Influence of Combinatorial Histone Modifications on Antibody and Effector Protein Recognition
ABSTRACT Increasing evidence suggests that histone posttranslational modifications (PTMs) function in a combinatorial fashion to regulate the diverse activities associated with chromatin. Yet how these patterns of histone PTMs influence the adapter proteins known to bind them is poorly understood. In addition, how histone-specific antibodies are influenced by these same patterns of PTMs is largely unknown. Here we examine the binding properties of histone-specific antibodies and histone-interacting proteins using peptide arrays containing a library of combinatorially modified histone peptides. We find that modification-specific antibodies are more promiscuous in their PTM recognition than expected and are highly influenced by neighboring PTMs. Furthermore, we find that the binding of histone-interaction domains from BPTF, CHD1, and RAG2 to H3 lysine 4 trimethylation is also influenced by combinatorial PTMs. These results provide further support for the histone code hypothesis and raise specific concerns with the quality of the currently available modification-specific histone antibodies.
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ABSTRACT: The lysine methyltransferase (KMT) SETMAR is implicated in response to and repair of DNA damage but its molecular function is not clear. More broadly, enzyme/substrate relationships are largely unknown for the dozens of predicted KMTs and hundreds or thousands of proteins modified by lysine methylation. SETMAR has been associated with di-methylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates we utilized a strategy based on quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 (K130) of mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates mono-methylation at this position. Further, we show that SETMAR methylates snRNP70 K130 in cells. As snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 03/2015; DOI:10.1074/jbc.M115.641530 · 4.60 Impact Factor
Dataset: Mol Cell 2012 UNC Online suppl
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ABSTRACT: Covalent post-translational modifications on histones impact chromatin structure and function. Their misfunction, along with perturbations or mutations in genes that regulate their dynamic status, has been observed in several diseases. Thus, targeting histone modifications represents attractive opportunities for therapeutic intervention and biomarker discovery. The best approach to address this challenge is to paint a comprehensive picture integrating the growing number of modifications on individual residues and their combinatorial association, the corresponding modifying enzymes, and effector proteins that bind modifications. Furthermore, how they are imposed in a distinct manner during the cell cycle and on specific histone variants are important dimensions to consider. Firstly, this report highlights innovative technologies used to characterize histone modifications, and the corresponding enzymes and effector proteins. Secondly, we examine the recent progress made in understanding the dynamics and maintenance of histone modifications on distinct variants. We also discuss their roles as potential carriers of epigenetic information. Finally, we provide examples of initiatives to exploit histone modifications in cancer management, with the potential for new therapeutic opportunities.09/2014; 6:76. DOI:10.12703/P6-76