We have focused on those aspects of eukaryotic DNA replication that appear to be unique to the metazoa: the mechanism by which metazoa regulate the number and locations of initiation sites during cell proliferation and development.

Eukaryotic DNA Replication
DePamphilis, Li, Kong, Bogan, Sun, Zhang; in collaboration with Coleman
In previous studies, we were the first to map origins of bi-directional replication (OBRs) to specific chromosomal sites of 0.4 to 2 kb in mammalian cells, and we demonstrated that pre-replication complexes are assembled at or close to these sites when hamster cells transit from metaphase to G1 phase during cell division. Moreover, we discovered that one of the six subunits (Orc1) of the “origin recognition complex” (ORC) that is responsible for determining where replication begins is selectively released from chromatin before or during metaphase and then rebinds to chromatin during the M to G1 transition, resulting in the appearance of pre-replication complexes at specific genomic sites. This discovery suggested that ORC determines not only where but also when replication begins by preventing assembly of pre-replication complexes until mitosis is complete and a nuclear membrane has reformed. Such an “ORC cycle” would represent a novel mechanism for regulating cell division and the premier step in regulating the onset of DNA replication.

During the past year, we have addressed two critical questions: whether ORC recognizes specific, genetically required sequences in complex eukaryotic replication origins, and whether ORC subunits cycle on and off chromatin during the cell division cycle.

Fission yeast (S. pombe), like mammals, contains large, AT-rich replication origins that lack a recognizable consensus sequence, but that nevertheless bear sequences required for replication. We have discovered that the SpOrc4 subunit was solely responsible for selection of initiation sites in S. pombe. Others had shown that Orc4 contains a special AT-hook motif that binds to AT-rich DNA sequences. We found that S. pombe ORC binds to specific sites within S. pombe replication origins that are genetically required for origin activity and that site selection is determined solely by the Orc4p subunit. Consistent with the fact that Orc4 contains nine AT-hook motifs that others have shown to bind to AT-rich sequences, these sites consist of clusters of A or T residues on one strand but are devoid of either alternating A and T residues or GC-rich sequences. We have further shown that Orc4p binds specifically to only one of the four required sequences in ARS3001, where it initiates assembly of a pre–RC and initiates bi-directional DNA replication. Thus, S. pombe replication origins may provide an appropriate paradigm for replication origins in higher eukaryotes.

Binding of ORC to DNA (chromatin) is the first step in assembly of a pre-replication complex. In contrast to yeast, in which all six ORC subunits are stably bound to chromatin throughout the cell cycle, mammalian Orc1 is selectively released from chromatin during S phase. Moreover, the Orc1 that is released during S phase is rapidly ubiquitinated and in some cases degraded. Other ORC subunits remain stably bound to chromatin and are not substrates for ubiquitination. During the M to G1 transition, Orc1 rebinds tightly to hamster ORC/chromatin sites to allow assembly of pre-replication 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, also providing a mechanism for reprogramming replication origins during animal development or after DNA damage, in which the Orc1 subunit could be degraded. Thus, in contrast to yeast, mammals use a novel pathway to regulate initiation of DNA replication: ORC activity is regulated during each cell division cycle through selective dissociation and reassociation of Orc1 from chromatin-bound ORC.

In searching for the trigger that releases Orc1 from mammalian chromatin, we discovered that the entire Xenopus ORC rapidly binds to the somatic cell chromatin, initiates DNA replication, and is released as soon as Mcm proteins are bound to chromatin to form a pre-replication complex.

Gene Expression at the Beginning of Mammalian Development
DePamphilis, Vassilev, Kaneko, Zhang; in collaboration with Zhao
In an effort to identify specific cis-acting sequences and trans-acting factors that regulate DNA replication and gene expression at the beginning of mouse development, we previously microinjected plasmid DNA into the nuclei of oocytes, fertilized eggs, and two-cell embryos and then measured the ability of the cells either to replicate the DNA or express a reporter gene. The work 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 current goals 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 mTEAD2 led to the surprising discovery of another gene only 3.8 kb upstream of mTEAD2. 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 the two genes close to one another. mSgy contains three methionine codons that have the potential to 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 begins at a cluster of nucleotides about 150 bp upstream of the first Met codon by involving a TATA-less promoter contained within the first 0.9 kb upstream to produce a single, dominant mRNA of about 1.3 kb. The activity of the promoter is repressed by upstream sequences between -0.9 and -2.5 kb in cells that do not express mSgy, but the repression is relieved in cells that do express mSg. mSgy mRNA is 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. Given that mTEAD-2 and mSgy are not expressed in the same cells, the mSgy/mTEAD-2 locus provides a unique paradigm for differential regulation of gene expression during mammalian development.

We have recently identified the long sought-after co-activator of the TEAD family of transcription factors. 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 co-activator. 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 lympho-cytic cells that normally do not support TEAD-dependent transcription (e.g., MPC11) resulted in up to 300-fold induction of TEAD activity. Conversely, TEAD over-expression “squelched” YAP activity. Therefore, the Cterminal 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 YAP65 also binds Src/Yes protein tyrosine kinases, we propose that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals.