Article

Influence of Combinatorial Histone Modifications on Antibody and Effector Protein Recognition

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Current biology: CB (Impact Factor: 9.92). 01/2011; 21(1):53-8. DOI: 10.1016/j.cub.2010.11.058
Source: PubMed

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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    • "Peptides used were synthesized by UNC High-Throughput Peptide Synthesis and Peptide Array Facility as previously described (Fuchs et al., 2011; Rothbart et al., 2012), and quality of all used peptides were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and analytical HPLC (>98~100% in purity). ITC experiments were carried out at UNC Macromolecular Interactions Facility using a MicroCal AutoITC-200 system (GE Healthcare). "
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    • "This necessitates a detailed quality control and documentation of each antibody and each lot in order to give the user all relevant information for correct data interpretation, which is often not sufficiently provided. The urgency for better quality assessment and documentation of antibodies used in chromatin research has been widely recognized in the field (Bock et al. 2011a; Egelhofer et al. 2011; Fuchs et al. 2011; Nishikori et al. 2012; Peach et al. 2012; Hattori et al. 2013; Heubach et al. 2013), and the ENCODE Project Consortium has set up quality criteria for histone PTM antibodies (Egelhofer et al. 2011; Landt et al. 2012). According to these guidelines, antibodies must specifically detect modified histones in Western blots and fulfill one or more of the following secondary criteria: (1) specific binding to modified peptides in dot blot assays; (2) mass spectrometric detection of the modification in precipitated chromatin; (3) loss of signal upon knockdown of the corresponding histone modifying enzyme; (4) reproducibility of ChIP-seq; (5) similarity of ChIP-seq results of two different Ó 2014 Kungulovski et al. "
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    • "Array preparation, binding conditions, and data analysis were as previously described (Fuchs et al., 2011; Rothbart et al., 2012). "
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