PROJECT SUMARY/ABSTRACT This proposed project is in response to the need for more extensive toxicological evaluation of environmental chemicals and for predictive models to assess associated risks of chemical exposure to humans. There is also need for innovative methods for evaluating the effects of chemicals on pluripotent stem cells and the differentiation process. A novel chromatin structure bearing both repressive and activating histone modifications ("bivalent domains") that direct gene expression is highly enriched in embryonic stem (ES) cells as compared to differentiated cells. Bivalently marked histones silence developmental genes in ES cells while keeping them "primed" for activation upon initiation of specific differentiation programs. The unique characteristics of chromatin and poised status of bivalently marked genes in ES and induced pluripotent stem (iPS) cells may render them particularly sensitive targets for epigenetic effects resulting from chemical exposure. To expand the scope of toxicological evaluation of chemicals, an objective of this project is the development of an assay platform designed to monitor histone modifications in stem cells using a biomarker panel of developmentally-relevant genes characterized by the bivalent histone signature. Using chromatin immunoprecipitation (ChIP) and PCR arrays, promoter regions of a panel of human stem cell transcription factors, Polycomb and Trithorax target genes, and markers of cell lineage progression will be screened to identify promoters exhibiting bivalent histone marks in stem cells. A subset of these consensus genes that exhibit the greatest response to chemical inhibitors of histone modifying enzymes will be identified and validated as biomarkers for detecting an "epigenetic response" in stem cells. In addition, a "Matrix ChIP" assay platform that allows for all steps to be performed in a single 96-well plate will be established and validated. To accomplish these goals the following specific aims are proposed: 1) Employ ChIP using antibodies against specific opposing histone marks (i.e., H3K4me3, H3K27me3, and H3K9ac) and PCR arrays to: a) identify a consensus panel of bivalently marked genes relevant to stem cell differentiation in several pluripotent stem cell lines and b) use canonical chemical inhibitors of histone methylation, acetylation, and deacetylation to define a signature subset of "epigenetically responsive" bivalently marked genes;2) Validate the ability of the signature biomarker panel to detect chemical-induced effects on the stem cell epigenome using a panel of reference chemicals;and 3) Establish and validate a 96-well Matrix ChIP assay platform for evaluating the effects of chemicals on epigenetic marks located at bivalently marked genes in stem cells. This project will result in a set of bivalently marked biomarker genes useful for evaluating effects of chemicals on histone modifications relevant to stem cell development and a validated 96-well plate method for measuring histone markers. The long term objective is to adapt the assay to a higher throughput platform for rapid and efficient screening of effects of environmental toxicants on the human epigenome that could predispose an individual to disease.
There is a need for more extensive toxicological evaluation of environmental chemicals and for predictive models to assess associated risks of chemical exposure to humans. The overall goal of this project is to develop an assay to evaluate the effects of chemicals directly on specific patterns of biochemical modification (epigenetic marks) of proteins located at regulatory domains of genes that control stem cell growth and maturation into different organs of the body. The long term objective is to adapt the assay to a high throughput platform for rapid and efficient screening of effects of environmental chemicals on the human epigenome that could predispose an individual to disease, including cancer.