A high-throughput approach towards deciphering the histone code

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DESCRIPTION (provided by applicant): A major challenge in current molecular biology is to understand how histone post- translational modifications work in concert to regulate DNA-templated processes in the cell. Work from many laboratories has established that chromatin structure plays a fundamental role in every aspect of DNA function, including gene transcription, DNA replication, recombination and repair. At the heart of chromatin structure is the nucleosome core particle, which is comprised of DNA and histone proteins. Significantly, a vast number of covalent modifications such as acetylation, methylation, ubiquitylation and phosphorylation exist on histones. How they all function is still not clear, but growing evidence suggests that they work in the form of a 'histone code' to regulate chromatin-based events. The 'histone code' hypothesis was proposed to explain how the variety of modifications found on histones function (Strahl & Allis, 2000). It stated that single and/or combinatorial modifications, on one or more histones, recruits effector proteins containing specialized domains that bind the modification(s). While the idea was formally proposed in 2000, there has been limited progress in deciphering the full extent of the histone code and the proteins that bind to them. This limited progress is primarily due to a lack of a technical approach that allows for high-throughput screening of effector proteins that bind to modified histones. To address this problem, we will develop a comprehensive library of modified histone peptides that can be used to rapidly and efficiently screen for proteins which bind unique modification patterns. Our approach will be to make high-content peptide arrays that will be probed with human proteins known to associate with chromatin. The general technology of protein/peptide arrays already exists; however, our particular approach has not been tried before - but is essential to do if we are to "crack" the underlying basis of the histone code. The impact of these studies, if successful, would be enormous on the biological and biomedical community. This is due to the fact that histone modifications impact all processes requiring access to DNA. As such, mutations or deregulation of histone modifying enzymes cause a number of human diseases including cancer. Thus, our fundamental understanding of human disease may depend on understanding the complexity and language of the histone code. Defects in chromatin organization, DNA packaging and its accessibility is a major cause of human disease, including cancer and numerous developmental defects. These studies will reveal how DNA-based activities are regulated, which will address the underlying cause of these public health concerns.