In previous work, we have used injection of plasmid DNA into the nuclei of mouse oocytes, fertilized eggs (1-cell embryos) and 2-cell embryos in an effort to identify cis-acting sequences and trans-acting factors that are required to activate DNA replication and gene expression at the beginning of mammalian development. DNA replication first occurs in 1- cell mouse embryos, and zygotic gene expression in 2-cell mouse embryos. These results, together with studies on endogenous gene expression and nuclear transplantation, revealed several features that regulate DNA replication and gene expression at the beginning of mammalian development, identified specific regulatory sequences and factors, and demonstrated the developmental acquisition of specific regulatory functions [Nothias et al. J. Biol. Chem. 270: 22077-80 (1995); Majumder & DePamphilis, BioEssays 17: 879-89 (1995)]. Our current research focuses on specific DNA sequences that bind TEAD transcription factors and provide a powerful enhancer activity in cleavage stage embryos. Mice encode four TEAD genes with the same DNA binding domain and affinity for DNA. All four mTEAD genes are expressed at later embryonic stages, and virtually every tissue expresses at least one family member, but only mTEAD-2 is expressed during the first 7 days of embryonic development. Thus, mTEAD-2 is one of the first transcription factors known to be expressed during zygotic gene activation, presumably turning on the next cascade of genes in the developmental program. During the past year:1) We have shown that mice contain only four TEAD genes. We have cloned all mTEAD gemes as the first step toward understanding how they are regulated so differently throughout mouse development, and what their different functions may be.2) We have shown that all four mTEAD proteins bind to the same specific DNA sequence with the same affinity.3) We have created embryonic stem cells in which one copy of mTEAD-2 has been deleted.4) We are in the process of making knock-out mice for mTEAD-2 in an effort to determine it function in vivo. We anticipate that it will be required for pre-implantation development.5) We have identify sequences required for expression of TEAD-2.6) We have identified a novel gene located only 3.5 kb upstream of mTEAD-2 and transcribed in the opposite direction. Surprisingly, this new gene is expressed only in the adult testes. Thus, we are trying to identify the DNA sequences that regulate this gene so dramatically different from mTEAD-2.