Rbf1-independent termination of E2f1-target gene expression during early Drosophila embryogenesis

The initiation and maintenance of G1 cell cycle arrest is a key feature of animal development. In the Drosophila ectoderm, G1 arrest first appears during the seventeenth embryonic cell cycle. The initiation of G1(17) arrest requires the developmentally-induced expression of Dacapo, a p27-like Cyclin E-Cdk2 inhibitor. The maintenance of G1(17) arrest requires Rbf1-dependent repression of E2f1-regulated replication factor genes, which are expressed continuously during cycles 1-16 when S phase immediately follows mitosis. The mechanisms that trigger Rbf1 repressor function and mediate G1(17) maintenance are unknown. Here we show that the initial downregulation of expression of the E2f1-target gene RnrS, which occurs during cycles 15 and 16 prior to entry into G1(17), does not require Rbf1 or p27(Dap). This suggests a mechanism for Rbf1-independent control of E2f1 during early development. We show that E2f1 protein is destroyed in a cell cycle-dependent manner during S phase of cycles 15 and 16. E2f1 is destroyed during early S phase, and requires ongoing DNA replication. E2f1 protein reaccumulates in epidermal cells arrested in G1(17), and in these cells the induction of p27(Dap) activates Rbf1 to repress E2f1-target genes to maintain a stable G1 arrest.     

Developmentally regulated histone modifications in <Emphasis Type="Italic">Drosophila</Emphasis> follicle cells: initiation of gene amplification is associated with histone H3 and H4 hyperacetylation and H1 phosphorylation

We have used gene amplification in Drosophila follicle cells as a model of metazoan DNA replication to address whether changes in histone modifications are associated with replication origin activation. We observe that replication initiation is associated with distinct histone modifications. Acetylated lysines K5, K8, and K12 on histone H4 and K14 on histone H3 are specifically enriched during replication initiation at the amplification origins. Strikingly, H4 acetylation persists at an amplification origin well after replication forks have progressed significantly outward from the origin, indicating that H4 acetylation is associated with origin regulation and not histone deposition at the replication forks. Origin recognition complex subunit 2 (orc2) mutants with severe amplification defects do not abolish H4 acetylation, whereas the dup/cdt1 mutant delays the appearance of acetylation foci, and mutants in rbf result in temporal persistence. These data indicate that core histone acetylation is associated with origin activity. Furthermore, follicle cells undergoing gene amplification exhibit high levels of histone H1 phosphorylation. The patterns of H1 phosphorylation provide insights into cell cycle states during amplification, as H1 kinase activity in follicle cells is responsive to high Cyclin E activity, and it can be abolished by overexpressing the retinoblastoma homolog, Rbf, that represses Cyclin E. These data suggest that amplification origins are able to initiate when the cells are in a late S-phase, when the genome is normally not licensed for replication.     

Specificity of E2F1, E2F2, and E2F3 in mediating phenotypes induced by loss of Rb.

The Rb/E2F pathway plays a critical role in the control ofcellular proliferation. Here, we report that E2F1, E2F2, and E2F3 make major individual contributions toward the in vivo phenotypic consequences of Rb deficiency. In the developing lens of Rb(-/-) embryos, loss of E2F1, E2F2, or E2F3 reduces the unscheduled proliferation of fiber cells, with the loss of E2F3 having the most pronounced effect. In Rb-deficient retinas, all three E2Fs contribute equally to the ectopic proliferation of postmitotic neuronal cells. In contrast, E2F1 is unique in mediating apoptosis in both Rb(-/-) lenses and retinas. In the central nervous system, loss of E2F1 or E2F3 can almost completely eliminate the ectopic DNA replication and apoptosis observed in Rb(-/-) embryos, and loss of E2F2 partially reduces the unscheduled DNA replication and has no effect on apoptosis. These results provide clear evidence for functional specificity among E2Fs in the control of Rb-dependent proliferation and apoptosis in a tissue-specific manner.     

MdmX Represses E2F1 Transactivation

Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.     

MdmX represses E2F1 transactivation

Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.     

Analysis of origin recognition complex in saccharomyces cerevisiae by use of Degron mutants

Origin recognition complex (ORC), a six-protein complex (Orc1p-Orc6p), may deeply involve in initiation of chromosomal DNA replication. However, since most temperature-sensitive orc mutants of Saccharomyces cerevisiae show the accumulation of cells with nearly 2C DNA content, the exact stage at which ORC acts is not fully understood. In this study, we constructed a heat-inducible degron mutant for each ORC subunit. As well as each targeted subunit, other subunits of ORC were also rapidly degraded under non-permissive conditions. In the orc5 degron mutant, incubation under the non-permissive conditions caused accumulation of cells with nearly 2C DNA content, and phosphorylation of Rad53p. When Orc5p (ORC) is depleted, this inhibits G1/S transition and formation of a pre-replicative complex (pre-RC). For pre-RC to form, and G1/S transition to proceed, Orc5p (ORC) must be present in late G1, rather than early G1, or G2/M. Block and release experiments revealed that Orc5p (ORC) is not necessary for S and G2/M phase progression. We therefore propose that ORC is necessary for the G1/S transition and pre-RC formation, and accumulation of cells with nearly 2C DNA content seen in various orc mutants is due to inefficient pre-RC formation, and/or induction of checkpoint systems.     

Plk1 phosphorylation of Orc2 promotes DNA replication under conditions of stress

Polo-like kinase 1 (Plk1) plays pivotal roles in mitosis; however, little is known about its function in S phase. In this study, we show that inhibition of Plk1 impairs DNA replication and results in slow S-phase progression in cultured cancer cells. We have identified origin recognition complex 2 (Orc2), a member of the DNA replication machinery, as a Plk1 substrate and have shown that Plk1 phosphorylates Orc2 at Ser188 in vitro and in vivo. Furthermore, Orc2-S188 phosphorylation is enhanced when DNA replication is under challenge induced by ultraviolet, hydroxyurea, gemcitabine, or aphidicolin treatment. Cells expressing the unphosphorylatable mutant (S188A) of Orc2 had defects in DNA synthesis under stress, suggesting that this phosphorylation event is critical to maintain DNA replication under stress. To dissect the mechanism pertinent to this observation, we showed that Orc2-S188 phosphorylation associates with DNA replication origin and that cells expressing Orc2-S188A mutant fail to maintain the functional pre-replicative complex (pre-RC) under DNA replication stress. Furthermore, the intra-S-phase checkpoint is activated in Orc2-S188A-expressing cells to cause delay of S-phase progress. Our study suggests a novel role of Plk1 in facilitating DNA replication under conditions of stress to maintain genomic integrity.