Melvin L. DePamphilis, PhD, Head, Section on Eukaryotic Gene Regulation
Alex Vassilev, PhD, Staff Scientist
Kotaro Kaneko, PhD, Postdoctoral Fellow
Daochun Kong, PhD, Postdoctoral Fellow
Cong-jun Li, PhD, Postdoctoral Fellow
David Li, MD, PhD, Postdoctoral Fellow
Tapas Saha, PhD, Postdoctoral Fellow
Wei-hsin Sun, PhD, Postdoctoral Fellow
Xiaohong Zhang, BA, Research Assistant

In the field of eukaryotic DNA replication, we have focused on how the metazoa regulate the number and locations of initiation sites during cell proliferation and development. In previous studies, we mapped origins of bidirectional replication (OBRs) to specific chromosomal sites of 0.4 to 2 kb in mammalian cells, demonstrated that prereplication complexes are assembled at or close to these sites when cells transit from metaphase to G1 phase during cell division, and discovered that one of the six subunits (Orc1) of the "origin recognition complex" (ORC), which is responsible for determining where replication begins, is selectively released from chromatin before or during metaphase and then rebound to chromatin during the M to G1 transition, resulting in the appearance of prereplication complexes at specific genomic sites. Our research suggested that, by preventing assembly of prereplication complexes until mitosis is complete and a nuclear membrane has reformed, ORC determines not only where but also when replication begins. Such an "ORC cycle" would represent a novel mechanism for regulating cell division and the premier step in regulating the onset of DNA replication. The other focus of our research is to identify specific cis-acting sequences and trans-acting factors that regulate DNA replication and gene expression at the beginning of mouse development.

Eukaryotic DNA replication

Kong, Saha, Vassilev, Zhang, DePamphilis; in collaboration with Coleman

During the past year, we have sought to determine whether ORC recognizes specific, genetically required sequences in complex eukaryotic replication origins and whether its activity is regulated as a function of the cell division cycle. The current paradigm for eukaryotic replication origins is the budding yeast Saccharomyces cerevisiae, whose replication origins consist of approximately 0.1 kb regions that contain a required consensus sequence to which ORC binds and an easily unwound DNA region where DNA synthesis begins. In contrast, replication origins in mammalian cells are much larger and lack a required consensus sequence. Moreover, even though replication origins do contain required sequences and ORC is bound to specific sites in vivo, site-specific ORC binding has not been observed in vitro. To understand the function of large complex eukaryotic replication origins, we used the fission yeast Schizosaccharomyces pombe, which, like mammalian cells, contains large, AT-rich replication origins that lack a recognizable consensus sequence but nevertheless contain sequences required for replication. Others had shown that the N-terminal half of SpOrc4 contains nine AT-hook motifs that strongly bind to AT-rich DNA sequences. We found that, both in vitro and in vivo, SpORC 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 SpOrc4 subunit. These sites consisted of asymmetric A:T-rich sequences (clusters of A or T residues on one strand) but were devoid of either alternating A and T residues or GC-rich sequences. DNA-binding specificity was independent of both ATP and the remaining five SpORC subunits. We further found that SpOrc4p binds specifically to only two of the four required sequences in the S. pombe replication origin ARS3001. A pre-RC is assembled adjacent to the strongest ORC binding site, with the latter becoming the site at which bidirectional DNA replication begins. The second, and weaker, SpOrc4 DNA-binding site may facilitate pre-RC assembly, and the fourth appears to bind to a non-ORC protein that is required for origin activation. Finally, we were able to show that, during its replication in frog egg extract, Xenopus laevis ORC preferentially binds to the same AT-rich sites in sperm chromatin as are targeted by SpOrc4. The results suggest that fission yeast replication origins provide a more appropriate paradigm for the complex replication origins in multicellular animals than those of S. cerevisiae. These and other results from our and other laboratories imply that site-specific DNA replication in multicellular organisms results from both genetic and epigenetic parameters. ORC preferentially binds to asymmetric A:T-rich sequences, but, during animal development, some of the sites are selectively activated while others are suppressed.

DePamphilis ML. Eukaryotic DNA replication origins: reconciling disparate data. Cell 2003;114:274-275.

DePamphilis ML. The "ORC Cycle": a novel pathway for regulating eukaryotic DNA replication. Gene

2003;310:1-15.

Kong D, Coleman TR, DePamphilis ML. Xenopus origin recognition complex (ORC) initiates DNA

replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC. EMBO J 2003;22:3441-3450.

Kong D, DePamphilis ML. Site-specific ORC binding, pre-replication complex assembly, and DNA

synthesis at S. pombe replication origins. EMBO J 2002;21:5567-5576.

Regulation of ORC activity

Li, Sun, DePamphilis; in collaboration with Coleman

Binding of ORC to DNA (chromatin) is the first step in assembly of a prereplication 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 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 prereplication complexes, sites that are located at specific genomic loci referred to as "origins of bidirectional 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, providing a mechanism for reprogramming replication origins during animal development or as a result of DNA damage in which the Orc1 subunit could be degraded.

In searching for the trigger that releases Orc1 from mammalian chromatin, we discovered two parameters that regulate the affinity of ORC for DNA in a cell cycle-dependent manner. First, the affinity of Xenopus ORC for DNA depends on chromatin structure. With sperm chromatin, XlORC remains bound when pre-RCs are assembled, but its affinity becomes salt-sensitive, and it is released only during mitosis. With somatic cell chromatin, XlORC is released as soon as the preRC is assembled. These results clearly demonstrated the existence of an "ORC cycle" that can regulate initiation of DNA replication during the cell cycle.

In mammalian cells, ORC activity is regulated during cell division by selectively releasing the Orc1 subunit during S phase and then preventing it from rebinding stably to chromatin until cyclin B is degraded, mitosis is completed, and a nuclear membrane is assembled. We have found that Orc1 is selectively and stably bound to a protein kinase during the G2/M-phase of proliferating hamster cells. We identified this Orc1-associated protein kinase as Cdk1(Cdc2)/cyclin A by its cell cycle specificity, ATP-binding, size, antibody recognition, immunoprecipitation with either anti-Orc1 or anti-Cdk1 antibodies, and sensitivity to inhibitors. It phosphorylated Orc1 in vitro, thereby accounting for the hyperphosphorylated Orc1 in M phase cells that was dephosphorylated during the M to G1 transition. Orc1 in M phase cells did not associate with either Orc2 or chromatin, but inhibition of Cdk activity resulted in rapid binding of Orc1 to chromatin. Thus, in mammals, premature rebinding of Orc1 to ORC/chromatin sites during mitosis appears to be prevented by the activity of Cdk1/cyclin A.

Li C-J, DePamphilis ML. Mammalian Orc1 protein is selectively released from chromatin and ubiquitinated

 during the S to M transition in the cell division cycle. Mol Cell Biol 2002;22:105-116.

Sun W-H, Coleman TR, DePamphilis ML. Cell cycle dependent regulation of the association between

origin recognition proteins and somatic cell chromatin. EMBO J 2002;21:1437-1446.

Gene expression at the beginning of mammalian development

Vassilev, Kaneko, Zhang, Li, DePamphilis

To investigate gene expression at the beginning of mouse development, we have previously microinjected plasmid DNA into the nuclei of oocytes, fertilized eggs, and two-cell embryos and then measured the ability of these cells to either replicate the DNA or express a reporter gene. Our work led to the discovery of a novel transcription factor, mTEAD-2, that is expressed at the onset of zygotic gene expression (ZGE), at which point 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. We identified the long sought-after coactivator of the TEAD family of transcription factors as YAP65; YAP65 interacts 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. Intriguingly, while TEAD is concentrated in the nucleus, YAP65 accumulates in the cytoplasm as a complex with the cytoplasmic localization protein 14-3-3. Given that TEAD-dependent transcription is limited by YAP65 and that YAP65 binds to Src/Yes protein tyrosine kinases, we propose that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals. 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 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 gene called Soggy (mSgy) that is transcribed in the direction opposite to mTEAD-2, thus placing the regulatory elements of the two genes in proximity. Soggy (Sgy) and Tead2, two closely linked genes with CpG islands, are coordinately expressed in mouse preimplantation embryos and embryonic stem (ES) cells but are differentially expressed in differentiated cells. Analysis of established cell lines revealed that Sgy gene expression could be fully repressed by methylation of the Sgy promoter and that DNA methylation acted synergistically with chromatin deacetylation. Differential gene expression correlated with differential DNA methylation, resulting in sharp transitions from methylated to unmethylated DNA at the open promoter in both normal cells and tissues as well as in established cell lines. However, neither promoter was methylated in normal cells and tissues even when its transcripts were undetectable. Moreover, the Sgy promoter remained unmethylated as Sgy expression was repressed during ES cell differentiation. Therefore, DNA methylation was not the primary determinant of Sgy/Tead2 expression. Nevertheless, Sgy expression was consistently restricted to basal levels whenever downstream regulatory sequences were methylated, suggesting that DNA methylation restricts but does not regulate differential gene expression during mouse development.

DePamphilis ML, ed. Gene Expression at the Beginning of Animal Development. Advances in Developmental Biology
and Biochemistry series, vol. 12 (Wassarman PM, series ed.). Amsterdam: Elsevier Science, 2002.

DePamphilis ML. Regulation of gene expression at the beginning of mammalian development. In: Encyclopedia of Molecular Cell Biology and Molecular Medicine. Weinheim, Germany: Wiley-VCH Verlag, 2003; in press.

DePamphilis ML, Kaneko KJ, Vassilev A. Activation of zygotic gene expression in mammals. In: DePamphilis ML, ed.
Gene Expression at the Beginning of Animal Development. Advances in Developmental Biology and Biochemistry series, vol. 12 (Wassarman PM, series ed.). Amsterdam: Elsevier Science, 2002;55-84.

Kaneko KJ, Rein T, Guo Z-S, Latham K, DePamphilis ML. DNA methylation may restrict but does not determine differential gene expression at the Sgy/Tead2 locus during mouse development. Mol Cell Biol 2003; in press.

COLLABORATOR

Thomas Coleman, PhD, Fox Chase Cancer Center, Philadelphia PA 

For more information and for illustrative figures, visit http://depamphilislab.nichd.nih.gov