Assay Development for Measuring Chemical Effects on the Epigenome in Stem Cells

DESCRIPTION provided by applicant This proposed project is in response to the need for more extensive toxicological evaluation of environmental chemicals and for better 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 Maintenance of stemness and the stem cell differentiation processes is regulated by networks of genes controlled at least in part by the stem cell epigenome Disruption of this finely tuned regulatory circuit by exposure to certain environmental agents can lead to adverse health effects Chromatin in pluripotent stem cells is characterized by several unique properties including a novel structure highly enriched in embryonic stem ES cells in which nucleosomes bear juxtaposed repressive and activating histone modifications andquot bivalent domainsandquot that direct gene expression Bivalently marked histones silence developmental genes in ES cells while keeping them andquot primedandquot for activation upon initiation of specific differentiation programs The unique characteristics of chromatin and poised status of bivalently marked genes in stem cells may render them particularly sensitive targets for epigenetic effects resulting from chemical exposure To expand the biological landscape of toxicological evaluation of chemicals the objective of this project is to develop a medium throughput assay platform to monitor pertinent histone modifications at a panel of developmentally relevant genes in human embryonic stem hES cells A comprehensive toxicogenomics evaluation of gene expression changes will be performed using a training set of chemicals representing various classes of chemicals known to affect enzymes that modulate histone acetylation and methylation including some known teratogens Correlating changes in specific active and repressive histone marks at the promoters of consensus differentially expressed genes will then be identified to define a biomarker signature indicative of an andquot epigenetic responseandquot in stem cells The signature will be validated by blind testing of a set of chemicals using a ChIP assay platform that allows for all immunoprecipitation steps to be performed in a single well plate The following specific aims are proposed to accomplish these goals conduct expression profiling and ChIP evaluation of hES cells exposed to chemicals known to influence histone acetylation methylation using a training set of compounds to construct a predictive transcriptome based signature of epigenetic impact on developmental processes establish methodology for preparing sheared chromatin from hES cells directly in the chemical exposure plate and for conducting ChIP in a well assay plate develop and implement bioassay standard procedures and quality control criteria and test the epigenetics based signature derived from the training set of chemicals with a blinded set of test chemicals This project will result in a medium throughput platform for rapid and efficient screening of effects of environmental toxicants on the human epigenome that could lead to developmental defects or predispose an individual to disease PUBLIC HEALTH RELEVANCE 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 andquot epigenetic marksandquot of proteins located at regulatory domains of genes that control stem cell growth and maturation into different organs of the body The assay platform is intended for rapid and efficient early screening of effects of environmental toxicants that could lead to developmental defects and or predispose an individual to disease or cognitive disorders