The Quantitative Proteomics core will provide high-end mass spectrometry based proteomic support to the P01 Project team members to help identify and quantify protein expression and post-translational modifications (PTMs) on histone and non-histone proteins from various cell lines and brain tumors. Histones are small proteins that help organize eukaryotic DNA information into nucleosomes, the repeating building blocks of chromatin. Gene regulation modulated via chromatin remodeling is achieved by the addition of PTMs to histones or by the incorporation of specialized histone variant members. Dysregulation of these pathways have been identified in many human disorders and cancers. Most histone related research work is centered on the use of antibodies that recognize a single histone PTM (Western blot, immunofluorescence, etc.). However, these types of experiments have drawbacks such as cross-reactivity, potentially limited specificity, and cannot be used to discover novel histone PTMs. Additionally, these antibodies often have issues with the recognition of their intended single modification site in modification-dense regions, a phenomenon known as epitope occlusion. Here we will use our novel mass spectrometry based proteomics platforms and integrated bioinformatics to comprehensively interrogate the PTM profiles of histones extracted from model cell lines, primary cells and tumor tissue harboring histone mutations. These experiments will allow for precise measurement of the abundance of over 100 different histone modification and variant proteoforms. Additionally, the Garcia lab has produced arguably the best platform for analysis of combinatorial histone modifications to determine crosstalk relationships between different modified sites. Studies aimed to isolate chromatin from specific cancer related genes will also be performed to understand the local histone environment contributing to tumoregenesis. We will also perform experiments to identify proteins that bind to specific cancer enriched histone PTMs (e.g. histone readers), or non-histone proteins that are modified themselves and may affect chromatin networks that modulate crucial gene expression programs.
Mutation to histone genes have been found in different types of cancers. This research will help define altered chromatin pathways due to these mutations, thus laying down the foundation for potential development of novel targeted drug therapy.
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