Epigenetics is the study of how environmental cues can alter interpretation of the genome to yield phenotypic differences in the absence of a DNA mutation. Modifications to DNA and to histone tails influence the heritable state of chromatin and are associated with transcription, replication, repair, recombination, and gene silencing. The histone/epigenetic code hypothesis states the combinatorial nature of histone post- translational modifications (PTMs) plays a role in distinct biological outputs such as the regulation of gene expression. One of the long-term goals of the Tackett laboratory is to develop technologies for the identification of combinatorial histone PTMs in the positional context of the chromosome and determine how they regulate gene expression. Current technology such as chromatin immunoprecipitation (ChIP) allows researchers to genomically position only a single histone PTM to a known sequence of DNA. In this NIH postdoctoral fellowship proposal, we outline the development of technology for the isolation of a particular region of a chromosome, identification of combinations of PTMs on single histone molecules within this purified chromosomal region, identification of non-histone proteins associated with this purified chromosomal region and controlling for non-specifically enriching proteins. Gene transcription assays will be used to functionally examine our findings. This suite of techniques will be referred to as ChAP-MS for chromatin affinity purification with mass spectrometry. We hypothesize that specific patterns of combinatorial histone PTMs provide for binding of key regulatory proteins that modulate gene expression, and that development of technologies to identify this will allow epigenetists to identify targets for diseases like cancer that alter normal gene expression. We will use Saccharomyces cerevisiae as a model system to identify the combinatorial pattern of histone PTMs at a specific gene locus, associated proteins, and associated gene expression. The following aims will be pursued:
Specific Aim 1 : Optimize chemical cross-linking technology to prevent dynamic chromatin dissociation during isolation and purification of a specific chromosomal region.
Specific Aim 2 : Engineer a S. cerevisiae strain for the isolation of a particular chromosomal section that will be subjected to mass spectrometric identification of combinatorial histone PTMs and associated proteins.
Histone post-translational modifications (PTMs) activate or repress transcription leading to disease and cancer. The identification of combinatorial histone PTMs will allow epigenetists to identify targets for diseases like cancer that alter normal gene expression.
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