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Preimplantation development is a period of dynamic epigenetic change that begins with remodeling of egg and sperm genomes, and ends with implantation. During this time, parental-specific imprinting marks are maintained to direct appropriate imprinted gene expression. We previously demonstrated that H19 imprinting could be lost during preimplantation development under certain culture conditions. To define the lability of genomic imprints during this dynamic period and to determine whether loss of imprinting continues at later stages of development, imprinted gene expression and methylation were examined after in vitro preimplantation culture. Following culture in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed and undermethylated. However, only a subset of individual cultured blastocysts (∼65%) exhibited biallelic expression, while others maintained imprinted H19 expression. Loss of H19 imprinting persisted in mid-gestation conceptuses. Placental tissues displayed activation of the normally silent allele for H19, Ascl2, Snrpn, Peg3 and Xist while in the embryo proper imprinted expression for the most part was preserved. Loss of imprinted expression was associated with a decrease in methylation at the H19 and Snrpn imprinting control regions. These results indicate that tissues of trophectoderm origin are unable to restore genomic imprints and suggest that mechanisms that safeguard imprinting might be more robust in the embryo than in the placenta.

Cloning by somatic cell nuclear transfer requires that epigenetic information possessed by the donor nucleus be reprogrammed to an embryonic state. Little is known, however, about this remodeling process, including when it occurs, its efficiency, and how well epigenetic markings characteristic of normal development are maintained. Examining the fate of epigenetic information associated with imprinted genes during clonal development offers one means of addressing these questions. We examined transcript abundance, allele specificity of imprinted gene expression, and parental allele-specific DNA methylation in cloned mouse blastocysts. Striking disruptions were seen in total transcript abundance and allele specificity of expression for five imprinted genes. Only 4% of clones recapitulated a blastocyst mode of expression for all five genes. Cloned embryos also exhibited extensive loss of allele-specific DNA methylation at the imprinting control regions of the H19 and Snprn genes. Thus, epigenetic errors arise very early in clonal development in the majority of embryos, indicating that reprogramming is inefficient and that some epigenetic information may be lost.

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G 1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.E2F is a transcriptional regulator that plays a pivotal role in the regulation of cellular proliferation (reviewed in reference 59; 8). Many E2F-responsive genes have been identified, and their products are components of either cell cycle control (e.g., cyclin E, cyclin A, and cdc2) or DNA synthesis (e.g., dihydrofolate reductase, thymidine kinase, or DNA polymerase ␣) machinery. In each case, E2F is thought to restrict the expression of these genes to the point of the cell cycle at which their products act (38).E2F is regulated by the retinoblastoma protein (pRB) (3, 5, 12), a tumor suppressor that is functionally inactivated in a large proportion of all human tumors (reviewed in reference 67). Consistent with its antiproliferative role, pRB blocks the ability of E2F to activate transcription (32, 33). In addition, overexpression studies have indicated that the resultant pRB-E2F complex can act as a transcriptional repressor, in which E2F provides the sequence-specific DNA binding activity and pRB inhibits transcription by sequestering adjacent transcription factors (2,10,62,69,70). This idea suggests that E2F participates in both the activation and the inhibition of cellular proliferation. Consistent with this hypothesis, homozygous deletion of the murine E2F-1 gene causes atrophy in some tissues and tumors in others (26, 73).The growth-inhibitory properties of pRB are regulated by its cell cycle-dependent phosphorylation (reviewed in reference 7). Phosphorylation is catalyzed by one or more of the cell cycle-dependent kinases...

An intriguing characteristic of imprinted genes is that they often cluster in large chromosomal domains, raising the possibility that gene-specific and domain-specific mechanisms regulate imprinting. Several common features emerged from comparative analysis of four imprinted domains in mice and humans: (a) Certain genes appear to be imprinted by secondary events, possibly indicating a lack of gene-specific imprinting marks; (b) some genes appear to resist silencing, predicting the presence of cis-elements that oppose domain-specific imprinting control; (c) the nature of the imprinting mark remains incompletely understood. In addition, common silencing mechanisms are employed by the various imprinting domains, including silencer elements that nucleate and propagate a silent chromatin state, insulator elements that prevent promoter-enhancer interactions when hypomethylated on one parental allele, and antisense RNAs that function in silencing the overlapping sense gene and more distantly located genes. These commonalities are reminiscent of the behavior of genes subjected to, and the mechanisms employed in, dosage compensation.

The E2F family of proteins is required to establish the correct cell-cycle-dependent transcription of genes that direct the process of cell division. All previously identified E2F proteins can act in a similar manner; depending on whether or not they are associated with the cell cycle inhibitors the retinoblastoma protein (pRB), p107, or p130, they can either repress or activate the transcription of E2F-responsive genes. We now report the cloning and characterization of another E2F family member, E2F-6, whose structure is reminiscent of the dominant inhibitors of other transcription factor families. The dimerization and DNA binding properties of E2F-6 are similar to those of the other E2F family members. However, it is not regulated by pRB, p107, or p130, and it is unable to activate transcription. Instead, it can act to repress the transcription of E2F responsive genes by countering the activity of the other E2F complexes via a pRB-, p107-, or p130-independent mechanism.

Tumor development is dependent upon the inactivation of two key tumor-suppressor networks, p16Ink4a -cycD/cdk4-pRB-E2F and p19Arf -mdm2-p53, that regulate cellular proliferation and the tumor surveillance response. These networks are known to intersect with one another, but the mechanisms are poorly understood. Here, we show that E2F directly participates in the transcriptional control of Arf in both normal and transformed cells. This occurs in a manner that is significantly different from the regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts (MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function was previously undetermined. E2f3 loss is sufficient to derepress Arf, triggering activation of p53 and expression of p21 Cip1. Thus, E2F3 is a key repressor of the p19 Arf -p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the activation of p53 and p21Cip1 in E2f3-deficient MEFs. Arf loss also rescues the known cell cycle re-entry defect of E2f3 −/− cells, and this correlates with restoration of appropriate activation of classic E2F-responsive genes. Our data also demonstrate a direct role for E2F in the oncogenic activation of Arf. Specifically, we observe recruitment of the endogenous activating E2Fs, E2F1, and E2F3a, to the Arf promoter. Thus, distinct E2F complexes directly contribute to the normal repression and oncogenic activation of Arf. We propose that monitoring of E2F levels and/or activity is a key component of Arf's ability to respond to inappropriate, but not normal cellular proliferation. The development of mammalian tumors is dependent upon the disruption of two key biological activities, the control of cellular proliferation and the apoptotic response (Hanahan and Weinberg 2000). Remarkably, the Ink4a/Arf locus encodes two distinct tumor-suppressor proteins, p16Ink4a and p19 Arf (p14 Arf in humans), that influence one or both of these processes Sherr 2001). p16 Ink4a is a core component of the cell cycle control machinery (Sherr and Roberts 1999). It controls the activity of the G 1 kinase, cyclinD · cdk4/6, and consequently, the phosphorylation status of the pocket protein family. This family includes the retinoblastoma protein (pRB) tumor suppressor and its relatives, p107 and p130. In the unphosphorylated state, the pocket proteins bind to the E2F family of transcription factors and prevent the expression of genes that are essential for entry into, and passage through the cell cycle (Trimarchi and Lees 2002). This inhibition occurs through two distinct mechanisms. pRB binds to the activating E2Fs, E2F1, E2F2, and E2F3a, and blocks their transcriptional activity. At the same time, the repressive E2Fs, E2F4, and E2F5 recruit p107 or p130 and their associated histone deactylases to E2F-responsive promoters. Under these conditions, the cell is blocked in G 0 /G 1 . Mitogenic signaling activates cell cycle re-entry by ...

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