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 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.
