SUMMARY/ABSTRACT Understanding the molecular regulation of early mammalian development is of fundamental importance and may inform new therapeutic avenues in Reproductive Health and Regenerative Medicine. Our knowledge of early development is very limited and largely derived from studies of single copy protein-coding genes. However, single copy protein-coding genes comprise only a minor fraction (~2%) of our genome. About half of the mouse and human genomes is derived from Transposable Elements (TEs), which are sequences repeated throughout the chromosomes and in some cases capable of moving to different locations in the genome. Despite being generally silenced in differentiated cell types, certain classes of TEs are highly expressed in mouse and human pre-implantation embryos and pluripotent stem cells. The role of TEs in early mammalian development remains essentially unknown. Our preliminary data in mouse embryonic stem cells and embryos support a new model in which the alternative transcriptional programs of totipotency and pluripotency are regulated by distinct classes of TEs. We will test the hypothesis that RNA derived from one family of TEs acts in cis as a long non-coding RNA to bind specific sites in the chromatin and promote pluripotency in part by repressing another family of TEs. Elements of this second family of TEs, in turn, act as promoters for genes that drive expression of the totipotency program. In Aim 1, we will dissect the antagonistic role of specific TEs in mouse embryonic stem cells; in Aim 2, we will use cutting-edge technology to dissect the molecular mechanism of action of a critical family of TEs in embryonic stem cells; in Aim 3, we will define the molecular role of this TE in early mouse development. This proposal is anticipated to cast pre-implantation development in an entirely new light, uncovering specific TEs as key mediators of the totipotency-to-pluripotency transition and deciphering their mechanism of action. This knowledge is expected to have important implications for understanding and treating infertility, and for using pluripotent stem cells as a platform for studying and treating degenerative diseases.