Regulation of E2F and Rb Activity

Rb/E2F complexes have been shown to be central targets of factors that influence the cell cycle. Rb contains numerous phosphorylation sites which can by phosphorylated by the G1 phase cyclinD/cdk4 complexes and by the Gl/S phase cyclinE/cdk2 and cyclinA/cdk2 complexes. Hyper-phosphorylation of Rb by cyclin/cdk complexes results in a decreased ability to interact both with its target E2F and with co-repressors such as histone deacetylase enzymes. Growth stimulating factors and growth inhibitory factors generally affect the transcription, translation and stabilities of D and E type of cyclins as well as cdk inhibitors. The net effect of these growth signaling pathways controls the E2F/Rb/

corepressor complex formation through regulating the phosphorylation of the Rb family of proteins, with the hypo-phosphorylated Rb being active and the hyper-phosphorylated forms of Rb being inactive.

In addition to the phosphorylation status of Rb, E2F activity can also be regulated by a large number of other factors directly. Regulation of E2F activity has been primarily studied for E2F 1. The histone acetylase enzymes P300/CBP and P/CAF can directly bind to E2F 1 and can acetylate E2F 1 in vitro (Martinez-Balbas et al., 2000). In vivo, the N-terminal lysines of E2F 1 are found to be acetylated, and this acetylation increases the DNA binding activity of E2F as well as its half life. The increased stability and DNA binding contribute to increased transcriptional activity of acetylated E2F 1. This acetylation can be reversed by histone deacetylase enzymes recruited by Rb. In addition, E2F 1 stability can also be increased specifically following activation of the DNA damage signaling response. Treatment of cells with DNA damaging agents leads to activation of the ATM kinase, which can specifically phosphorylate E2F 1, resulting in increased protein accumulation (Lin et al., 2001). In contrast, E2F activity was found to be negatively regulated by the kinase activity of cyclinA/cdk2 (Krek et al., 1994). Cyclin A binds to the N terminus of E2F 1 and can phosphorylate both E2F 1 and DP in vitro, resulting in decreased DNA binding activity, and in vivo phosphorylation of DP is dependent on cyclin A binding to E2F 1.

Mechanisms of Transcriptional Control

E2F and Rb control transcription through a variety of mechanisms. While E2F alone can activate transcription, binding by Rb not only blocks transcriptional activation but also leads to active repression. Inhibition of Rb function would be predicted to both remove active repression and to allow transactivation by E2F. The relative importance of de-repression versus activation may depend on the specific target. For genes at which active repression is important, mutation of the E2F binding site would be predicted to increase the basal level of transcription. Such target genes include B-myb and cyclin E, which show premature expression when the E2F binding site is mutated. In contrast, for genes at which activation is important, mutation of the E2F binding site would be expected to result in loss of appropriate expression. Examples of these genes include DHFR and thymidine kinase (reviewed in Mundle and Saberwal, 2003).

The effects of Rb and E2F on gene expression have been primarily evaluated using reporter assays of transiently transfected plasmids. Although a great deal has been learned from these experiments, they may not necessarily be an accurate reflection of events in a chromosomal setting. Additional experiments have used chromatin immunoprecipitation (ChIP) to examine the proteins localized at endogenous sites on chromatin or have examined patterns of endogenous gene expression to further understand the mechanisms by which E2F and Rb control transcription.

The ability of Rb to actively repress transcription was suggested by the observation that E2F binding sites conferred increased reporter activity in Rb negative cells but decreased reporter activity in Rb positive cells (Weintraub et al., 1992). Fusion of Rb to the Gal4 DNA binding domain demonstrated that it could actively repress transcription of a reporter gene independently of E2F. Moreover, this repression was inhibited by cyclin E expression, indicating that hyperphosphorylated Rb could not repress transcription (Weintraub et al., 1995).

Recruitment of co-repressors contributes to Rb-dependent repression. Rb was first shown to interact with histone deacetylase (HDAC) 1 through its pocket domain. The HDAC inhibitor trichostatin can abolish Rb-dependent repression from a promoter containing E2F binding sites, and over-expression of HDAC and Rb results in a cooperative decrease in expression of the E2F target cyclin E, providing genetic evidence for the role of histone deacetylation in Rb's repression of normal chromatin (Brehm et al., 1998). Although HDAC and E2F both bind to Rb's pocket domain, they can simultaneously interact with hypophosphorylated Rb, allowing HDAC to be recruited to E2F sites. Rb's pocket domain has been shown to interact with histone deacetylase (HDAC) enzymes 1-3 through their LXCXE sequences. Interestingly, Rb that has been phosphorylated by cyclinD/cdk4 cannot bind to HDACs, which allows the expression of targets such as cyclin E (Zhang et al, 2000).

This mode of repression by Rb is in direct contrast to activation by E2F, which can bind to the histone acetylases (HATs) p300/CBP and P/CAF, making the DNA more accessible to transcription factors. The coordination of RB/E2F binding, the acetylation status of E2F-dependent promoters and induction of transcription has been shown in a number of studies examining the proteins localized to the promoters of E2F-dependent genes at different stages of the cell cycle. As compared to quiescent cells, in late G1 there is a loss of the pocket proteins and repressive E2Fs (E2F 4/pl30), which are replaced by activating E2Fs. At the same time, there is a switch from HDACs to HATs. This is accompanied by hyperacetylation of histones H3 and

H4 and the initiation of E2F-dependent gene expression (reviewed in Frolov and Dyson, 2004).

A role for chromatin remodeling by Rb as a part of its repressive activity was first identified by the interaction of Rb with BRG1, a component of the human SWI/SNF complex. BRG1 interacts with the unphosphorylated pocket domain, and in BRG1 deficient cells its re-expression is capable of conferring a growth arrested, flat cell phenotype (Dunaief et al., 1994). However, it has since been shown that the Rb binding site of BRG1 is not conserved in the Drosophila BRG homolog, and mutation of human BRG1 so that it could no longer bind Rb did not affect its ability to arrest cells in Gl, suggesting that the genetic interaction between BRG1 and Rb does not reflect a direct biochemical interaction. Instead, it appears that BRG1 induces expression of a cyclinE/cdk2 inhibitor, resulting in decreased phosphorylation of Rb, thus allowing it to remain in an active state (Kang et al., 2004).

Rb may also repress transcription through its ability to recruit histone methylase activity. Endogenous Rb associates with SUV39H1, an enzyme which methylates lysine 9 on histone H3. In reporter assays SUV39H1 can repress transcription when Rb is targeted to the promoter, and it can cooperate with Rb to repress endogenous cyclin E expression. Furthermore, HP1, a protein which binds methylated lysine 9 and is associated with transcriptionally silent regions of chromatin, can bind Rb. The nucleosome at the cyclin E promoter is associated with methylated H3 and HPI, but only in Rb positive cells, suggesting that Rb is responsible for this chromatin modification. (Nielsen et al., 2001)

A further mechanism of Rb repression may be its ability to recruit the DNA methyltransferase enzyme DNMT1. Rb physically directly interacts with DNMT1, and DNMT1 shows cooperative effects on repression of reporter genes. However, DNA was not methylated at these sites, suggesting that the effect of DNMT1 may not be through its enzymatic activity but instead might help recruit other co-repressors.

Although a large number of general mechanisms of repression and activation have been studied, more recently effort has been directed towards elucidating the mechanism by which various E2F and Rb family members carry out their distinct functions. Since the crystallization of an E2F 4/DP DNA-bound heterodimer suggested that all the DNA-contacting residues are conserved among E2F family members (Zheng et al., 1999), it is unlikely that specificity is conferred by variations in the DNA sequence of the E2F binding sites. An alternative hypothesis is that specificity of E2F family members is determined by their interaction with other transcription factors. A number of papers have examined the role of the marked box domain of E2F family members. This domain, directly downstream of the DNA binding domain, was first found to be important in the interaction of E2F 1 with the adenoviral protein E4, and it allowed for a synergistic effect of these two proteins on the activation of the E2 promoter. It was hypothesized that this region might also bind to other cellular transcription factors (Jost et al., 1996). The importance of this domain for specificity was initially suggested in studies examining why E2F 1, but not the other activating E2Fs, was capable of inducing apoptosis. Construction of chimaeric proteins containing E2F 1 and E2F 3 sequences indicated that the marked box domain and adjacent regions of E2F 1 were critically involved in the ability of activating E2Fs to induce apoptosis (Hallstrom and Nevins, 2003).

To further examine this region, the marked box domain of E2F 3 was used as bait in a yeast 2-hybrid screen to identifying interacting proteins (Giangrande et al., 2003; Giangrande et al., 2004). Although this domain shows 55% homology between E2F 1 and E2F 3, one of the proteins from the screen, the ubiquitous, E box binding transcription factor TFE-3, specifically bound only to E2F 3. The promoter of p68, a polymerase a subunit with both an E-box and E2F sites proximal to the promoter, was used to examine the interaction between these proteins. E2F 3 and TFE-3 were shown to synergistically activate p68 and to directly bind to one another through the E2F 3 marked box domain. In fact, the synergistic activation required this interaction, since an E2F 3 chimera with the E2F 1 marked box domain could not activate transcription. In the absence of E2F 3, TFE-3 was unable to bind to the p68 promoter. Conversely, in the absence of TFE-3, E2F 3 showed delayed binding, which was due to the presence of another weakly compensating E-box binding protein, USF1. USF1 was also capable of binding specifically to the E2F 3 marked box domain and could synergistically activate p68 expression. These findings could be extended to some, but not all, E2F 3 specific promoters that contained both E2F and E box sites proximal to the promoter, suggesting that additional factors also contribute to control of expression.

In a separate approach, another group found that the repressive E2Fs, E2F 4 and 5, could specifically interact with the SMAD transcription factors (Chen et al., 2002). In response to TGFp signaling, phosphorylation of SMAD 2 and 3 allows them to bind to SMAD4, and this complex can either activate or repress transcription of target genes. In studying TGFP-mediated repression, it was found that the TGFp response element of c-myc contained a consensus E2F site adjacent to the SMAD binding site. Mutation of either site led to decreased SMAD binding and loss of TGF(3 responsiveness. Chromatin immunoprecipitation experiments demonstrated that SMADs 2, 3, and 4, as well as E2Fs 4 and 5 and pi07 were found at the TGFp response element, but that the other RB family members and activating E2Fs were absent. In fact, SMAD3 could directly bind to both E2F 4 and 5 as well as pi07. This interaction was required for repression of c-myc in response to TGF(3 since c-myc expression did not decrease in E2F 4-/-; E2F 5-/- or pl07-/- cells. This repressive ability of E2Fs 4 and 5 may therefore in some cases be due to their ability to be recruited to the promoter by their specific interaction with other transcription factors.

Finally, recent work in Drosophila has also contributed to further understanding of the differences between activating and repressive E2Fs. In an attempt to understand the specific function of dE2F2, the repressive E2F, native complexes were purified from embryo extracts (Korenjak et al., 2004). In addition to either RBF or RBF2, these complexes contained components of the dMyb complex, a known transcriptional regulator. The dMyb complex showed no co-staining with actively transcribed regions of chromatin, consistent with its association with repressive E2Fs. Depletion of these dMyb subunits by RNAi resulted in up-regulation of genes normally controlled exclusively by dE2F2 repression, demonstrating that dMyb is required for dE2F2 repression. Rather than having a role in the cell cycle, these E2F target genes are involved in differentiation, often in expression of sex-specific genes. The Myb/Rb/E2F interaction also appears to be evolutionarily conserved: Rb and components of the Myb complex form part of the synMuv class of genes in C. elegans that control vulval development, and, although less studied, the human Myb homologs also appear to bind to Rb directly.

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