FFPE histone enrichment for antibody and mass spectrometry analysis of post translational modifications in epigenetic biomarker discovery

PROJECT SUMMARY Many environmental stresses known to cause chronic or acute disease induce changes in epigenetic mechanisms regulating chromatin structure and gene expression One manifestation of this epigenetic dysregulation is changes in global levels of histone modifications such as acetylation methylation phosphorylation etc Global changes in several histone H and H post translational modifications PTMs have been observed in autoimmune inflammatory neurological and neoplastic diseases for which in the latter are predictive of clinical outcome for several primary tumor types This strong association with disease raises the possibility that histone modifications have potential as a new class of epigenetic biomarker for a wide range of disease types and in environmental exposure risk management Archived formalin fixed paraffin embedded FFPE clinical samples are extremely valuable for biomarker identification since they are often accompanied with important information regarding patient history treatment courses and clinical outcome However evaluation of histone PTM as disease biomarkers in FFPE samples has not been possible as the extensive crosslinking of proteins in FFPE material makes protein extraction very challenging and current PTM detection methods require large amounts of fresh or frozen samples This Phase I proposal intends to develop histone enrichment methods from FFPE samples while preserving histone PTMs by adaptation of chromatin extraction methods developed for FFPE ChIP seq Chromatin extraction methods in effect result in histone extraction albeit in the context of genomic DNA which can be removed Aim efforts will develop a histone enrichment procedure compatible with downstream antibody based detection methods including multiplexing bead based ELISAs that can quantitate multiple PTMs in the same sample Aim efforts will focus on method development compatible with mass spectrometry histone PTM quantitation Although mass spectrometry requires additional material the method allows for the unbiased profiling of diverse modifications including discovery of novel PTMs and bypasses the need for characterized antibodies Initial experiments will be performed using environmental exposure target tissues lung liver and kidney isolated from mice following sham or histone deacetylase inhibitor treatment with comparisons made between fresh frozen and FFPE samples to demonstrate histone PTM preservation Following successful development of these histone enrichment methods for mouse FFPE samples a proof of principle experiment to establish the applicability of the method to human samples will be performed with matched tumor and healthy clinical samples from a glioma patient Successful completion of these efforts will enable the mining of large cohorts of archived clinical samples for potential histone PTM biomarkers in future Phase II efforts Exposure to environmental stresses that result in chronic and acute diseases are known to cause changes in gene expression patterns and expression associated histone marks This proposal intends to develop a method for isolation of histones from archived clinical samples to enable evaluation of disease associated changes in histone mark patterns as biomarkers for diagnostic and prognostic applications