Our goal is to understand how multicellular animals regulate DNA replication and gene expression at the beginning of animal development. To this end, we focus on four objectives. We wish, first, to determine how and when cells regulate the activity of the “origin recognition complex” (ORC) and, second, to discover communication links between DNA replication and other proteins that regulate cell growth and development. Such links are needed to maintain homeostasis by telling the cell when replication has begun, when it is complete, and when it is aberrant. We discovered Tead2, its co-activator Yap65, and a closely linked gene Dkkl1. Our third objective is thus to identify the roles of Tead:Yap65 transcription factor complexes in regulating transcription. Our fourth objective is to identify the roles of Dickkopf-like 1 (Dkkl1) in regulating the formation and differentiation of stem cells.
The "ORC cycle," a novel pathway for regulating eukaryotic DNA replication
Ghosh, Li, Noguchi, Radichev, Saha,1 Vassilev, Ullah
In 2000, we reported the first clear evidence that six-subunit ORC activity in mammalian cells is regulated by cell-cycle changes in the affinity of the largest subunit (Orc1) for chromatin. Since then, evidence from several laboratories has confirmed our concept and extended it to show that mammalian Orc1 is selectively ubiquitinated and phosphorylated during the transition from S- to M-phase while ORC subunits 2 to 5, which constitute a stable core complex, remain tightly bound to chromatin throughout cell division (see Figure 17.1). Moreover, various manifestations of the ORC cycle appear in frogs, flies, and yeast. In frogs, we showed that the entire XlORC is released from somatic cell chromatin following assembly of pre-replication complexes in G1-phase. In flies, others have shown that DmOrc1 is selectively degraded during mitosis. In yeast, still others have shown that Orc2 and Orc6 are phosphorylated when cells enter S-phase, thus reducing ORC activity. Therefore, there is a universal control point in eukaryotic cell division cycles: the same cyclin-dependent protein kinase that regulates the onset of mitosis (Cdk1) also prevents premature assembly of functional ORC/chromatin sites until mitosis is complete and a nuclear membrane is present.
More recently, we discovered that Orc1 could be selectively eluted from chromatin as cells entered S-phase. Furthermore, we identified a role for S-phase–specific ubiquitination in selectively destabilizing the Orc1 subunit. Other laboratories working with human cells subsequently reported similar results, and it is now clear that a ubiquitin-dependent process selectively degrades HsOrc1 during S-phase. In contrast, although Orc1 in hamster cells is subject to mono-ubiquitination and a reduction in its affinity for chromatin during S-phase, it is not selectively degraded. To explore this further, we isolated Orc1 complexes from HeLa cells that constitutively expressed an epitope-tagged Orc1 protein. The results revealed that CcnA is bound specifically to Orc1, allowing Cdk1 to bind during S-phase. Orc1 mutants that do not bind to CcnA are not selectively degraded during S-phase and do not support cell proliferation when siRNA suppresses endo-genous Orc1 expression. The results demonstrate that cyclin A regulates both the stability and function of Orc1 and suggest that cell cycle–specific degradation (or modification) of Orc1 is required for DNA replication to proceed.
We also discovered a second mechanism that prevents assembly of a functional ORC until completion of mitosis: the selective association of Orc1 with Cdk1 (Cdc2)/cyclin A during the G2-/M-phase of cell division. The association accounted for the appearance in M-phase cells of hyperphosphorylated Orc1 that was subsequently dephosphorylated during the transition from M- to G1-phase. Rebinding of Orc1 to chromatin follows the same time course as degradation of cyclin B, suggesting that exit from mitosis triggers Orc1 binding to chromatin. In fact, inhibition of Cdk activity in metaphase cells resulted in rapid binding of Orc1 to chromatin. We conclude that the same cyclin-dependent protein kinase that initiates mitosis in mammalian cells concomitantly inhibits assembly of functional ORC-chromatin sites. Presumably, the same mechanism exists in human cells where Cdk1/cyclin A prevents any residual or nascent Orc1 from prematurely binding to chromatin.
To determine directly the effects of ubiquitination and phosphorylation on Orc1 activities, we transiently expressed individual ORC subunits in mammalian cells. Surprisingly, unmodified Orc1 rapidly induced p53-independent apoptosis, and we observed perinuclear accumulations rather than uniform distribution throughout the nucleus. Remarkably, co-expression of Orc1 with Orc2, the only ORC subunit that did not induce apoptosis, prevented Orc1 induction of apoptosis and restored Orc’s uniform nuclear localization, suggesting that assembly of ORC:chromatin sites during the M- to G1-phase transition of cell division neutralizes the toxic effects of individual subunits. Apoptosis was also suppressed by either the addition of a single ubiquitin to Orc1 or the quasi-hyperphosphorylation of Orc1 (conversion of S/T to D at Cdk phosphorylation sites). However, these modifications caused Orc1 to localize to the cytoplasm where it cannot participate in the assembly ORC:chromatin sites, thus confirming that these Orc1 modifications would indeed suppress ORC activity during the S- and G2-/M-phases. Furthermore, failure to carry out the modifications during cell proliferation could induce apoptosis.
DePamphilis ML. Cell cycle dependent regulation of the origin recognition complex. Cell Cycle 2005;4:70-79.
DePamphilis ML. Eukaryotic DNA replication origins and the proteins that recognize them. Chemtracts Biochem Mol Biol 2004;17:115-126.
DePamphilis ML, Li C-J. DNA replication: eukaryotic origins and the origin recognition complex. In: Lennarz WJ, Lane MD, eds. Encyclopedia of Biological Chemistry. Oxford: Elsevier, 2004;753-760.
Li C-J, Vassilev A, DePamphilis ML. A role for Cdk1(Cdc2)/Cyclin A in preventing the mammalian origin recognition complex’s largest subunit (Orc1) from binding to chromatin during mitosis. Mol Cell Biol 2004;24:5875-5886.
Saha T, Ghosh S, Vassilev A, DePamphilis ML. Induction of apoptosis by the origin recognition complex’s largest subunit (Orc1) and its regulation by cell cycle dependent events. J Cell Sci 2005 (in press).
Tead2 and Dkkl1, two closely linked genes that are differentially expressed during mammalian development
Vassilev, Kaneko, Kohn, Yagi, Zhang; in collaboration with Guo, Latham, Liu, Rein
Mammals express four highly conserved Tead(TEF) transcription factors that recognize a canonical M-CAT motif (5´-CATTCCT-3´) found in promoters specific for transcription in muscle as well as similar motifs found in SV40 and polyomavirus enhancers. Tead-1(TEF-1) is required for gene expression in cardiac muscle cells as well as for mouse cardiac development by day 10 of embryo development. Tead-4(TEF-3) appears to play a specific role in activating skeletal muscle genes. Tead-3(TEF-5) is expressed primarily in the placenta and in cardiac muscle. In adult mice, Tead-2(TEF-4) is expressed strongly in heart and lung tissues, in the granulosa cells of the ovary, and weakly in several other tissues. However, we showed that Tead2 is the only Tead gene expressed in mouse embryos during the first seven days of development, suggesting that it plays a unique role at the beginning of mammalian development by allowing pre-implantation mouse embryos to use Tead-dependent promoters and enhancers that we had shown previously to function in pre-implantation embryos and embryonic stem cells.
While searching for Tead2-regulatory elements, we discovered a novel single-copy gene that is now called Dickkopf-like 1 (Dkkl1) [formerly called Soggy (Sgy)]. It is located 3.8 kb upstream of the Tead2 mRNA start site and transcribed in the opposite direction. Dkkl1, found only in mammals where it is always closely linked to Tead2, is related to a group of secreted proteins that are antagonists of Wingless (Wnt) signal transduction pathways. Thus, Dkkl1- and Tead2-regulatory elements lie close to one another and provide an example of two closely spaced, divergently transcribed genes. They also provide a unique paradigm for differential regulation of gene expression during mammalian development.
Both Dkkl1 and Tead2 are among the first genes to be expressed at the beginning of mouse development. In pre-implantation embryos, the Dkkl1 gene is selectively expressed in trophoblast stem cells and concomitantly repressed in embryonic stem cells (see Figure 17.2). Thus, Dkkl1 seems to be required in the placental lineage where it appears in the trophectoderm and eventually in the trophoblast giant cells involved in implantation; nonetheless, it is toxic to the embryonic lineage. In adult mammals, Dkkl1 is expressed predominantly, although not exclusively, during the formation of the male germ cells where it eventually localizes in the acrosome of mature sperm. Following capacitation, some Dkkl1 protein migrates to the surface of the sperm where it may be involved in fertilization. Thus, Dkkl1 is involved in two seemingly unrelated functions: production of sperm and production of trophoblast cells and their derivatives.
DePamphilis ML. Mammalian development, regulation of gene expression. In: Meyers RA, ed. Encyclopedia of Molecular Cell Biology and Molecular Medicine, volume 6. Weinheim, Germany: Wiley-VCH, 2004;483-506.
Intine RV, Dundr M, Vassilev A, Schwartz E, Zhou Y, Zhao Y, DePamphilis ML, Maraia RJ. Nonphosphorylated human La antigen interacts with nucleolin at nucleolar sites involved in rRNA biogenesis. Mol Cell Biol 2004;24:10894-10904.
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 2004;24:1968-1982.
Kohn MJ, Kaneko KJ, DePamphilis ML. DkkL1 (Soggy), a dickkopf family member, localizes to the acrosome during mammalian spermatogenesis. Mol Reprod Dev 2005;71:516-522.
Park J-M., Kohn MJ, Bruinsma M, Vech C, Intine RV, Fuhrmann S, Grinberg A, Mukherjee I, Love PE, Ko MS, DePamphilis ML, Maraia RJ. La antigen (SS/B) is required for mouse development and for the establishment of embryonic stem cells. Mol Cell Biol 2005 (in press).
1Tapas Saha, PhD, former Postdoctoral Fellow
COLLABORATORS
Zong-Sheng Guo, PhD, Cancer Institute, University of Pittsburgh Medical School, Pittsburgh, PA
Bruce Howard, MD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD
Keith Latham, PhD, Fels Institute for Cancer Research and Molecular Biology, Temple University Medical School, Philadelphia, PA
Chengyu Liu, PhD, Transgenic Mouse Core Facility, NHLBI, Bethesda, MD
Richard Maraia, MD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD
Theo Rein, PhD, Max Planck Institut für Psychiatrie, Munich, Germany
Alfred Yergey, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD
For more information, visit http://depamphilislab.nichd.nih.gov or contact depamphm@mail.nih.gov.