In previous studies, microinjection of plasmid DNA into the nuclei of oocytes, fertilized eggs, and two-cell embryos identified cis-acting sequences and trans-acting factors that regulate DNA replication and gene expression at the beginning of mouse development. These investigations led to the discovery of a novel transcription factor, mTEAD-2, that is expressed at the onset of zygotic gene expression (ZGE), where it is capable of strongly stimulating transcription from promoters or enhancers that contain its sequence-specific binding site. mTEAD-2 is the only member of the TEAD family of transcription factors that is expressed in mouse embryos during the first seven days of development. Our objectives are to identify the factors that regulate mTEAD-2 gene expression, to elucidate mechanisms by which mTEAD-2 regulates gene expression, and to identify the role of mTEAD-2 in mammalian development.

Investigation of the regulatory region of mTEAD-2 led to the surprising discovery of another gene only 3.8 kb upstream of mTEAD-2. The new gene is a single-copy, testis-specific gene called Soggy (mSgy) that is transcribed in the direction opposite to mTEAD-2, thus placing the regulatory elements of these two genes in proximity. mSgy contains three methionine codons that could potentially act as translation start sites, but most mSGY protein synthesis in vitro was initiated from the first Met codon to produce a full-length protein, suggesting that mSGY normally consists of 230 amino acids (26.7 kDa). Transcription began at a cluster of nucleotides about 150 bp upstream of the first Met codon using a TATA-less promoter contained within the first 0.9 kb upstream to produce a single, dominant mRNA of about 1.3 kb in length. The activity of this promoter was repressed by upstream sequences between -0.9 and about 2.5 kb in cells that did not express mSgy, but this repression was relieved in cells that did express mSgy. mSgy mRNA was detected in embryos only after day 15, and in adult tissues only in the developing spermatocytes of seminiferous tubules, suggesting that mSgy is a spermatocyte-specific gene. Since mTEAD-2 and mSgy were not expressed in the same cells, the mSgy/mTEAD-2 locus provides a unique paradigm for differential regulation of gene expression during mammalian development.
The long sought-after coactivator of the TEAD family of transcription factors has been recently identified. TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with a novel TEAD-binding domain at the N-terminus of YAP65, a powerful transcriptional coactivator. YAP65 interacted specifically with the C-terminus of all four TEAD proteins. Both this interaction and sequence-specific DNA binding by TEAD were required for transcriptional activation in mouse cells. Expression of YAP in lymphocytic cells that normally do not support TEAD-dependent transcription (e.g., MPC11) resulted in up to a 300-fold induction of TEAD activity. Conversely, TEAD overexpression squelched YAP activity. Therefore, the C-terminal acidic activation domain in YAP is the transcriptional activation domain for TEAD transcription factors. However, while TEAD was concentrated in the nucleus, excess YAP65 accumulated in the cytoplasm as a complex with the cytoplasmic localization protein, 14-3-3. Given that TEAD-dependent transcription was limited by YAP65 and that YAP65 binds Src/Yes protein tyrosine kinases, it seems reasonable to propose that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals.

Initiation of DNA Replication in Mammalian Chromosomes
Bogan, DePamphilis, Kong, Li
In eukaryotes, site specificity is determined by a six-member "origin recognition complex" that binds to specific sites along the genome. In the fission yeast, S. pombe, it has been possible to show that Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, 2, 3, 5, and 6 proteins (ORC-5) did not alter either Orc4p binding to origin DNA or Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.

FIGURE 37

In mammals, the existence of a novel regulatory pathway in the initiation of DNA replication has been identified. Orc1 and Orc2 are both tightly bound to chromatin during late G1 phase, and late G1 phase nuclei contain active ORC/chromatin sites by virtue of the fact that they can initiate DNA replication at specific genomic sites when incubated in an Orc-depleted Xenopus egg extract. In contrast to yeast, where all six ORC subunits are stably bound to chromatin throughout the cell cycle, the affinity of mammalian Orc1 for chromatin is selectively reduced during S phase, such that lysis of cells in 0.1 percent Triton X-100, 0.15 M NaCl, and 1 mM Mg++ATP releases Orc1, but not Orc2, into the chromatin unbound fraction. Moreover, the Orc1 that is released during S phase is rapidly ubiquitinated with only one or two ubiquitin adducts. In contrast, Orc2 is not a substrate for ubiquitination. During the S to M transition, Orc1 is deubiquitinated. During the M to G1 transition, Orc1 rebinds tightly to hamster ORC/chromatin sites to allow assembly of prereplication complexes. The sites are located at specific genomic loci referred to as "origins of bi-directional replication." The role of ubiquitination is to sequester Orc1 during S phase and thus prevent reinitiation at replication origins during a single cell division cycle. However, if Ub-Orc1 is released into the cytosol, it is then polyubiquitinated and degraded by the 26S proteasome pathway, perhaps providing a mechanism for reprogramming replication origins during animal development or as a result of DNA damage. Thus, in contrast to yeast, mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin bound ORC.