Epigenetic mechanisms regulate gene expression potential and alterations in epigenetic marks, such as DNA methylation and histone modifications, have been associated with a variety of diseases. Tissue samples (both research and clinical) are often preserved by formalin fixation and paraffin embedment (FFPE), which allows for long term storage in minimally controlled environments. However there can be significant variability in the preparation of FFPE samples and the preservation conditions are harsh, making FFPE samples challenging for sophisticated downstream analyses. Clinical FFPE samples are often accompanied by valuable information including, histology, treatment course and patient outcome, thus they are an incredibly valuable source for linking basic and clinical research. The goal of this Phase I proposal is to provide the feasibility studies to use or newly developed transposase associate chromatin immunoprecipitation (TA-ChIP) to localize DNA methylation and histone modification patterns in DNA and chromatin isolated from FFPE samples. TA-ChIP uses antibody/oligonucleotide conjugates to target a transposase (Tn5) to genomic regions carrying a particular mark of interest (e.g. DNA methylation or histone modification). Upon antibody binding the transposase cuts the nearby DNA and pastes the oligonucleotides that are attached to the antibody into the DNA. Primers corresponding to these oligonucleotides can then be used for amplification and generation of next-generation libraries for genome wide sequencing. Due to the cut and paste abilities of the transposase several key steps of the traditional ChIP-seq procedure are eliminated thereby streamlining the protocol and a minimizing loss. The work described in this Phase I proposal outlines the experiments we will perform to obtain transposase compatible genomic DNA and chromatin from FFPE samples, determine optimal tagmentation conditions for genomic DNA and chromatin as well as determine whether TA-CHIP can enrich for DNA methylation and histone marks on a region specific and global scale.
Aim 1 will examine DNA methylation patterns thus focusing on genomic DNA while aim 2 examines histone marks (H3K4me3, H3K27me3 and H3K9me3) and thus focuses on chromatin. Understanding the epigenetic alterations that are present in FFPE samples will allow us to not only correlate epigenetic marks with disease, but also potentially in stratifying disease state as to help predict treatment response and recurrence. If successful, these efforts will be translated into commercialization of the reagents necessary for extracting transposase compatible DNA and chromatin and the protocols necessary for these extractions as well as performing TA-ChIP ion FFPE samples. If successful, we will submit a phase II proposal in which we aim to expand the range of antibodies that could be used to study FFPE samples and also attempt to multiplex a combination of antibodies into a single ChIP reaction. Furthermore, we would also apply this technology to samples in which the preparation and preservation protocols varied widely in attempt to determine the key factors in retaining high quality DNA and chromatin during the preservation procedure.
Formalin Fixation and paraffin embedding is the gold standard for preservation and long-term storage of tissue samples. However, the variability and harshness in preparing FFPE samples can make downstream analyses difficult. The aims outlined in this proposal will provide the feasibility studies necessary to determine whether our novel transposase assisted ChIP, which is a streamlined ChIP protocol that requires less material, can be applied to FFPE samples to localize epigenetic marks. Given the vast amounts of clinical data often associated with FFPE samples, and the role that epigenetic mechanisms play at all disease stages, being able to study epigenetic marks in FFPE tissue will be incredibly clinically valuable.
