SBIR-STTR Award

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.

Public Health Relevance Statement:


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 ("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.

NIH Spending Category:
Biotechnology; Genetics; Regenerative Medicine; Stem Cell Research

Project Terms:
Acetylation; Agreement; Antibodies; assay development; Automation; base; Binding (Molecular Function); Biochemical; Biological Assay; Biological Markers; Cell Differentiation process; cell growth; Cell Line; Cell Lineage; Cell Maturation; Cells; Characteristics; Chemical Exposure; Chemicals; Chromatin; chromatin immunoprecipitation; Chromatin Structure; Consensus; Deacetylation; derepression; design; Developmental Gene; Disease; DNA; DNA purification; DNA-Protein Interaction; Embryo; Embryonic Development; embryonic stem cell; environmental chemical; Enzymes; Epigenetic Process; epigenome; Evaluation; Exhibits; Exposure to; Gene Expression; Gene Targeting; Genes; Goals; histone modification; Histones; Human; human embryonic stem cell line; human stem cells; Immunoprecipitation; improved; Individual; induced pluripotent stem cell; inhibitor/antagonist; innovation; Malignant Neoplasms; Maps; Measures; meetings; Methods; Methylation; Modification; Monitor; novel; Nucleosomes; Organ; Pathway interactions; Pattern; Pluripotent Stem Cells; Polycomb; predictive modeling; Predisposition; programs; Promoter Regions (Genetics); Promotor (Genetics); Proteins; public health relevance; Publishing; Reproducibility; response; Risk; Role; Sampling; screening; stem; Stem Cell Development; stem cell differentiation; Stem cells; Testing; Toxic Environmental Substances; transcription factor; Transcription factor genes; Tube

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 ("bivalent domains") that direct gene expression. 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 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 "epigenetic response" 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 96-well plate. The following specific aims are proposed to accomplish these goals: 1) conduct expression profiling and ChIP evaluation of hES cells exposed to chemicals known to influence histone acetylation/methylation using a training set of ≥40 compounds to construct a predictive transcriptome-based signature of epigenetic impact on developmental processes; 2) establish methodology for preparing sheared chromatin from hES cells directly in the chemical exposure plate and for conducting ChIP in a 96-well assay plate; 3) develop and implement bioassay standard procedures and quality control criteria; and 4) 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 Statement:


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 ("epigenetic marks") 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.

NIH Spending Category:
Genetics; Human Genome; Regenerative Medicine; Stem Cell Research; Stem Cell Research - Embryonic - Human

Project Terms:
Affect; assay development; base; Binding (Molecular Function); Biochemical; Biological; Biological Assay; Biological Markers; blind; Blinded; Cardiovascular Diseases; Cell Differentiation process; cell growth; Cell Maturation; Cell Nucleus; Cells; Characteristics; Chemical Exposure; Chemicals; Chromatin; chromatin immunoprecipitation; chromatin protein; Chromatin Structure; Cognition Disorders; Cognitive; Complex; Congenital Abnormality; Consensus; daughter cell; Defect; Development; Developmental Gene; Developmental Process; differential expression; Disease; Embryonic Development; embryonic stem cell; environmental agent; environmental chemical; Enzymes; Epigenetic Process; epigenome; Evaluation; Exposure to; Gene Expression; Gene Expression Profile; Genes; Goals; Health; Heterochromatin; Histone Acetylation; histone methylation; histone modification; Histones; Human; human embryonic stem cell; human stem cells; Immunoprecipitation; Individual; innovation; Late Effects; Lead; Learning; Life; Maintenance; Malignant Neoplasms; Measures; Memory impairment; Mental disorders; Metabolic Diseases; Methodology; Methods; Methylation; Modification; Molecular Profiling; Monitor; nervous system disorder; novel; Nucleosomes; Organ; Pattern; Pharmacologic Substance; Phase; Play; pluripotency; Pluripotent Stem Cells; predictive modeling; Predisposition; Procedures; Process; programs; Promotor (Genetics); Property; Proteins; public health relevance; Quality Control; research study; response; Risk; Role; screening; Spontaneous abortion; Stem Cell Development; stem cell differentiation; Stem cells; stemness; Stimulus; Structure; Teratogens; Testing; Toxic Environmental Substances; Toxicogenomics; Training; transcription factor; Ursidae Famil