ABSTRACT Studies in humans and laboratory animals have shown that ancestral exposure to chemicals in the environment results in increased risk to a variety of diseases, including breast cancer, obesity, and autism, that can be transmitted from the exposed to subsequent generations. Therefore, identifying the mechanisms underlying the transgenerational transmission of environmentally induced epiphenotypes is critical for human health and for basic biology, since explaining how epigenetic alterations can resist reprograming and be restored in subsequent generations in the absence of the original stimulus challenges our current understanding of epigenetics. Work in our lab indicates that exposure of F0 pregnant female mice to BPA during reprograming of the F1 embryo germline results in obesity in the F2-F6 generations. Transgenerational transmission of obesity correlates with activation of proximal and distal cis-regulatory elements (CREs) in the Fto locus of the gametes by recruitment of CTCF, FOXA1, estrogen receptor (ESR1) and androgen receptor (AR). These CREs interact with each other and with the promoters of the Fto, Irx3, and Irx5 genes in sperm of BPA but not control mice. Irx3 and Irx5 negatively regulate the differentiation of appetite controlling POMC and AgRP/NPY neurons in the hypothalamus. Their over-expression in mice ancestrally exposed to BPA leads to an increase of AgRP neurons, leptin resistance, increased food intake, and obesity. Simultaneous deletion of the FOXA1 and CTCF sites in the Fto proximal CRE reverses all these effects after BPA exposure, suggesting that BPA- induced occupancy of the CTCF/FOXA1 sites plays a causal role in transgenerational inheritance of obesity epiphenotypes. Here we propose to gain a detailed understanding of the molecular processes affected by BPA exposure in the germline and early embryo. We will study the interplay between transcription factor (TF) occupancy, deposition of histone modifications and histone variants, DNA methylation, and gene expression immediately after exposure to BPA of the primordial germ cells (PGCs) at day E13.5. We will then follow BPA- induced epigenetic alterations in the F1 male and female germlines during their development, including meiosis, up to the formation of the mature gametes in the adult organism. We will analyze changes in TF occupancy in the F2 zygote after fertilization, during pre-implantation development, and in tissues of the epiblast containing recently committed PGCs to understand the mechanisms by which changes induced in F1 PGCs are maintained or re-established in F2 PGCs. Mouse strains in which binding sites for specific TFs and eRNAs have been deleted will be analyzed to determine the causal relationships in their recruitment and their role in the establishment of enhancer feedback loops resulting in transgenerational inheritance. These results will fill an important gap in our knowledge of the fundamental principles by which widely used endocrine d...