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 H3 and H4 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 1 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 2 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.
Public Health Relevance Statement: 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.
Project Terms: Acetylation; Acute Disease; Antibodies; Antibody Specificity; Archives; Autoimmune Process; base; Biological Assay; Biological Markers; Biological Models; Biological Preservation; biomarker discovery; biomarker identification; Brain; Bypass; Cardiovascular system; ChIP-seq; Chromatin; Chromatin Structure; Chronic Disease; cigarette smoke; Clinical; cohort; Collaborations; Dana-Farber Cancer Institute; Detection; Development; Diagnostic; Disease; Environmental Exposure; Enzyme-Linked Immunosorbent Assay; epigenetic marker; Epigenetic Process; Evaluation; experimental study; Exposure to; Feasibility Studies; Formalin; Freezing; Future; Gene Expression; Gene Expression Profile; Genomic DNA; Glioma; Global Change; Health; Heavy Metals; Histone Deacetylase Inhibitor; Histone H2A; Histone H3; Histone H4; histone modification; Histones; Human; Immunologics; Inflammatory; Institutes; interest; Kidney; Knowledge; Liver; Lung; Malignant Neoplasms; Mass Spectrum Analysis; Mental disorders; Metabolic; method development; Methods; Methylation; Mining; Modification; Mus; neoplastic; Neurologic; novel; Outcome; Paraffin Embedding; Patients; Pattern; Phase; Phosphorylation; Post Translational Modification Analysis; Post-Translational Protein Processing; predict clinical outcome; Primary Neoplasm; Procedures; Process; prognostic; Prognostic Marker; protein crosslink; Proteins; Reagent; Recording of previous events; Risk Management; Saints; Sampling; Small Business Innovation Research Grant; Spleen; Stress; Therapeutic; Tissues; tumor; Ultraviolet Rays; Western Blotting
