E2F
Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G1/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. Mutations have been constructed that disrupt C pocket activity. When overproduced, the RB C-terminal fragment does not induce terminal growth arrest but can inhibit G1/S progression, and this activity is abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduce but do not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolish RB's ability to cause an increase in the percentage of cells in G1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G1 through mechanisms that are independent of each other. In contrast, long-term growth arrest requires combined activities from both regions of RB. In addition, E2F and LXCXE binding are not the only mechanisms through which RB inhibits cell growth. The C pocket also contributes to RB-mediated growth suppression (Whitaker, 1998).
Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). The E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Additionally, phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind the LXCXE proteins, c-Abl, and E2F, even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB (Knudsen, 1997).
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. 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.
E2F-4 accounts for the majority of endogenous E2F species. 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 G1 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 (Verona, 1997).
Overexpression of human cyclin E shortens G1 causing a premature entry into S. The retinoblastoma protein is a target for the cyclin E-cdk2 complex. Coexpression of Rb with cyclin E induces Rb hyperphosphorylation and overrides the ability of Rb to suppress G1 exit. Hypophosphorylated Rb can interact with the transcription factor E2F during G1 and this complex can then bind to DNA and repress transcription of E2F target genes. Conversely, hyperphosphorylation of Rb prevents its interaction with E2F, releasing E2F from an inhibitory constraint and enabling it to promote gene expression (Sherr, 1993 and references).
The Retinoblastoma-related protein p107, like the p21 family of cdk inhibitors, can inhibit the phosphorylation of target substrates by cyclin A/cdk2 and cyclin E/cdk2 complexes. The associations of p107 and p21 with cyclin/cdk2 rely on a structurally and functionally related interaction domain. Interactions between p107 and p21 are mutually exclusive: p21 causes a dissociation of p107/cyclin/cdk2 complexes to yield p21/cyclin/cdk2 complexes. The activation of the p107-bound cyclin/cdk kinases leads to dissociation of p107 from the transcription factor E2F. It has been suggested that p107 functions similarly to Rb, causing growth arrest of sensitive cells in the G1 phase of the cell cycle. The p107 molecule can be dissected into two domains, either of which is able to independently block cell cycle progression. One domain corresponds to the sequences needed for interaction with transcription E2F, and the other corresponds to the interaction domain for cyclin A or cyclin E complexes (Zhu, 1995 a and b).
The activity of the E2F transcription factor is controlled by physical association with the retinoblastoma
protein (pRB) and two related proteins, p107 and p130. The pRB family members are thought to
control different aspects of E2F activity, but it has been unclear what the respective functions of these
proteins might be. To dissect the specific functions of pRB, p107, and p130, a study was carried out to determine how the expression of E2F-regulated genes changes in cultures of primary cells lacking each of these family members. Whereas no changes are found in the expression of E2F-target genes in cells
lacking either p107 or p130, deregulated expression of E2F targets was seen in cells lacking pRB and
in cells lacking both p107 and p130. Surprisingly, the genes that are deregulated in these two settings
are completely different. cyclin E and p107 are derepressed in pRb mutants. B-myb (see Drosophila Myb oncogene-like), cdc2, E2F-1, TS, RRM2 and cyclin A2 are derepressed in p107mutant/p130mutant cells. These findings show that pRB and p107/p130 indeed provide different functions in E2F regulation and identify target genes that are dependent on pRB family proteins for
their normal expression. Because deregulation of E2F activity is thought to play an important role in promoting the proliferation of Rb mutant tumor cells, it is surprising that far more extensive changes in E2F activity are found in p107/p130 double mutants than in Rb mutants. Perhaps the set of E2F targets genes that are misexpressed in Rb mutants might be more important for cell proliferation than the E2F targets that are misexpressed in p107/p130 double mutants. An alternative explanation for the result is suggested by mutational studies showing that E2F binding is not sufficient for cell cycle arrest and growth suppression properties of RB. In addition to E2F at least 39 pRB-associated proteins have been described. It is possible that the role of pRB in E2F regulation may only be important for tumor suppressor activity in conjunction with other functions that are carried out specifically by pRB but not by p107 and p130 (Hurford, 1997).
The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene
suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in
vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To
determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis, the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural
stem cells derived from Rb-deficient mice was examined. Although Rb -/- neural precursor cells are able to
differentiate in vitro, these cells exhibit a significant delay in terminal mitosis relative to
wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found
in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural
precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A
N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other
Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts
with the putative Rb target E2F3 in neural precursor cells, these results indicate that it clearly cannot
restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3
activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In
these studies it is shown that Rb is essential for the regulation of E2F 1 and 3 activity as well as the
onset of terminal mitosis in neural precursor cells (Callaghan, 1999).
Electrophoretic mobility shift assays were used to analyse the pattern of E2F transcription factor complexes containing pRB and related 'pocket' proteins associated with changes in growth of monkey CV-1 cells. Little change is noted in pRB/E2F complexes following growth arrest or serum stimulation. Serum starvation induces the formation of a novel slowly-migrating p130/E2F complex, termed C7, which is comparable to one reported previously in terminally differentiated C2C12 mouse cells and thought to contain one or more additional unidentified proteins. After serum stimulation, C7 complex disappears in S-phase but returns during mitosis. A major E2F complex containing p107 appears during S-phase but is undetectable at other times. It appears likely that regulation of pRB, p107 and p130 occurs by several mechanisms: Binding of such proteins with p130/E2F occurs via a site in the 'pocket' similar to that utilized by adenovirus E1A
proteins. Such proteins could function as additional regulators of E2F-driven transcription (Corbeil, 1997).
E2F1 overexpression has been shown to induce apoptosis in cooperation with p53. Using Saos-2 cells, which are null for p53 and lack functional Rb, it can be shown that E2F1 overexpression can also induce apoptosis in the absence of p53 and retinoblastoma protein (Rb). E2F1-induced apoptosis can be specifically inhibited by Rb but not mdm2, which is known for its ability to inhibit p53-induced apoptosis. Through the study of the apoptotic function of a set of E2F1 mutants, it has become clear that the transactivation and the apoptotic function of E2F1 are uncoupled. Transactivation-defective E2F1 mutants can induce apoptosis as effectively as wild-type E2F1. In contrast to E2F1 transactivation, the DNA-binding activity of E2F1 is essential for its apoptotic function, as the DNA-binding-defective mutants failed to induce apoptosis. Therefore Rb may inhibit E2F1-induced apoptosis by mechanisms other than the suppression of the transactivation of E2F1. This hypothesis was supported by the observation that although Rb overexpression can specifically repress the apoptosis induced by wild-type E2F1 and an Rb-binding-competent E2F1 mutant, it fails to inhibit the apoptosis induced by mutants that are defective or reduced in Rb binding and transactivation. All of these points argue for a novel function for E2F1 and Rb in controlling apoptosis. The results also indicate that transcriptional repression rather than the transactivation function of E2F1 may be involved in its apoptotic function. It is thought that the Rb-E2F1 complex actively represses genes involved in apoptosis (Hsieh, 1997).
Certain E2F transcription factor species play a pivotal role in regulating cell-cycle progression. The activity of E2F1, a protein with neoplastic transforming
activity when unregulated, is tightly controlled at the transcriptional level during G0 exit. In addition, during this interval, the stability of endogenous E2F1
protein increases markedly. E2F1 stability also is dynamically regulated during myogenic differentiation and in response to gamma irradiation. One or
more retinoblastoma family proteins likely participate in the stability process, because simian virus 40 T antigen disruptes E2F1 stability regulation during
G1 exit in a manner dependent on its ability to bind to pocket proteins. Thus, endogenous E2F1 function is regulated by both transcriptional and
posttranscriptional control mechanisms (Martelli, 1999).
Rb inhibits progression from G1 to S phase of the cell cycle. It associates with a number of cellular proteins;
however, the nature of these interactions and their relative significance in cell cycle regulation are still
unclear. Evidence is presented that Rb must normally interact with the E2F family of transcription factors to
arrest cells in G1, and that this arrest results from active transcriptional repression by the Rb-E2F complex,
not from inactivation of E2F. Thus, a major role of E2F in cell cycle regulation is assembly of this repressor
complex. Active repression by Rb-E2F mediates the G1 arrest triggered by both TGFbeta and p16INK4a and also mediates contact inhibition (Zhang, 1999).
E2F-1 has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response has led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. Although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic
functions, consistent with its putative role as a tumor suppressor (Phillips, 1997).
Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture.
A major target of the RB protein is the S-phase-inducing transcription factor E2F1. RB binds directly
to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To
induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are
components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical
interaction between RB and hbrm/BRG-1. In transfected cells RB can simultaneously contact both
E2F1 and hbrm, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found
within the same complex in vivo. RB and hbrm cooperate to repress E2F1 activity in
transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent
upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site.
However, the bromodomain seems dispensable for this activity. Taken together, these results point out an
unexpected role as corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in
repressing E2F1 activity could be an underlying mechanism for the observed cooperation between hbrm and RB to induce G1 arrest. The domain of hbrm that binds RB has transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm but also regulates its activity (Trouche, 1997).
Complexes of the cyclin-dependent kinase, cdk4 (Drosophila homolog: Cyclin-dependent kinase 4/6), and each of three different D-type cyclins phosphorylate the Retinoblastoma protein. Cyclins D2 and D3 but not D1 bind Rb in intact cells. Introduction of cdk4, together with Rb and D-type cyclins, induce Rb hyperphosphorylation and dissociation of cyclins D2 and D3. The transcription factor E2F-1 also binds Rb, and coexpression of cyclin D-cdk4 complexed triggers Rb phosphorylation and prevents its interaction with E2F-1. Thus D-type cyclins play a dual role as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates (Kato, 1993)
Cyclin D1 controls the timing of S phase onset in mammalian cells, acting as a positive regulator of the transcription factor E2F. Cyclin D1 overexpression leads to the activation of the dihydrofolate reductase gene promoter, acting through the E2F binding site in the promoter. P16INK4 represses this interaction, and this repression can be released by overexpression of cdk4. Thus cyclin D1 and its associated kinase have a direct role in cell cycle regulation of E2F activity and consequently of S phase-specific gene expression. E2F binding sites bind complexes containing the retinoblastoma protein, while in Rb-deficient cell lines overexpression of cyclin D1 fails to activate E2F-dependent transcrition, suggesting that Rb may be involved in promoter activation (Schulze, 1994).
Considerable evidence points to a role for G1 cyclin-dependent kinase (CDK) in allowing the accumulation of E2F transcription factor activity and induction of the S phase of the cell cycle. Numerous experiments have also demonstrated a critical role for both Myc and Ras (See Drosophila Ras1) activities in allowing cell-cycle progression. Inhibition of Ras activity blocks the normal growth-dependent activation of G1 CDK, prevents activation of the target genes of E2F, and results in cell-cycle arrest in G1. Ras is essential for entry into the S phase in Rb+/+ fibroblasts but not in Rb-/- fibroblasts, establishing a link between Ras and the G1 CDK/Rb/E2F pathway. However, although expression of Ras alone will not induce G1 CDK activity or S phase, coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the induction of S phase, coincident with the loss of the p27 cyclin-dependent kinase inhibitor (CKI). These results suggest that Ras, along with the activation of additional pathways, is required for the generation of G1 CDK activity, and that activation of cyclin E-dependent kinase in particular depends on the cooperative action of Ras and Myc (Leone, 1997).
Free E2F-1 and E2F-4 transcription factors are unstable; their degradation is mediated by the ubiquitin-proteasome pathway. Both E2F-1 and E2F-4 are rendered
unstable by an epitope in the carboxyl terminus of the proteins, in close proximity to their pocket protein interaction surface. E2F-1 interaction with pRb and E2F-4 interaction with p107 or p130 protects these E2Fs from degradation, causing the complexes to be stable. The increased stability of E2F-4 pocket protein complexes may contribute to the maintenance of active transcriptional repression in quiescent cells. Surprisingly, adenovirus transforming proteins, which release pocket protein-E2F complexes, also inhibit breakdown of free E2F. These data reveal an additional level of regulation of E2F transcription factors by targeted proteolysis, which is inhibited by pocket protein binding and adenovirus early region 1 transforming proteins (Hateboer, 1996).
E2F-1 plays a crucial role in the regulation of cell-cycle progression at the G1-S transition. When overproduced, it is both an oncoprotein and a potent inducer of apoptosis; therefore, its transcriptional activity is subject to multiple controls. Among these multiple controls are binding by the retinoblastoma gene product (pRb), activation by cdk3, and S-phase-dependent down-regulation of DNA-binding capacity by cyclin A-dependent kinase. E2F-1 is actively degraded by the ubiquitin-proteasome pathway. Efficient degradation depends on the availability of selected E2F-1 sequences. Unphosphorylated pRb stabilized E2F-1, protecting it from in vivo degradation. pRb-mediated stabilization is not an indirect consequence of G1 arrest, but rather depends upon the ability of pRb to interact physically with E2F-1. Thus, in addition to binding E2F-1 and transforming it into a transcriptional repressor, pRb has another function, protection of E2F-1 from efficient degradation during a period when pRb/E2F complex formation is essential to regulating the cell cycle. In addition, there may be a specific mechanism for limiting free E2F-1 levels. The failure of such a mechanism could compromise cell survival and/or homeostasis (Hofmann, 1996).
Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as
monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. Cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observations suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late
G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region (Zerfass-Thome, 1997).
The interaction between pRB and E2F is critical for control of the cell cycle and apoptosis. pRB contains two distinct E2F binding sites. The previously identified E2F binding site on pRB is necessary for stable association with E2Fs on DNA. A second E2F interaction site is located entirely within the C-terminal domain of pRB and is specific for E2F1. E2F1/pRB complexes formed through this site have low affinity for DNA, but the interaction is sufficient for pRB to regulate E2F1-induced apoptosis, and E2F1 loses the ability to interact with this site following DNA damage. These results show that pRB interacts with individual E2F proteins in different ways and suggest that pRB's regulation of E2F1-induced apoptosis is physically separable from its transcriptional control of other E2F proteins (Dick, 2003).
Previous work has provided evidence for E2F-dependent transcription control of both G1/S- and G2/M-regulated genes. Analysis of the G2-regulated cdc2 and cyclin B1 genes reveals the presence of both positive- and negative-acting E2F promoter elements. Additional elements provide both positive (CCAAT and Myb) and negative (CHR) control. Chromatin immunoprecipitation assays identify multiple interactions of E2F proteins that include those previously shown to activate and repress transcription. E2F1, E2F2, and E2F3 were found to bind to the positive-acting E2F site in the cdc2 promoter, whereas E2F4 binds to the negative-acting site. Binding of an activator E2F is dependent on an adjacent CCAAT site that is bound by the NF-Y transcription factor and binding of a repressor E2F is dependent on an adjacent CHR element, suggesting a role for cooperative interactions in determining both activation and repression. Finally, the kinetics of B-Myb interaction with the G2-regulated promoters coincides with the activation of the genes, and RNAi-mediated reduction of B-Myb inhibits expression of cyclin B1 and cdc2. The ability of B-Myb to interact with the cdc2 promoter is dependent on an intact E2F binding site. These results thus point to a role for E2Fs, together with B-Myb, which is an E2F-regulated gene expressed at G1/S, in linking the regulation of genes at G1/S and G2/M (Zhu, 2004).
Using genome-wide analysis of transcription factor occupancy, this study investigated the mechanisms underlying three mammalian growth arrest pathways that require the pRB tumor suppressor family. It was found that p130 and E2F4 cooperatively repress a common set of genes under each growth arrest condition and showed that growth arrest is achieved through repression of a core set of genes involved not only in cell cycle control but also mitochondrial biogenesis and metabolism. Motif-finding algorithms predicted the existence of nuclear respiratory factor-1 (NRF1) binding sites in E2F target promoters, and genome-wide factor binding analysis confirmed these predictions. NRF1 (Drosophila homolog: Erect wing), a factor known to regulate expression of genes involved in mitochondrial function, is a coregulator of a large number of E2F target genes. These studies provide insights into E2F regulatory circuitry, suggest how factor occupancy can predict the expression signature of a given target gene, and reveal pathways deregulated in human tumors (Cam, 2003).
To understand cell cycle control mechanisms in early development and how they change during differentiation, embryonic stem cells were used to model embryonic events. The results demonstrate that as pluripotent cells differentiate, the length of G(1) phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb-E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the down-regulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F-pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F-pRb pathway (White, 2005).
In keratinocytes, the cyclin/CDK inhibitor p21WAF1/Cip1 is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function has been associated with down-regulation of Wnt signaling. This study shows that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independent of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control (Devgan, 2005).
Thus in keratinocytes, p21 functions as a transcriptional regulator that associates physically to the promoter of the Wnt4 gene. While increased p21 expression suppresses both Wnt3 and Wnt4 expression and endogenous p21 is required for the effective down-modulation of both genes by Notch1, in the skin of p21-/- mice and in primary p21-/- keratinocytes under basal conditions, only Wnt4 is up-regulated. This is likely a reflection of the fact that, biochemically, association of the endogenous as well as overexpressed p21 protein to the Wnt4 promoter is readily observed, while association to the Wnt3 promoter, if it occurs, is much weaker and harder to demonstrate. By ChIP assays, it was found that E2F-1 binds the same region of the Wnt4 promoter as p21, and that the two proteins can be recovered in association at this promoter. These findings are consistent with an elegant model, whereby E2F-1-p21 association provides a bridging mechanism for bringing p21 to target promoters. Importantly, however, in the cells used, p21 binding is specific for the Wnt4 promoter and does not occur at the promoter of another 'classical' E2F-1 target gene such as PCNA, the expression of which is unaffected by increased p21 expression. Concomitantly, p21 binding at the Wnt4 promoter is linked to curtailed recruitment of c-Myc and p300. By exogenous expression and promoter activity studies, p21 was previously reported to associate with the c-Myc protein suppressing its activity. The data are consistent with such a mechanism taking place at the Wnt4 promoter. Even in this case, however, there is an important element of selectivity, in that expression of other classical c-Myc target genes (such as that for ornithine decarboxylase), remains unaffected by p21 expression in keratinocytes (our unpublished observations). Thus, the findings are overall consistent with the emerging crucial role of chromatin configuration and promoter context in control of gene expression, with a physical and functional interplay between p21 and the specific transcription regulatory apparatus of individual genes such as that for Wnt4 (Devgan, 2005).
In summary, the common biological function of Notch1 and p21 as negative regulators of keratinocyte self renewal and tumorigenesis can be explained, in part, by one being a mediator of the other in transcriptional suppression of Wnt family members with consequent down-modulation of ß-catenin signaling. More specifically, p21 is directly involved in transcription regulation of the Wnt4 target gene, the control of which at the integrated chromatin level, remains an exciting topic for future studies (Devgan, 2005).
The E2F transcription factor plays a major role in cell cycle regulation, differentiation and apoptosis, but it is not clear how it is regulated by
non-mitogenic signaling cascades. Two kinases involved in signal transduction have opposite effects on E2F function: the
stress-induced kinase JNK1 inhibits E2F1 activity whereas the related p38 kinase reverses Rb-mediated repression of E2F1. JNK1
phosphorylates E2F1 in vitro, and co-transfection of JNK1 reduces the DNA binding activity of E2F1; treatment of cells with TNFalpha has
a similar effect. Fas stimulation of Jurkat cells is known to induce p38 kinase and a pronounced increase in Rb phosphorylation is found
within 30 min of Fas stimulation. Phosphorylation of Rb correlates with a dissociation of E2F and increased transcriptional activity. The
inactivation of Rb by Fas is blocked by SB203580, a p38-specific inhibitor, as well as a dominant-negative p38 construct; cyclin-dependent
kinase (cdk) inhibitors as well as dominant-negative cdks have no effect. These results suggest that Fas-mediated inactivation of Rb is
mediated via the p38 kinase, independent of cdks. The Rb/E2F-mediated cell cycle regulatory pathway appears to be a normal target for
non-mitogenic signaling cascades and could be involved in mediating the cellular effects of such signals (Wang, 1999).
In T lymphocytes, the hematopoietic cytokine interleukin-2 (IL-2) uses phosphatidylinositol 3-kinase (PI 3-kinase)-induced signaling
pathways to regulate E2F transcriptional activity, a critical cell cycle checkpoint. PI 3-kinase also regulates the activity of p70(s6k: see Drosophila RPS6-p70-protein kinase), the
40S ribosomal protein S6 kinase, a response that is abrogated by the macrolide rapamycin. This immunosuppressive drug is known to
prevent T-cell proliferation, but the precise point at which rapamycin regulates T-cell cycle progression has yet to be elucidated.
Moreover, the effects of rapamycin on IL-2 and PI 3-kinase activation of E2Fs have not been characterized; neither has the role of p70(s6k) in such activation. The present results show that IL-2- and PI 3-kinase-induced pathways for the regulation of E2F transcriptional activity
include both rapamycin-resistant and rapamycin-sensitive components. Expression of a rapamycin-resistant mutant of p70(s6k) in T
cells can restore rapamycin-suppressed E2F responses. Thus, the rapamycin-controlled processes involved in E2F regulation appear
to be mediated by p70(s6k). However, the rapamycin-resistant p70(s6k) can not rescue rapamycin inhibition of T-cell cycle entry,
consistent with the involvement of additional, rapamycin-sensitive pathways in the control of T-cell cycle progression. The present
results thus show that p70(s6k) is able to regulate E2F transcriptional activity and provide direct evidence for the first time for a link
between IL-2 receptors, PI 3-kinase, and p70(s6k) that regulate a crucial G1 checkpoint in T lymphocytes (Brennan, 1999).
The E2F-1 transcription factor is regulated during cell cycle progression and induced by cellular stress, such as DNA damage. Checkpoint kinase 2 (Chk2: see Drosophila loki) regulates E2F-1 activity in response to the DNA-damaging agent etoposide. A Chk2 consensus phosphorylation site in E2F-1 is phosphorylated in response to DNA damage, resulting in protein stabilization, increased half-life, transcriptional activation and localization of phosphorylated E2F-1 to discrete nuclear structures. Expression of a dominant-negative Chk2 mutant blocks induction of E2F-1 and prevents E2F-1-dependent apoptosis. Moreover, E2F-1 is resistant to induction by etoposide in tumor cells expressing mutant chk2. Therefore, Chk2 phosphorylates and activates E2F-1 in response to DNA damage, resulting in apoptosis. These results suggest a role for E2F-1 in checkpoint control and provide a plausible explanation for the tumor suppressor activity of E2F-1 (Stevens, 2003).
The humpty dumpty (humdy) mouse mutant exhibits failure to close the neural tube and optic fissure, causing exencephaly and retinal coloboma, common birth defects. The humdy mutation disrupts Phactr4, an uncharacterized protein phosphatase 1 (PP1) and actin regulator family member, and the missense mutation specifically disrupts binding to PP1. Phactr4 is initially expressed in the ventral cranial neural tube, a region of regulated proliferation, and after neural closure throughout the dorsoventral axis. humdy embryos display elevated proliferation and abnormally phosphorylated, inactive PP1, resulting in Rb hyperphosphorylation, derepression of E2F targets, and abnormal cell-cycle progression. Exencephaly, coloboma, and abnormal proliferation in humdy embryos are rescued by loss of E2f1, demonstrating the cell cycle is the key target controlled by Phactr4. Thus, Phactr4 is critical for the spatially and temporally regulated transition in proliferation through differential regulation of PP1 and the cell cycle during neurulation and eye development (Kim, 2007).
Despite biochemical and genetic data suggesting that E2F and pRB (pocket protein) families regulate transcription via
chromatin-modifying factors, the precise mechanisms underlying gene regulation by these protein families have not yet been defined in a
physiological setting. This study investigates promoter occupancy in wild-type and pocket protein-deficient primary cells. Corepressor complexes consisting of histone deacetylase (HDAC1) and mSin3B are specifically recruited to endogenous
E2F-regulated promoters in quiescent cells. These complexes dissociated from promoters once cells reached late G1, coincident with
gene activation. Interestingly, recruitment of HDAC1 complexes to promoters depends absolutely on p107 and p130, and requires an intact E2F-binding site. In
contrast, mSin3B recruitment to certain promoters does not require p107 or p130, suggesting that recruitment of this corepressor can occur via E2F-dependent and
-independent mechanisms. Remarkably, loss of pRB has no effect on HDAC1 or mSin3B recruitment. p107/p130 deficiency triggers a dramatic loss of E2F4
nuclear localization as well as transcriptional derepression, which is suggested by nucleosome mapping studies to be the result of localized hyperacetylation of
nucleosomes proximal to E2F-binding sites. Taken together, these findings show that p130 escorts E2F4 into the nucleus and, together with corepressor complexes
that contain mSin3B and/or HDAC1, directly represses transcription from target genes as cells withdraw from the cell cycle (Rayman, 2002).
E2F-mediated gene repression plays a key role in regulation of neuron survival
and death. However, the key molecules involved in such regulation and the
mechanisms by which they respond to apoptotic stimuli are largely unknown.
This study shows that p130 is
the predominant Rb family member associated with E2F in
neurons, that its major partner for repression of pro-apoptotic genes is E2F4,
and that the p130-E2F4 complex recruits the chromatin modifiers HDAC1 and
Suv39H1 to promote gene silencing and neuron survival. Apoptotic stimuli induce
neuron death by sequentially causing p130 hyperphosphorylation, dissociation of
p130-E2F4-Suv39H1-HDAC complexes, altered modification of H3 histone and gene
derepression. Experimental suppression of such events blocks neuron death while
interference with the synthesis of E2F4 or p130, or with the interaction of
E2F4-p130 with chromatin modifiers, induces neuron death. Thus, neuron survival
and death are dependent on the integrity of E2F4-p130-HDAC/Suv39H1 complexes (Liu, 2005).
Active repression of E2F-regulated genes by Rb family members is achieved by
recruitment of chromatin-modifying proteins to complexes with E2F.
Thus, a key finding here is that the mechanism by which
p130-E2F4 complexes promote neuron survival is via gene repression that requires
their interaction with the chromatin-modifying corepressors HDAC and Suv39H1.
Moreover, apoptotic stimuli induce death by causing loss of such
interactions. In support of this, p130 and p130-E2F4 fusion proteins mutated to
abolish interaction with HDAC and Suv39H1 promote neuron death, while
phosphorylation-resistant E2F4-p130 fusion proteins that do not lose association
with HDAC or Suv39H1 under apoptotic conditions are protective. Although these
studies identified HDAC1 as a partner for p130 in neuronal cells, preliminary
findings indicate that other HDAC family members may also be involved in
p130-mediated gene repression and neuron survival (Liu, 2005).
How might p130-tethered HDAC1 and Suv39H1 promote gene repression and neuron
survival? One target for these enzymes is the N-terminal tail of histone H3. In
the absence of corepressors, H3 is phosphorylated on Ser 10, and this
facilitates acetylation of Lys 14. These modifications promote transcription and
are essential for cell cycle progression in mitotically competent cells.
When tethered to chromatin by Rb-E2F complexes, Suv39H1
methylates H3 residue Lys 9. This, in
turn, inhibits phosphorylation of Ser 10 and, in concert with HDACs, favors
deacetylation of Lys 14. Such changes lead to condensation of local chromatin
and gene silencing. Consistent with this mechanism, it was
observed that levels of Ac-p-H3 associated with the endogenous B-myb promoter
are low in viable neuronal cells and greatly increase in response to an
apoptotic stimulus. In support of the involvement of HDAC and Suv39H1, it was found
that a death stimulus abolishes the association between p130 and HDAC and
substantially diminishes levels of p130-associated HMT activity (Liu, 2005).
An issue raised by these studies is the target of E2F-mediated gene repression
that regulates neuron survival and death. A variety of observations support the
closely related B-myb and C-myb genes in such a role. The promoters for these
genes contain E2F-binding sites, and their expression is subject to
E2F-dependent repression. Apoptotic stimuli, including p130
down-regulation, induce Myb reporter activity in neuronal cells, and the
findings in this study show that apoptotic stimuli lead to loss of repressive complexes
containing E2F4-p130-HDAC-Suv39H1 from the endogenous B-myb promoter as well as
changes in associated chromatin consistent with gene derepression. Moreover,
B-myb and C-myb transcripts and proteins are significantly induced by apoptotic
stimuli, and overexpression of B-myb and C-myb promotes neuron death.
Finally, down-regulation of B-myb and C-myb with anti-sense and siRNA
constructs protects neurons from apoptotic stimuli (Liu, 2005).
These findings strongly support a repression/derepression model for regulation of
neuron survival/death by E2F4-p130 and associated chromatin modifiers. They
identify both the molecules and mechanisms by which p130 promotes silencing of
E2F-responsive genes in viable neurons and by which apoptotic stimuli lead to
derepression of E2F-responsive genes and death (Liu, 2005).
p130 has been implicated as a promoter of quiescence in nonneuronal cells and
may well contribute to the post-mitotic state of neurons. Thus, stimuli that
lead to dissolution of p130 complexes and that thereby trigger the derepression
of pro-apoptotic genes such as Mybs, might concomitantly stimulate neurons to
attempt cell cycle re-entry. Such a situation may explain not only why a variety
of cell cycle markers are observed in neurons affected by injury and
neurodegenerative disorders, but also why treatments targeted at suppressing
derepression of E2F-regulated genes in neurons may have a therapeutic benefit in
preventing neuron degeneration. In this regard, the present findings provide several additional
molecular targets for such an approach (Liu, 2005).
The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPalpha (see Drosophila Slbo), normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). Evidence that aging switches the liver-specific pathway of C/EBPalpha growth arrest to repression of E2F transcription. An age-specific C/EBPalpha-Rb-E2F4 complex has been identified that binds to E2F-dependent promoters and represses these genes. The C/EBPalpha-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. These results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy, providing a possible mechanism for the age-dependent loss of liver regenerative capacity (Iakova, 2003).
Target genes of E2F
Continued: see E2F: Evolutionary homologs part 3/3 | Return: E2F: Evolutionary homologs part 1/3
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