Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol alpha expression during early S phase.
(33/1127)
E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol alpha, is regulated by interactions with Rb and by cell-cycle dependent alterations in E2F-1 abundance. We have shown that depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA expression downregulates pol alpha and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol alpha and E2F-1 gene expression, we utilized immortalized mouse fibroblasts derived from wild-type and PARP knockout (PARP-/-) mice as well as PARP-/- cells stably transfected with PARP cDNA [PARP-/-(+PARP)]. After release from serum deprivation, wild-type and PARP-/-(+PARP) cells, but not PARP-/- cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h. Whereas [3H]thymidine incorporation remained negligible in PARP-/- cells, in vivo DNA replication maximized after 18 h in wild-type and PARP-/-(+PARP) cells. To investigate the effect of PARP on E2F-1 promoter activity, a construct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells. E2F-1 promoter activity in control and PARP-/-(+PARP) cells increased eightfold after 9 h, but not in PARP-/- cells. PARP-/- cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP-/-(+PARP) cells. RT - PCR analysis and pol alpha activity assays revealed the presence of pol alpha transcripts and a sixfold increase in activity in both wild-type and PARP-/-(+PARP) cells after 20 h, but not in PARP-/- cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity, which then positively regulates both E2F-1 and pol alpha expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum. (+info)
E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells.
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We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here that overexpression of E2F-1, E2F-2 and E2F-4, or a chimeric E2F-4 tethered to a nuclear localization signal cannot reactivate postmitotic skeletal muscle cells (myotubes). This is not due to lack of transcriptional activity, as demonstrated on both a reporter construct and a number of endogenous target genes. Although cyclin E was strongly overexpressed in E2F-transduced myotubes, it lacked associated kinase activity, possibly explaining the inability of the myotubes to enter S phase and accumulate cyclin A. Although E2F is not sufficient to trigger DNA synthesis in myotubes, its activity is necessary even in the presence of E1A, as dominant-negative DP-1 mutants inhibit E1A-mediated cell cycle reentry. Our data show that, to reactivate myotubes, E1A must exert other functions, in addition to releasing E2F. They also establish mouse myotubes as an experimental system uniquely suited to study the most direct E2F functions in the absence of downstream cell cycle effects. (+info)
Regulation of the G1/S transition phase in mesangial cells by E2F1.
(35/1127)
It has been established that E2F transcription factors are essential for the regulation of the cell cycle. The E2Fs play an important role in G1/S transition phase, as they regulate the activation of several genes whose products are required for DNA synthesis. E2Fs bind to the retinoblastoma protein family and their transcriptional activities are suppressed in the G0 and early G1 phases. The E2F family consists of a group of five closely related proteins (E2F1 through E2F5). Proliferation of the mesangial cell is a common feature of many glomerular diseases, but the regulation of mesangial cell cycle has not been clarified, nor has the participation of the E2F family in mesangial cells. To elucidate the mechanisms of G1/S transition phase in mesangial cells, we investigated the roles of the E2F family in the mesangial cell cycle. In primary cultured mesangial cells, the protein expression of E2F1 through E2F3 was induced by fetal calf serum (FCS) stimulation. E2F1 especially was strongly induced by mitogenic stimulation. The E2F4 protein was abundantly expressed in the quiescent state and was slightly increased by FCS stimulation. We considered E2F1 to be representative of the E2F family, and used adenovirus-mediated gene transfer to investigate the function of E2F1 to show that overexpression of E2F1 promoted cell cycle progression as measured by a flow cytometer. Furthermore, we investigated the effect of E2F1 overexpression to cyclin D1 and cyclin E expression. Because we previously reported that the regulation of G1 cyclins is a key factor in the G1/S transition phase in mesangial cells, we showed that overexpression of E2F1 induced protein expression of cyclin D1 and cyclin E and increased promoter activity. Thus, we conclude that E2F1 plays an important role in the G1/S transition phase and acts on the mesangial cell cycle through two distinct pathways: (1) E2F1 directly transcribes an S-phase gene, and (2) E2F1 promotes cell cycle progression via the induction of cyclin D1 and cyclin E. (+info)
Rb and prohibitin target distinct regions of E2F1 for repression and respond to different upstream signals.
(36/1127)
E2F transcription factor is subject to stringent regulation by a variety of molecules. We recently observed that prohibitin, a potential tumor suppressor protein, binds to the retinoblastoma (Rb) protein and represses E2F transcriptional activity. Here we demonstrate that prohibitin requires the marked box region of E2F for repression; further, prohibitin can effectively inhibit colony formation induced by overexpression of E2F1 in T47D cells. Prohibitin was also found to interact with the signaling kinase c-Raf-1, and Raf-1 could effectively reverse prohibitin-mediated repression of E2F activity. Agents such as E1A, p38 kinase, and cyclins D and E had no effect on prohibitin-mediated repression of E2F1, but all of these molecules could reverse Rb function. Similarly, stimulation of the immunoglobulin M signaling pathway in Ramos cells could inactivate prohibitin, but this had no effect on Rb function. Serum stimulation of quiescent Ramos cells inactivated Rb and prohibitin with different kinetics; further, while the serum-dependent inactivation of Rb was dependent on cyclin-dependent kinase activity, the inactivation of prohibitin was not. We believe that prohibitin is a novel regulator of E2F function which channels specific signaling cascades to the cell cycle regulatory machinery. (+info)
Distinct cellular factors regulate the c-myb promoter through its E2F element.
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Most E2F-driven promoters are transiently activated around the G(1)/S transition. Although the promoter for the c-myb proto-oncogene harbors an E2F element, it is induced early in G(1) following entry into the cell cycle. Furthermore, this promoter remains active throughout subsequent cell cycles. Since E2F sites function as repressor elements during G(1) (due to the association of pRb with E2F factors), we investigated whether the E2F element in the c-myb promoter is regulated differently than E2F elements in promoters that are repressed during G(1). By gel shift analysis, the E2F element from the c-myb promoter was found to form a unique complex, referred to as E2Fmyb-sp, which was not observed with E2F elements from several other promoters. Antibodies to DP-1, E2F1 to -5, p107, or pRb failed to either supershift or block E2Fmyb-sp complex formation. Methylation interference experiments indicate that the DNA contact residues for the E2Fmyb-sp complex are distinct from but overlapping with residues required for the binding of E2F proteins. In addition to the identification of E2Fmyb-sp, we have found that SP-1 binds to the c-myb E2F element. Functional studies revealed that E2Fmyb-sp and/or SP-1 are required to achieve full activation of the c-myb promoter in different cell types and to maintain elevated expression of the c-myb promoter during G(1) in NIH 3T3 cells. These studies demonstrate that E2F elements can be regulated differently through the binding of unique sets of proteins. (+info)
Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor.
(38/1127)
Normal cells do not divide indefinitely due to a process known as replicative senescence. Human cells arrest growth with a senescent phenotype when they acquire one or more critically short telomeres as a consequence of cell division. Recent evidence suggests that certain types of DNA damage, chromatin remodeling, and oncogenic forms of Ras or Raf can also elicit a senescence response. We show here that E2F1, a multifunctional transcription factor that binds the retinoblastoma (pRb) tumor suppressor and that can either promote or suppress tumorigenesis, induces a senescent phenotype when overexpressed in normal human fibroblasts. Normal human cells stably arrested proliferation and expressed several markers of replicative senescence in response to E2F1. This activity of E2F1 was independent of its pRb binding activity but dependent on its ability to stimulate gene expression. The E2F1 target gene critical for the senescence response appeared to be the p14(ARF) tumor suppressor. Replicatively senescent human fibroblasts overexpressed p14(ARF), and ectopic expression of p14(ARF) in presenescent cells induced a phenotype similar to that induced by E2F1. Consistent with a critical role for p14(ARF), cells with compromised p53 function were immune to senescence induction by E2F1, as were cells deficient in p14(ARF). Our findings support the idea that the senescence response is a critical tumor-suppressive mechanism, provide an explanation for the apparently paradoxical roles of E2F1 in oncogenesis, and identify p14(ARF) as a potentially important mediator of the senescent phenotype. (+info)
Roles of E2F1 in mesangial cell proliferation in vitro.
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Roles of E2F1 in mesangial cell proliferation in vitro. BACKGROUND: The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. METHODS: To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. RESULTS: E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. CONCLUSIONS: In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins. (+info)
Association of Pur alpha and E2F-1 suppresses transcriptional activity of E2F-1.
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Protein-protein interaction can play an important role in the control of several biological events including gene transcription, replication and cell proliferation. E2F-1 is a DNA-binding transcription factor which, upon interaction with its target DNA sequence, induces expression of several S phase specific genes allowing progression of the cell cycle. Evidently, the activity of this protein is modulated by its cellular partner, pRb, which in the hypophosphorylated form, binds to E2F-1 and inactivates its transcriptional ability. In this study, we have demonstrated that expression of a sequence-specific single-stranded DNA binding protein, Pur alpha, in cells decreases the ability of E2F-1 to exert its transcriptional activity upon the responsive promoter derived from DHFR. Results from band shift experiments revealed that while Pur alpha does not recognize the double-stranded DNA fragment containing the E2F-1 binding site, it has the ability to inhibit E2F-1 interaction with its target DNA sequence. Results from GST pull-down assays and the combined immunoprecipitation/Western blot analysis of nuclear extracts revealed a direct association of E2F-1 with Pur alpha in the absence of the DNA molecule containing the E2F-1 binding site. The association of Pur alpha with E2F-1 may increase the stability of E2F-1, as a higher level of E2F-1 was detected in cells coexpressing Pur alpha and E2F-1. The importance of these observations with respect to the role of Pur alpha in the control of cell cycle progression is discussed. (+info)