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

Identification of protein lysine methylation readers with a yeast three-hybrid approach

Abstract Background Protein posttranslational modifications (PTMs) occur broadly in the human proteome, and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylation sites among histone and nonhistone proteins have been characterized; however, the list of readers associated with them is incomplete. Results This study introduces a modified yeast three-hybrid system (Y3H) to screen for methyllysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like the chromodomains of CBX1 or MPP8 as prey. We next conducted an unbiased screen using a library composed of human-specific open reading frames. It led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei. Conclusions Our approach introduces a cost-effective method for screening reading domains binding to histone and nonhistone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the power of the Y3H assay which will allow for proteome-wide screens of PTM readers

Deposition, structure, physical and invitro characteristics of Ag-doped beta-Ca-3(PO4)(2)/chitosan hybrid composite coatings on Titanium metal

Pure and five silver-doped (0-5Ag) beta-tricalcium phosphate [beta-TCP, beta-Ca-3(PO4)(2)]/chitosan composite coatings were deposited on Titanium (Ti) substrates and their properties that are relevant for applications in hard tissue replacements were assessed. Silver, beta-TCP and chitosan were combined to profit from their salient and complementary antibacterial and biocompatible features. The beta-Ca-3(PO4)(2) powders were synthesized by co-precipitation. The characterization results confirmed the Ag+ occupancy at the crystal lattice of beta-Ca-3(PO4)(2). The Ag-doped beta-Ca-3(PO4)(2)/chitosan composite coatings deposited by electrophoresis showed good antibacterial activity and exhibited negative cytotoxic effects towards the human osteosarcoma cell line MG-63. The morphology of the coatings was observed by SEM and their efficiency against corrosion of metallic substrates was determined through potentiodynamic polarization tests. (C) 2016 Elsevier B.V. All rights reserved

Mapping of Protein-Protein Interaction Sites by the 'Absence of Interference' Approach.

Protein–protein interactions are critical to most biological processes, and locating protein–protein interfaces on protein structures is an important task in molecular biology. We developed a new experimental strategy called the ‘absence of interference’ approach to determine surface residues involved in protein–protein interaction of established yeast two-hybrid pairs of interacting proteins. One of the proteins is subjected to high-level randomization by error-prone PCR. The resulting library is selected by yeast two-hybrid system for interacting clones that are isolated and sequenced. The interaction region can be identified by an absence or depletion of mutations. For data analysis and presentation, we developed a Web interface that analyzes the mutational spectrum and displays the mutational frequency on the surface of the structure (or a structural model) of the randomized protein†. Additionally, this interface might be of use for the display of mutational distributions determined by other types of random mutagenesis experiments. We applied the approach to map the interface of the catalytic domain of the DNA methyltransferase Dnmt3a with its regulatory factor Dnmt3L. Dnmt3a was randomized with high mutational load. A total of 76 interacting clones were isolated and sequenced, and 648 mutations were identified. The mutational pattern allowed to identify a unique interaction region on the surface of Dnmt3a, which comprises about 500–600 Å2. The results were confirmed by site-directed mutagenesis and structural analysis. The absence-of-interference approach will allow high-throughput mapping of protein interaction sites suitable for functional studies and protein docking

Structural, Mechanical, Imaging and in Vitro Evaluation of the Combined Effect of Gd³⁺ and Dy³⁺ in the ZrO₂–SiO₂ Binary System

Mechanical strength and biocompatibility are considered the main prerequisites for materials in total hip replacement or joint prosthesis. Noninvasive surgical procedures are necessary to monitor the performance of a medical device in vivo after implantation. To this aim, simultaneous Gd³⁺ and Dy³⁺ additions to the ZrO₂–SiO₂ binary system were investigated. The results demonstrate the effective role of Gd³⁺ and Dy³⁺ to maintain the structural and mechanical stability of cubic zirconia (<i>c</i>-ZrO₂) up to 1400 °C, through their occupancy of ZrO₂ lattice sites. A gradual tetragonal to cubic zirconia (<i>t</i>-ZrO₂ → <i>c</i>-ZrO₂) phase transition is also observed that is dependent on the Gd³⁺ and Dy³⁺ content in the ZrO₂–SiO₂. The crystallization of either ZrSiO₄ or SiO₂ at elevated temperatures is delayed by the enhanced thermal energy consumed by the excess inclusion of Gd³⁺ and Dy³⁺ at <i>c</i>-ZrO₂ lattice. The addition of Gd³⁺ and Dy³⁺ leads to an increase in the density, elastic modulus, hardness, and toughness above that of unmodified ZrO₂–SiO₂. The multimodal imaging contrast enhancement of the Gd³⁺ and Dy³⁺ combinations were revealed through magnetic resonance imaging and computed tomography contrast imaging tests. Biocompatibility of the Gd³⁺ and Dy³⁺ dual-doped ZrO₂–SiO₂ systems was verified through in vitro biological studies