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Kolln-reisiek, Germany

Bock I.,Jacobs University Bremen | Dhayalan A.,Jacobs University Bremen | Kudithipudi S.,Jacobs University Bremen | Brandt O.,INTAVIS Bioanalytical Instruments | And 2 more authors.
Epigenetics | Year: 2011

Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers, which are directed towards modified histone tails. The arrays contained 384 peptides from eight different regions of the N-terminal tails of histones, viz. H3 1-19, 7-26, 16-35 and 26-45, H4 1-19 and 11-30, H2A 1-19 and H2B 1-19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profiles of the tested antibodies. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition, some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed toward the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies. © 2011 Landes Bioscience.

Heubach Y.,Reutlingen University | Planatscher H.,Reutlingen University | Sommersdorf C.,Reutlingen University | Maisch D.,INTAVIS Bioanalytical Instruments | And 4 more authors.
Proteomics | Year: 2013

Antibodies that recognize PTMs of histones play a central role in epigenetic proteomic research. Modification-specific antibodies are employed in chromatin immunoprecipitation, for Western blotting and during the immunoprecipitation steps for MS-based global proteomic analyses. Knowledge about the antibodies' off-target binding is essential for the interpretation of experimental data. To address this challenge we developed a fast and cost efficient system for generating peptide bead arrays. We employed this method to establish a bead-based peptide array containing 384 peptides displaying phosphorylated, acetylated, methylated, and citrullinated N-terminal regions of histones H2A, H2B, H3 and H4 and controls. We profiled the binding of 40 PTM-specific antibodies important for epigenetic proteomic research. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zhang Y.,Jacobs University Bremen | Jurkowska R.,Jacobs University Bremen | Soeroes S.,Max Planck Institute for Biophysical Chemistry | Rajavelu A.,Jacobs University Bremen | And 7 more authors.
Nucleic Acids Research | Year: 2010

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 bio- logical 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. © The Author(s) 2010. Published by Oxford University Press.

Agency: Cordis | Branch: FP7 | Program: MC-ITN | Phase: PEOPLE-2007-1-1-ITN | Award Amount: 1.90M | Year: 2008

The focus of SIRENs network partners within this present project is to train young biologists in using integrated approaches to study and understand SIgnals and REgulatory Networks (SIREN) in early plant embryogenesis at their internationally acknowledged European research sites. The multidisciplinary, interface-oriented approach will give fundamental insight into the regulatory system of early plant embryogenesis and synthesize a plausible descriptive and predictive mathematical model. Early plant embryogenesis represents the first time in plant life that new cell fates are established, and is an excellent model for de novo establishment of meristems, plant stem cell niches. Plant early embryogenesis research has been notoriously difficult to address experimentally. SIRENs network partners together represent the European critical mass in plant embryogenesis research and now join their knowledge and different technologies such as flow-sorting and microarray technology, sensitive proteomics technology, high-throughput mRNA ISH technology and SPIM microscopy within an exclusive technological platform. The recent identification of a number of key regulators of early embryogenesis provide a unique starting point for an integrated, multidisciplinary and intersectorial approach that will identify the regulatory networks that underlie early plant embryogenesis. Ultimately, these networks will be abstracted by mathematical modeling. Knowledge gained from studying this biological problem will be of high interest for agro-industry in optimizing plant biomass. Furthermore, the integrated approach can serve as a model for similar studies on other aspects of plant growth and development. Last but not least, SIRENs trainees will get an excellent career perspective through the wide range of technical and methodical skills and knowledge learnt within SIREN.

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