Nucleic Acids Research Advance Access published online on November 6, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp899
Methods Online |
A homogeneous method for investigation of methylation-dependent protein–protein interactions in epigenetics
1NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, 2The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, P.O. Box 389, Smithville, TX 78957, USA, 3The Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Headington, OX3 7DQ and 4The Botnar Research Centre, Oxford Biomedical Research Unit, Oxford, OX3 7LD, UK
*To whom correspondence should be addressed. Tel: +1 301 217 5721; Fax: +1 301 217 5736; Email: asimeono{at}mail.nih.gov
Received August 6, 2009. Revised September 15, 2009. Accepted October 7, 2009.
Methylation of lysine residues on the tails of histone proteins is a major determinant of the transcription state of associated DNA coding regions. The interplay among methylation states and other histone modifications to direct transcriptional outcome is referred to as the histone code. In addition to histone methyltransferases and demethylases which function to modify the methylation state of lysine sidechains, other proteins recognize specific histone methylation marks essentially serving as code readers. While these interactions are highly specific with respect to site and methylation state of particular lysine residues, they are generally weak and therefore difficult to monitor by traditional assay techniques. Herein, we present the design and implementation of a homogeneous, miniaturizable, and sensitive assay for histone methylation-dependent interactions. We use AlphaScreen, a chemiluminescence-based technique, to monitor the interactions of chromodomains (MPP8, HP1β and CHD1), tudor domains (JMJD2A) and plant homeodomains (RAG2) with their cognate trimethyllysine histone partners. The utility of the method was demonstrated by profiling the binding specificities of chromo- and tudor domains toward several histone marks. The simplicity of design and the sensitive and robust nature of this assay should make it applicable to a range of epigenetic studies, including the search for novel inhibitors of methylation-dependent interactions.