REGULATION OF MAMMALIAN CELL PROLIFERATION AND DEVELOPMENT
, Head, Section on Eukaryotic Gene Regulation
Kotaro Kaneko, PhD, Staff Scientist
Alex Vassilev, PhD, Staff Scientist
Kohji Noguchi, PhD, Visiting Scientist
Matthew Kohn, PhD, Postdoctoral Fellow
Isa Maria Stehle, PhD, Postdoctoral Fellow
Wenge Zhu, PhD, Postdoctoral Fellow
Soma Ghosh, PhD, Visiting Fellow
Zakir Ullah, PhD, Visiting Fellow
Rieko Yagi, PhD, Visiting Fellow
Ilian Radichev, BS, Graduate Student
Xiaohong Zhang, BA, Technical Assistant
DNA replication origins are determined by genetic as well as epigenetic features. Genetic features are embedded in the DNA sequence itself and are generally protein binding sites or genes that encode the structure for specific proteins and RNAs. Epigenetic features are not determined by DNA sequence but are nevertheless heritable from one generation to the next. Moreover, epigenetic features are reversible and include chromatin structure, nuclear organization, transcription, transcription factors, deoxyribonucleotide pools, DNA topology, and DNA methylation. Consequently, the number and locations of replication origins in the cells of multicellular organisms can change from an average of one in every 10 to 20 kb in the rapidly cleaving embryos of frogs, flies, and fish to one in every 50 to 300 kb in the differentiated cells of adult organisms. Developmental changes in origin density also occur during specific stages in animal development. Thus, metazoan genomes contain many potential initiation sites for DNA replication, but, during development, some of these sites are selectively activated while others are suppressed. When we introduced the initiation/suppression concept many years ago, we termed it the “Jesuit Model” because the Jesuits remind us that while many are called, few are chosen.
Genome duplication
Our goal is to discover how human cells determine where and when to initiate DNA replication and how the process is regulated. The importance of our goal cannot be underestimated. During normal animal development, some cells are programmed to arrest mitosis and to duplicate their genomes several times in order to produce a large number of copies of critical genes without undergoing apoptosis. As we age, however, the ability to regulate DNA replication diminishes. The result is genome instability and promiscuous cell division, the primary hallmarks of cancer. In fact, at least 122 human diseases result from aberrant DNA replication, from mutation in DNA replication or repair genes, or from infection by DNA viruses (DePamphilis, ed. Cold Spring Harbor Laboratory Press, 2006).
Several years ago, we discovered that the behavior of origin replication complexes (ORCs) in mammalian cells differs significantly from ORCs’ behavior in single-cell eukaryotes such as yeast. Yeast ORCs consist of a stable complex of six different subunits that remain bound to chromatin throughout cell division and target specific DNA sequences. In contrast, mammalian ORCs consist of a stable core complex ORC(2–5) of Orc2 through Orc5 that interacts weakly with Orc1 and Orc6. Nevertheless, the association of Orc1 with ORC(2–5) is essential for prereplication complex assembly and DNA replication. In vitro, however, metazoan ORCs exhibit little affinity for specific DNA sequences other than for asymmetric A:T-rich regions. Yet, in the differentiated cells of mammals and flies, ORCs are localized at specific genomic sites that are coincident with DNA replication origins. Thus, the ability of an ORC to activate a particular replication origin appears to depend on its ability to interact with DNA as it exists within the nucleus, an interaction that appears to be regulated by Orc1 (Figure 7.1).
Figure 7.1
The ORC cycle in mammalian cells (reviewed in DePamphilis et al., 2006b). ORC is bound to chromatin during G1 phase of the cell cycle, where it is part of a prereplication complex. When S phase begins, human Orc1 is selectively degraded by the 26S proteasome via a ubiquitin-dependent mechanism and reappears during the M- to G1-phase transition. During metaphase, Orc1 is hyperphosphorylated, an event that prevents ORC assembly. As cells exit metaphase, Orc1 is dephosphorylated and, together with other ORC subunits, binds to chromatin, an event that is facilitated by the Orc1 BAH domain.
During the past year, we established the basic features of the ORC cycle, a regulatory pathway that we previously proposed as restricting initiation of DNA replication events so that genomes are duplicated once and only once each time a cell divides. The largest subunit (Orc1) regulates association of the stable ORC(2–5) core complex with replication origins in vivo and does so through its BAH (bromo-associated homology) domain. The domain interacts with another as yet unidentified protein that is required for ORC binding to chromosomes as cells exit mitosis and begin a new cell division cycle. Moreover, if Orc1 is not bound to ORC(2–5), it can induce apoptosis (programmed cell death). However, if the unbound Orc1 is either monoubiquitinated or phosphorylated (two normal cell cycle–dependent modifications of Orc1), the modified Orc1 is localized in the cytoplasm where it cannot initiate replication, accounting for the fact that Orc1 is modified and in some cells degraded as cells enter S phase (the period of DNA replication). In addition, the modified forms of Orc1 do not induce apoptosis. Thus, cell cycle modifications regulate Orc1 activity, and Orc1 activity regulates ORC activity, which in turn regulates initiation of DNA replication.
DePamphilis ML, Blow JJ. Regulating initiation of DNA replication in the metazoa. In: DePamphilis ML, ed. DNA Replication and Human Disease. Cold Spring Harbor Laboratory Press, 2006a;313-34.
DePamphilis ML, Blow JJ, Ghosh S, Saha T, Noguchi K, Vassilev A. Regulating the licensing of DNA replication origins in metazoa. Curr Opin Cell Biol 2006b;18:231-9.
Noguchi K, Vassilev A, Ghosh S, Yates JL, DePamphilis ML. The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo. EMBO J 2006;25:5372-82.
Radichev I, Kwon SW, Zhao Y, DePamphilis ML, Vassilev A. Genetic analysis of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear localization of the origin recognition complex. J Biol Chem 2006;281:23264-73.
Saha T, Ghosh S, Vassilev A, DePamphilis ML. Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis. J Cell Sci 2006;119:1371-82.
Early mammalian development
The fertilization of an egg by a sperm activates the first round of genome duplication, after which the fertilized egg cleaves into a two-cell embryo. In mice (a laboratory model for human development), the two-cell embryo then activates the expression of about 300 genes that are required to continue development of the organism. Initially, all cells (blastomeres) produced by the cleavage events are “totipotent,” that is, they are capable of giving rise to the entire organism. But within five rounds of cell cleavage, a blastocyst appears, marking the beginning of cell differentiation. Blastocysts consist of a spherical monolayer of cells called the trophectoderm that gives rise to trophoblast stem (TS) cells and eventually to the placenta. The trophectoderm layer encompasses a group of cells called the inner cell mass that gives rise to embryonic stem (ES) cells and eventually the embryo.
Our goal is to identify genes whose expression is required in order for development to begin and then to determine the role played by those genes in establishing a living organism. We expect that the genes are essential for genome duplication, cell proliferation, and differentiation of the totipotent blastomeres into placental and embryonic cell lineages.
Genome duplication undergoes two dramatic changes during animal development. First, the number and location of replication origins can and do change at specific times during development. Second, the several concerted pathways that restrict genome duplication to once and only once per cell division are inactivated at a few specific stages during development to allow several cycles of genome duplication without intervening mitosis. This process is called endoreduplication and occurs in mammals when TS cells differentiate into trophoblast giant cells during the process of embryo implantation.
Figure 7.2
Differential expression of Tead2 and Dkkl1 mark divergent pathways to embryonic and placental tissues. The blastocyst is composed of two cells types, an outer layer of trophectodermal cells and the intermediate cell mass (ICM). The trophectoderm forms most of the extra-embryonic tissues and the placenta. Trophoblast stem (TS) cells can be derived from the trophectoderm cells that overlie the ICM. The ICM gives rise to the embryo. Embryonic stem (ES) cells can be derived from the ICM. Northern blotting-hybridization, RT-PCR, and EST database analyses reveal that the trophectoderm and its derivatives strongly express Dkkl1 (↑), whereas Tead2 expression remains unchanged (←→). In contrast, the ICM and its derivatives strongly express Tead2 (↑) and suppress expression of Dkkl1 (↓). One notable exception occurs during the production of male germ cells. Developing spermatocytes strongly express Dkkl1 in the absence of Tead2 expression such that this Dkkl1 is eventually localized to the acrosome of mature sperm.
During the past year, we identified the biological roles of three genes that are unique to vertebrates and are expressed at the very beginning of mammalian development (Figure 7.2). Our group discovered all three genes. One of the genes, Dkkl1, is unique to mammals. We found that it is expressed specifically during implantation of the embryo and during development of spermatocytes into sperm. Moreover, genetic inactivation of Dkkl1 in mice resulted in production of sperm that are defective in fertilization.
The second gene, Tead2, is one of a highly conserved family of four transcription factors that share a common DNA binding domain. Surprisingly, TEAD2 is not required until after implantation and the beginning formation of the nervous system. When we genetically inactivated Tead2, mice had difficulty in forming a neural tube. In developing vertebrates, the neural tube is the embryo’s precursor to the central nervous system, which comprises the brain and spinal cord. Failure to close the neural tube in mice is called exencephaly, which is related to anencephaly, a common human birth defect that can be prevented by folic acid.
The third gene is Tead4. We discovered that it and Tead2 are the only Tead genes that are expressed in pre-implantation mouse embryos and that genetic inactivation of Tead4 (but not of Tead2) arrests development before formation of a blastocyst. In fact, we discovered that Tead4 is required for specification of the trophectoderm linage, thus making it the earliest gene known so far on the pathway to formation of the placenta. It now appears that the totipotent blastomeres in mouse morula expressing the transcription factor TEAD4 become trophectoderm and eventually placenta, whereas those expressing the transcription factor OCT4 become the inner cell mass and eventually the embryo. Thus, Tead4 appears to be a master gene that sets into motion the first round of cell differentiation during mammalian development (Figure 7.3).
Figure 7.3
Transcription factors Tead4 and Oct4 appear to function as Master Genes in that they mark the beginning of a chain of events that specifies a unique cell lineage. Oct4 is the first gene whose expression is required for specification of the embryonic cell lineage (ICM and ES cells) that produces the embryo. Tead4 is the first gene required for specification of the trophectoderm cell lineage that produces TS cells, trophoblast giant (TG) cells, and the placenta. In the absence of Tead4 expression, all of the totipotent blastomeres produce OCT4 protein. Expression of Tead4 induces expression of Cdx2, which suppresses expression of Oct4 and thus causes the blastomere to differentiate into trophectoderm instead of ICM.
Another gene that is expressed at the beginning of mammalian development is La, which encodes a protein involved in the processing of RNA. In a collaborative effort with Richard Maraia and colleagues, we helped demonstrate that La is necessary for mammalian development by virtue of the fact that it is required for establishing embryonic stem cells.
Kaneko KC, Kohn MJ, Liu C, DePamphilis M. Transcription factor TEAD2 is involved in neural tube closure. Genesis 2007;45:577-87.
Kohn MJ, Kaneko KC, Yagi R, Litscher ES, Wassarman PM, DePamphilis M. Dickkopf-like 1–a protein unique to mammals that is associated both with formation of trophoblast stem cells and with spermatogenesis. In: Lau Y-FC, Chan WY, eds. Y Chromosome and Male Germ Cell Biology. World Scientific Publishers, 2007;185-99.
Park JM, Kohn MJ, Bruinsma MW, Vech C, Intine RV, Fuhrmann S, Grinberg A, Mukherjee I, Love PE, Ko MS, DePamphilis ML, Maraia RJ. The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells. Mol Cell Biol 2006;26:1445-51.
Yagi R, Kohn MJ, Karavanova I, Kaneko KC, Vullhorst D, DePamphilis M, Buonanno A. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development 2007;134:3827-36.
COLLABORATORS
Andres Buonanno, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
Irina Karavanova, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
Sung Won Kwon, PhD, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
Eveline S. Litscher, PhD, Mount Sinai School of Medicine, New York, NY
Chengyu Liu, PhD, Transgenic Facility, NHLBI, Bethesda, MD
Richard Maraia, MD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Paul M. Wassarman, PhD, Mount Sinai School of Medicine, New York, NY
John L. Yates, PhD, Roswell Park Cancer Institute, Buffalo, NY
Yingming Zhao, PhD, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
For further information, contact depamphm@mail.nih.gov.
