recognizes histone 3 lysine 9 trimethylation marks. Our findings provided the functional clue about the heterochromatin localization of ATRX for the first time. We have developed a general method, which we designated as Absence of Interference approach for rapid mapping of protein-protein interaction sites by using Dnmt3a and Dnmt3L yeast two hybrid interaction pair as a model system. The method is well suited for high throughput applications as well

Special Issue “Structure, Activity, and Function of Protein Methyltransferases”

Post-translational modifications (PTMs) largely expand the functional diversity of the proteome [...]</jats:p

Special Issue &ldquo;Structure, Activity, and Function of Protein Methyltransferases&rdquo;

Post-translational modifications (PTMs) largely expand the functional diversity of the proteome [...

Specificity Analysis-Based Identification of New Methylation Targets of the SET7/9 Protein Lysine Methyltransferase

SummaryWe applied peptide array methylation to determine an optimized target sequence for the SET7/9 (KMT7) protein lysine methyltransferase. Based on this, we identified 91 new peptide substrates from human proteins, many of them better than known substrates. We confirmed methylation of corresponding protein domains in vitro and in vivo with a high success rate for strongly methylated peptides and showed methylation of nine nonhistone proteins (AKA6, CENPC1, MeCP2, MINT, PPARBP, ZDH8, Cullin1, IRF1, and [weakly] TTK) and of H2A and H2B, which more than doubles the number of known SET7/9 targets. SET7/9 is inhibited by phosphorylation of histone and nonhistone substrate proteins. One lysine in the MINT protein is dimethylated in vitro and in vivo demonstrating that the product pattern created by SET7/9 depends on the amino acid sequence context of the target site