We propose to study DNA damage response and repair mechanisms with a novel approach that is focused on early signals encoded in the total modification state of the histone proteins and enabled by novel analytical capabilities recently developed in our lab. Histones are the primary protein component of chromatin fiber that make up chromosomes and play an important role in mediating access to DNA and in the recruitment of molecular machinery to DNA. Individual histone modifications (PTMs) have been shown to be involved in DNA damage response and repair and other processes. The mechanisms for how these PTMs transduce signals and how the combinations of PTMs function in concert remain unknown. Although there is strong and growing evidence of the importance of the combinations of modifications, or Histone Codes, a thorough study of their action has been inhibited by technical limitations. HeLa S3 cell cultures will be treated with chemicals that induce a couple of different DNA damage response and repair mechanisms. Fractions will be taken at multiple time points after damage induction, histones extracted and mixed in equal proportion with histones from an untreated cell culture grown in 15N, 13C rich heavy media. The histones will be separated by reverse phase HPLC into the individual histone proteins or sequence variants. Enzymatic digestion, e.g. GluC and AspN, will be used to produce relatively large peptides that contain most of the modification sites of each histone and the peptide containing the modifications will be purified from the digestion mixture. This sample which contains hundreds to millions of modified forms, or Histone Codes will be analyzed using a pH gradient nanoflow weak cation exchange- hydrophilic interaction chromatography-electron transfer dissociation mass spectrometry method to identify and quantitate each histone code. The untreated sample will provide an isotopically labeled internal standard for quantitation. We will further enrich for Histone Codes near damage sites and potentially track them through pathways with FLAG-tag chromatin precipitation, using the method above for reading the Histone Codes. With time permitted, we will perform targeted shRNA based gene knockdown to validate and further investigate the direct involvement of Histone Codes in the molecular mechanisms of this pathway.
DNA damage response and repair and the failure thereof are important in cancer. How the signals for DNA repair begin and propagate and the recruitment of the machinery that performs the repair are not understood and is the focus our study. This work may result in a better molecular understanding of cancer, more specific diagnostics and potentially epigenetic therapies for cancer.
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|Plazas-Mayorca, Mariana D; Bloom, Joshua S; Zeissler, Ulrike et al. (2010) Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown. Mol Biosyst 6:1719-29|