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: 10.99). 01/2011; 21(1):53-8.
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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    ABSTRACT: Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.
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