This project develops an automated protein preparation technology that uses magnetic microparticles to isolate histones in their native state from specific genomic loci of interest via the chromatin to which they are bound. After extraction, the targeted histones are purified for downstream analysis by quantitative mass spectrometry or ELISA. Histones and their post-translational modification are of high scientific and pharmaceutical importance due to their role in the causation and development of human disease, in particular cancer. The 'reverse ChIP'technology proposed here will fill a distinct need in epigenomics and disease research, and provide a new tool for the candidate drug screening of potential histone deacetylase (HDAC) inhibitors. Currently no such ability exists. We have used a synthetic transgene to demonstrate the approach of targeting chromatin segments with sequence- or SNP-specific primers and have recovered specifically modified histones in sufficient amount to permit post-translational analysis. We further make use of the fact that the nucleosome density of genomic chromatin is generally reduced for transcription factor binding sites. In conjunction with adjacent unique sequence elements, this provides us with convenient and highly relevant choices of target sites for the primer-based capture of any specific locus. The approach generates high-resolution combinatorial histone code information for any disease-associated target region in a streamlined and largely automated process. The information gained can lead to the identification of target biomarkers and Companion Diagnostics and significantly adds to currently available tools for protein detection, identification and quantification. The combined information of proteomic histone modification and of the underlying genomic sequence contributes to a better understanding of cancer at the molecular level, as well as of other human diseases such as autoimmune and neurological disorders. The chromatin capture and mass spectrometry tools we use for this purpose are both cutting edge and allow scaling up the technology in Phase II to provide a highly sensitive and high-throughput analysis pipeline. One first use will be identifying and characterizing histone modifications originating from specific disease- associated loci from tumor and normal biospecimens, such as the MYCN locus amplified in neuroblastoma. Mass spectrometry is a rapidly growing tool due to its versatility, sensitivity and throughput. Coupled with the automation of our straightforward chromatin isolation procedure we expect that sequence-specific histone extraction can quickly become an assay that is used in a clinical environment. We will first work on chromatin prepared from neuroblastoma cell lines, before applying the technology with our scientific collaborators to actual tumor versus normal samples of neuroblastoma.
Histone post-translational modifications (PTMs) are an intensely investigated field for improving human health. Alterations in these PTM patterns play distinct roles in the pathology of common diseases such as cancer, and the technology developed here will allow researchers and clinicians for the first time to correlate unique changes in the histone code to their underlying specific genomic sequence. This provides a comprehensive understanding of epigenetic signaling patterns at the molecular level and their role in the development and possible treatment of disease.
