Deciphering the Histone Code by Mass Spectrometry and High Dimensional Statistics

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DESCRIPTION (provided by applicant): The project aims to determine the precise relationship between combinations of post- translational modifications (PTMs) on chromatin and gene expression. It is known that PTMs on chromatin play a major role in memory formation and addiction. Individual histones may bear various combinations of PTMs, and the histone code hypothesis proposes that combinations of PTMs may have specific effects on gene regulation. By determining what changes in gene expression occur simultaneously with which combinations, it will be possible to better understand the molecular mechanisms underlying addiction, and suggest therapies to ameliorate them. To this end, Top Down mass spectrometry will be used to simultaneously measure combinations of marks in S. cerevisiae under conditions and strains that perturb them, and ultra-high-throughput sequencing will be used to measure gene expression. Statistical techniques based on regularization and variable selection will then be used to select groups of genes whose expression is most tightly correlated with changes in the relative abundance of particular subsets of marks. ChIP-seq will then be used to determine the location and mobility of nucleosomes bearing these particular combinations of marks. It will be determined if the relative abundances of mark combinations as measured by mass spectrometry are equal to the relative abundances of nucleosomes with those combinations as measured by ChIP-seq; this will determine whether the nucleosomes bearing these marks are bound or free. To support these aims, computational techniques will be developed to enable high-throughput data gathering and statistical analysis. Preliminary work suggests that methods developed using this model system are straightforwardly transferrable to work in human cell culture and primary tissue samples, providing a direct pathway for applying the results of these studies to future work on the chromatin remodeling mechanisms underlying addiction. PUBLIC HEALTH RELEVANCE: Addiction and memory formation are now known to involve changes in molecular marks on proteins called histones, which act as a scaffold for DNA. This project aims to determine the precise meaning of combinations of these marks, and their effects on gene regulation.