Retinoblastoma Family Genes

W Du and J Pogoriler

Ben May Institute for Cancer Research and Center for Molecular Oncology, University of Chicago, Chicago, IL, USA

The retinoblastoma gene Rb was the first tumor a decreased ability to interact with their target proteins suppressor gene cloned, and it is well known as a negative and to exert their biological functions. As growth- regulator of the cell cycle through its ability to bind the stimulating and growth-inhibitory factors generally transcription factor E2Fand repress transcription of affect the transcription, translation and stabilities of the genes required for S phase. Although over 100 other D and E type cyclins as well as their inhibitors, these proteins have been reported to interact with Rb, in most growth-signaling pathways regulate cell proliferation at cases these interactions are much less well characterized. least in part through regulating the phosphorylation of Therefore, this review will primarily focus on Rb and E2F the Rb family of proteins, with the hypo-phosphorylated interactions. In addition to cell cycle regulation, studies of Rb being active and preventing transition into S phase Rb and E2Fproteins in animal models have revealed (Chellappan et al., 1991). important roles for these proteins in apoptosis and The biological functions of Rb include tumor differentiation. Recent screens of Rb/E2Ftarget genes suppression, regulation of the cell cycle, differentiation have identified new targets in all these areas. In addition, and apoptosis. These functions of Rb are mediated by the mechanisms determining how different subsets of its interaction with a large number of cellular proteins. target genes are regulated under different conditions have Currently, over 100 proteins have been reported to only begun to be addressed and offer exciting possibilities interact with the Rb protein (Morris and Dyson, 2001), for future research. and most, if not all, of these interactions also involve the Oncogene (2006) 25, 5190–5200. doi:10.1038/sj.onc.1209651 pocket domain. The best-studied binding partners of Rb are the E2F transcription factors. Keywords: Rb; E2F; development; tumorigenesis; cell proliferation and differentiation E2F transcription factors in mammals The E2F transcription factors function as heterodimers that are composed of a subunit of the E2F gene family and a subunit of the DP gene family. In mammalian The Rb and E2Ffamilies of protein systems, there are eight E2F family members and two DP family members (for reviews, see Dyson, 1998; Mammalian Rb family of proteins Attwooll et al., 2004a). All the E2F and DP proteins, Rb, p107 (RBL1) and p130 (RB2) are members of a with the exception of the newly identified E2Fs 7 and 8, family of closely related proteins (Figure 1). Together, have both a conserved DNA-binding domain (DBD) these proteins are often referred to as the ‘pocket and a dimerization domain (Dim, see Figure 2). The proteins’ because their main sequence similarity resides E2F family members can be divided into four sub- in a domain, the pocket domain, which mediates groups. The first subgroup includes E2Fs 1–3 and is interactions with viral oncoproteins as well as cellular often referred to as the ‘activating E2Fs.’ In addition to proteins to exert the biological functions of this family. A the DNA-binding and dimerization sequences, these spacer region that is not conserved separates the pocket E2F proteins also contain a cdk-binding domain and a domain into the A and B pockets. The spacers of p107 nuclear localization sequence near the N-terminus, and and p130 but not Rb contain binding sites for cyclin/cdk a transcriptional activation domain and an Rb-binding complexes (Zhu et al., 1995). The Rb family of proteins sequence near the C-terminus (Figure 2). This subgroup also contains numerous phosphorylation sites which can of E2F proteins generally has a role in the activation of be phosphorylated by the G1 phase cyclinD/cdk4 com- E2F target genes at the G1/S transition and is important plexes and by the G1/S phase cyclinE/cdk2 and cyclinA/ for proper cell cycle progression. The second E2F cdk2 complexes (Hinds et al., 1992; Ewen et al., 1993; subgroup includes E2Fs 4 and 5 and is often referred Kato et al., 1993; Resnitzky et al., 1995; Du et al., 1996a). to as the ‘repressive E2Fs.’ This subgroup of E2F In general, the hyper-phosphorylated forms of Rb exhibit proteins still contains the Rb-binding sequence at the C-terminus but does not have the cdk binding or the nuclear localization sequences in the N-terminus Correspondence: Dr W Du, Ben May Institute for Cancer Research and Center for Molecular Oncology, University of Chicago, 924 E. and has nuclear export sequences instead (Dynlacht 57th Street, Chicago, IL 60637, USA. et al., 1994b; Muller et al., 1997; Gaubatz et al., E-mail: [email protected] 2001; Apostolova et al., 2002). Consistent with these Retinoblastoma family genes W Du and J Pogoriler 5191

Figure 1 The mammalian and Drosophila pocket protein families. The pocket protein family in mammals consists of Rb, p107 and p130, and in Drosophila contains RBF and RBF2. The pocket domain, responsible for most protein–protein interactions, consists of two conserved sequences, A and B. The spacer region between A and B is conserved between p107 and p130 (yellow box), and can bind to cyclin/cdk complexes. The activity of Rb proteins is controlled by phosphorylation at numerous phosphorylation sites (indicated for Rb and RBF by *). Figure 3 Interactions between the Rb and E2F proteins in mammals and Drosophila. The mammalian activating E2Fs, E2Fs 1–3, interact primarily with Rb. Similarly, the Drosophila- activating E2F, dE2F1, interacts only with RBF. The mammalian- repressive E2Fs, E2Fs 4 and 5, interact with p107 and p130. In addition, E2F 4 can also interact with Rb. Similarly, the Drosophila-repressive E2F, dE2F2, interacts with both RBF and RBF2.

pendent of the Rb family of proteins. These two subgroups of E2F will not be further addressed in this review. Interestingly, despite the sequence similarities among the Rb family members and among the conserved C-terminal Rb-binding domains of E2F, specific members of the Rb family preferentially interact with specific members of the E2F family. As shown in Figure 3, while Rb preferentially binds to E2Fs 1–4, p107 and p130 predominantly bind to E2Fs 4 and 5 Figure 2 The mammalian and Drosophila E2F protein families. (Classon and Harlow, 2002). The preferential binding of In mammals the E2F family is composed of E2Fs 1–8, DP1 and activating E2Fs by Rb but not by p107 or p130 DP2, while in Drosophila it consists of dE2F1, dE2F2 and dDP. All E2Fs have a conserved DNA-binding domain (DBD) and (except potentially underlies the observation that only Rb for E2Fs 7 and 8) a dimerization domain for binding to DP (Dim). mutations are frequently detected in cancers. The mammalian-activating E2Fs 1–3 contain a cyclin/cdk-binding domain (cdk) at the N-terminus and a strong activation domain at the C-terminus. This activation domain overlaps with the Rb- RB and E2F proteins in Drosophila binding domain (Rb) so that binding by Rb masks the activation There are two E2F (dE2F1 and dE2F2), one DP (dDP), domain. The repressive E2Fs 4 and 5 also bind the pocket proteins but lack the cyclin/cdk binding domain. E2F 6 shares only the and two Rb family (RBF and RBF2) genes in the DBD and dimerization domain with the rest of the family, while Drosophila genome (Dynlacht et al., 1994a; Ohtani and E2Fs 7 and 8 have two conserved DBDs, allowing them to function Nevins, 1994; Du et al., 1996a; Sawado et al., 1998; without DP to form a heterodimer. The DP proteins are distantly Stevaux et al., 2002). Interestingly, the two Drosophila related members of the family that share the DBD and dimerization domain, allowing them to form heterodimers with E2F E2F proteins behave like the first two subgroups of proteins. the mammalian E2F proteins: dE2F1 mainly functions as a transcription activator (Du, 2000; Frolov et al., 2001), comparable to the mammalian-activating structural features, this subgroup of E2Fs functions E2Fs 1–3, while dE2F2 primarily mediates active mainly as repressors of E2F target gene expression, and repression, similar to the mammalian-repressive E2Fs they translocate into the nucleus when in complex with 4 and 5 (Frolov et al., 2001). Furthermore, similar to the Rb family of proteins during the G0/G1 phases of the mammalian Rb protein that can bind to both the the cell cycle (Muller et al., 1997; Verona et al., 1997). activating and the repressive E2F proteins, RBF can E2Fs 6–8, representing the remaining two subgroups, bind to both dE2F1 and dE2F2 proteins in Drosophila also function as transcriptional repressors (Trimarchi (Frolov et al., 2001). In contrast, RBF2 can only bind et al., 1998, 2001; de Bruin et al., 2003a; Di Stefano dE2F2 (Stevaux et al., 2002) analogous to the mamma- et al., 2003; Attwooll et al., 2004b; Maiti et al., 2005). lian p107/p130 proteins that bind preferentially to However, in contrast to E2Fs 1–5, these E2F proteins do the repressive E2F proteins (see Figure 3). Thus, the not have the Rb-binding sequence at the C-terminus, Rb-E2F pathway is well conserved and is much simpler and therefore their function and regulation are inde- in Drosophila than in the mammalian systems.

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5192 The advantages of this simplified model system have (Royzman et al., 1999). As this allele of dE2F1 cannot been exploited in examining the in vivo functions of the activate transcription, it was proposed that E2F and Rb different classes of E2F and Rb family members as seen proteins may regulate DNA replication directly at the in many of the experiments described below. DNA replication origins. Indeed, RBF, dE2F1 and dDP were found to be in a complex with ORC proteins and were bound to the chorion replication origin in vivo (Bosco et al., 2001). Therefore, the E2F/RB complex Biological functions of the Rb and E2Ffamily of proteins may have a role in regulating DNA replication at the replication origin in addition to a role in regulating Studies of the Rb and E2F proteins using the Drosophila transcription. model system In contrast to de2f1, de2f2 mutant flies are viable As a result of the simplicity of the Drosophila Rb/E2F (Cayirlioglu et al., 2001). Unexpectedly, reducing the protein families, significant insights into the biological gene dosage of rbf,de2f2 ,ordDP all substantially functions of the E2F/Rb proteins have been derived suppress the phenotype of de2f1 null mutants (Du, 2000; from studies of this model system. Although there are Frolov et al., 2001), suggesting that the phenotype of two RBF genes in Drosophila, RBF2 is not an essential de2f1 mutants is at least in part due to the presence of gene, and rbf2 mutant flies show no obvious phenotypes an RBF/dE2F2 repressor complex. Characterization (Stevaux et al., 2005). In contrast, RBF appears to fulfill of the de2f1 and de2f2 double mutant or dDP mutant all of the cell cycle-related function of the Rb family flies revealed slower progression through S phase and of proteins in Drosophila. Characterization of the a defect in the G2/M transition (Frolov et al., 2001). phenotypes of embryos devoid of RBF revealed that Combining the de2f2 mutant with a novel allele of RBF is required for the repression of E2F target gene dE2F1 (dE2F1su89), which retains the transcription expression and for maintaining the first G1 cell cycle activation function but disrupts interaction with RBF, arrest during embryonic development (Du and Dyson, allowed the characterization of the active repression 1999). This establishment of the first G1 cell cycle arrest function of Rb/E2F complexes. Interestingly, the tissues in the developing embryos requires Dacapo, the only that are most affected by the complete removal of member of the p21/p27 family of cdk inhibitors in Rb/E2F complexes are tissues with extensive endo- Drosophila (de Nooij et al., 1996; Lane et al., 1996). replication (Weng et al., 2003). This is apparently due to Therefore, Rb and the p21/p27 family of cdk inhibitors a requirement for Rb/E2F complexes to downregulate function cooperatively to achieve a stable G1 cell cycle cyclin E levels after each round of S phase for the arrest during fly embryonic development. However, the initiation of additional rounds of endoreplication. exact functional relationship between the two families of In summary, it appears that the Rb proteins normally cell cycle regulators varies in different cell types. For function to maintain G1 arrest in conjunction with the example, in the developing photoreceptor neurons RBF p21/p27 family of cdk inhibitors and that active and Dacapo have redundant roles in maintaining repression by the Rb/E2F complex is required in G1 cell cycle arrest. Interestingly, in the absence of continuously endoreplicating tissues. Although E2F both RBF and Dacapo, the developing photoreceptor does not seem to be absolutely required for S phase neurons fail to exit the cell cycle and continue to entry, it is required for the normal progression through incorporate 5-bromodeoxyuridine (BrdU), in spite of S phase and for the G2/M transition. Furthermore, the fact that these cells begin to differentiate and to inappropriate activation of E2F, either by ectopic express post-mitotic markers (Firth and Baker, 2005). expression of dE2F1 or by mutation of rbf, does lead Analysis of the phenotype of flies with mutations of to ectopic S phase entry and increased apoptosis. These de2f1, the only activating class of E2F in Drosophila, effects of E2F on the cell cycle and apoptosis are likely showed that dE2F1 plays a critical role in the expression due to the fact that Rb/E2F regulates expression of of the classical E2F target genes, such as RNR2 and multiple cell cycle and apoptotic targets as described in proliferative cell nuclear antigen (PCNA), that are more detail below. important for DNA replication (Duronio et al., 1995). However, in de2f1 mutants, although the rate of BrdU incorporation was significantly reduced, it was not Studies of Rb family proteins in mice completely blocked (Royzman et al., 1997). This effect The in vivo roles of Rb and E2F in mammalian systems is in contrast to that of cyclin E mutant, which showed have been addressed by knockout mice of individual an all or none effect on BrdU incorporation (Knoblich family member. Because of the extensive functional et al., 1994; Duronio et al., 1998; Follette et al., 1998). overlap between the family members, such studies Therefore, during normal development dE2F1 is limit- generally revealed functions for individual Rb or E2F ing for the expression of replication factors but is not members in regulating proliferation, apoptosis and limiting for the expression of cell cycle regulators such as differentiation in specific tissues. The best characterized cyclin E. Interestingly, flies with an allele of de2f1 of the pocket protein knockouts are Rb-deficient mice. (de2f1i2), which retains the DNA binding and dimeriza- Rb null mice die at embryonic day 13.5 and demonstrate tion domain but lacks the transcriptional activation and ectopic S phases and extensive neuronal apoptosis in the Rb-binding domain, are viable, but female are sterile central nervous system (CNS) and lens, as well as defects due to increased gene amplification in follicle cells in the differentiation of muscles and red blood cells

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5193 (Jacks et al., 1992). Some of these defects appear to be is possible that some of these effects could be accounted due to the unrestrained activity of activating E2Fs, since for by a rescue of the RbÀ/À placenta. Although the removal of any of E2F 1, 2 or 3 could decrease ectopic exact mechanism is not clear, this genetic observation is S phase in the CNS, lens and retina (Tsai et al., 1998; consistent with the notion that Rb normally can hold Ziebold et al., 2001; Saavedra et al., 2002). In contrast, Id2 activity in check. In support of this idea, unrest- only loss of E2F 1 could suppress apoptosis in all ricted Id2 appears to prevent terminal differentiation in these tissues, while E2F 3 loss could suppress apoptosis fetal liver macrophages from RbÀ/À embryos, at least only in the CNS. However, recent reports indicate in part by preventing association of the transcription that many of these RbÀ/À defects are not cell auto- factor PU.1 with its binding sites (Iavarone et al., 2004). nomous but instead are secondary to placental abnor- Although Rb has no effect on PU.1-binding alone, it malities. Development of many tissues in RbÀ/À is able to reverse Id2 repression. An additional genetic embryos was remarkably restored when provided with interaction has been shown in tumor suppression: wild-type placenta. In particular, although ectopic Rb þ /À;Id2À/À pituitary glands show decreased proli- proliferation continued in the CNS, apoptosis was feration, premature differentiation, and decreased pitui- suppressed, and erythrocyte developmental delays were tary tumor incidence relative to Rb þ /À glands, almost completely suppressed. In contrast, both ectopic presumably due to loss of Id2’s inhibitory effect proliferation and apoptosis in the lens were unaffected, on transcription factors involved in differentiation and the skeletal muscle defect was not restored (de Bruin (Lasorella et al., 2005). However, this observed effect et al., 2003b; Wu et al., 2003). In light of these findings, may not reflect a direct physical interaction between Rb the suppression effects of the RbÀ/À phenotype by loss and Id2 since these two proteins may control different of the activating E2Fs will need to be revisited: does events in pituitary development. the removal of E2F 1 or 3 reduce CNS apoptosis In contrast to RbÀ/À mice, p107 or p130 null through an indirect effect of restoring placental deve- animals appear to develop normally, although lopment? It should be pointed out that a recent study p107À/À;p130À/À animals are not viable, apparently does show a cell intrinsic role of Rb in erythropoiesis due to defects in chondrocytes leading to bone abnormal- under stress conditions (Spike et al., 2004). ities (Cobrinik et al., 1996), suggesting some essential, Consistent with its role as a tumor suppressor, tissue-specific roles for these proteins. In addition, Rb þ /À mice develop pituitary and thyroid tumors although these proteins are less frequently found to be (Jacks et al., 1992). In agreement with the idea that the mutated in tumors, they do play a role in tumor Rb and the p21/p27 family of cdk inhibitors cooperate suppression in the absence of Rb. For example, the in cell cycle regulation, removal of p21 or p27 in an additional loss of p107 in RbÀ/À chimaeric mice is Rb þ /À background increases tumor development sufficient for development of retinoblastoma (Robanus- (Brugarolas et al., 1998; Park et al., 1999). Conversely, Maandag et al., 1998). More recently Rb þ /À;p107À/À the incidence of pituitary tumors was decreased by chimaeras were shown to develop a broad spectrum of eliminating E2F 1 (Yamasaki et al., 1998) or E2F 3 tumors, most of which showed loss of the remaining Rb (Ziebold et al., 2003), although removal of E2F 3 allele, indicating that p107 is an important tumor actually increased the incidence of thyroid cancer in suppressor in the absence of Rb (Dannenberg et al., Rb þ /À mice. This suggests that in at least some tumor 2004). Similarly, Rb þ /À;p130À/À and RbÀ/À;p130À/À types the ability of Rb to suppress tumorigenesis is chimaeras were also tumor prone. In particular, at least in part due to its inhibition of the activating RbÀ/À;p130À/À mice showed consistent development E2Fs. Surprisingly, removing the repressive E2F 4 also of retinoblastomas, pheochromocytomas and neuro- suppressed tumorigenesis in Rb þ /À mice (Lee et al., endocrine hyperplasia in the lungs, suggesting that p130 2002). It was suggested that removal of E2F 4 in RbÀ/À is particularly important in restraining tumor develop- tumor cells allowed p107 and p130 to associate with ment in these tissues (Dannenberg et al., 2004). E2Fs 1 and 3, thereby replacing Rb’s ability to repress In summary, studies in the mouse models have shed transcription. Consistent with these results, loss of E2F light on the role of the Rb family of proteins in normal 4 could repress inappropriate proliferation and E2F development and tumorigenesis. These phenotypes are target gene expression in RbÀ/À cells. These results likely the consequences of the roles of the Rb family of suggest that although E2F and Rb family members are proteins in the control of cell cycle, apoptosis, and classified based on their biochemical activities as shown differentiation as discussed in detail below. in Figure 3, their biological function may vary under specific in vivo conditions. In addition to E2F, Rb has been shown to interact Cell cycle roles of the Rb family of proteins genetically with the inhibitor of differentiation protein The Rb family of proteins are best known for their Id2. The Id family of basic helix–loop–helix proteins ability to regulate the cell cycle, and most studies point lack DNA-binding domains and serve as dominant- to E2F as the key target that mediates the cell cycle role negative regulators of the bHLH transcription factors of Rb proteins. Compound knockout of E2Fs 1–3 leads involved in differentiation. With the exception of the to cell cycle arrest of mouse embryonic fibroblasts skeletal muscle defects, removal of Id2 corrected many (MEFs) with an accumulation of the cdk inhibitor p21 of the defects of RbÀ/À mice (Lasorella et al., 2000). (Wu et al., 2001). This is similar to RNAi knock-down However, given the placental effects recently reported, it of dE2F1 in Drosophila SL2 cells (Frolov et al., 2003),

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5194 supporting the notion of a conserved role for the in senescent cells, suggesting a particular role for Rb in activating E2F proteins. Although simplified models inducing senescence (Narita et al., 2003). Interestingly, suggest that the Rb/E2F complexes present during quie- these E2F targets could not be upregulated in senescent scence or G0 are replaced with free E2Fs at the G1/S cells even with overexpression of E2F 1, while removal transition, the actual composition of these complexes of Rb or p16 with siRNA decreased heterochromatin appears to vary with the cell cycle (Classon and Harlow, foci and allowed expression of E2F target genes. 2002; Bracken et al., 2004). In quiescent cells, p130 is the main pocket protein in complex with the inhibitory E2Fs, while in cycling cells it is replaced by p107/E2F 4 The role of Rb in apoptosis in G0/G1 phases and by Rb/E2F 1–3 complexes in As indicated by the apoptosis in various tissues in S phase (Shirodkar et al., 1992). Although MEFs RbÀ/À mice, the Rb family of proteins has been found deficient in any one of the inhibitory E2Fs or in both to suppress apoptosis. Some of the ability of Rb to limit p107 and p130 proliferate normally, they show defects in apoptosis may be due to its ability to control the cell response to growth inhibitory signals. Triple knockout cycle: in response to DNA-damaging agents quiescent MEFs deficient in all three pocket proteins show cells are more resistant to apoptosis than dividing cells, increased entry into S phase, loss of contact inhibition, and overexpression of Rb is similar in effect to other and resistance to senescence and DNA-damage-induced modifications that arrest the cell cycle (Chau and Wang, G1 arrest (Dannenberg et al., 2000; Sage et al., 2000). 2003; Masselli and Wang, 2006). Similarly, models have In addition to control of the G1/S transition, Rb has proposed that the induction of apoptosis in RbÀ/À cells also been implicated in regulating other phase of the cell is indirectly due to their continued proliferation as they cycle as well as cell cycle checkpoints. For example, initiate differentiation, and that this conflict signals for overexpression of dE2F1 in the Drosophila developing apoptosis. wing accelerates both G1/S and G2/M transitions, while However, other studies indicate that Rb has a direct overexpression of RBF slowed all phase of the cell cycle, role in suppressing apoptosis. For example, Rb can with the greatest effect on S-phase duration (Neufeld be cleaved by caspases, and mice engineered to express et al., 1998). These cell cycle effects are likely due to the a caspase-resistant form of Rb show defects in tumor ability of RBF and dE2F1 to control the expression of necrosis factor (TNF)-induced apoptosis in several targets such as cyclin E and string (Drosophila cdc25), as tissues (Chau et al., 2002). Unlike wild-type Rb, well as multiple DNA replication factors that regulate caspase-resistant Rb could inhibit TNF-induced apop- the progression of different cell cycle phases. Recent tosis in fibroblasts treated with cycloheximide, even screens of Rb/E2F target genes by microarray have also though both populations arrested in G1. These results identified cell cycle checkpoint genes (Polager et al., further suggest that Rb has antiapoptotic effects 2002; Ren et al., 2002). For example, Mad2, a spindle independent of its ability to regulate gene expression. checkpoint gene, was recently identified as an E2F As proteins reported to interact with Rb such as c-Jun target. Removal of Rb or overexpression of E2F 1 was N-terminal kinase and c-ABL (Morris and Dyson, 2001; sufficient to upregulate expression of Mad2 throughout Chau and Wang, 2003) can also induce apoptosis, it the cell cycle. Although increased expression of a spindle is possible that their interaction with Rb might also checkpoint gene might be expected to inhibit tumor- be able to inhibit their pro-apoptotic signaling. Inter- igenesis, removal of Rb or overexpression of Mad2 was estingly, the caspase-resistant form of Rb is unable sufficient for abnormal chromosome accumulation and to prevent DNA-damage-induced apoptosis, suggesting delayed exit from mitosis. Significantly, partial suppres- that in addition to cell type, the role of Rb in suppression of Mad2 levels was sufficient to reverse the sing apoptosis may depend on the apoptotic stimulus. chromosomal abnormalities associated with removal of Regulation of E2F transcription does appear to be Rb (Hernando et al., 2004). relevant to Rb’s antiapoptotic effect in some tissues. Rb is also involved in senescence, a permanent, non- Many genes involved in apoptosis are direct targets of reversible exit from the cell cycle. While pocket protein E2F, overexpression of E2F 1 has been shown to induce members may compensate for one another to some apoptosis, and E2F 1À/À thymocytes are defective in extent, Rb appears to be particularly important for undergoing apoptosis (Field et al., 1996). Whether this senescence, and MEFs that are acutely inactivated for ability to induce apoptosis is restricted to E2F 1 or is Rb are able to re-enter the cell cycle, even from a shared by other activating E2Fs is still unclear. E2F senescent state (Sage et al., 2003), in spite of intact p107 targets related to apoptosis include APAF-1, caspases 3 and p130. Senescent cells show nuclear reorganization and 7 and p73 (Chau and Wang, 2003). Although some with foci of DNA accumulation that correlate with apoptosis appears to be independent of p53, E2F can transcriptionally inactive genes (Narita et al., 2003). activate the p53 pathway through upregulation of Arf These regions exhibit characteristics of heterochroma- and Pin-1, both of which contribute to p53 stabilization. tin: histone H3 methylated on lysine 9 and HP1 protein The ability of E2F to affect apoptosis would be expected accumulation. These modifications are associated with to inhibit tumor formation by leading to apoptosis of the promoters of E2F target genes only during senes- cells with excessive E2F activity. Consistent with this, cence and not in merely quiescent cells. Interestingly, E2F 1À/À mice are surprisingly tumor prone (Yamasaki although p107 and p130 were found at the promoters of et al., 1996), although they show a different tumor E2F target genes in quiescent cells, Rb was only detected spectrum from that of Rb þ /À mice. Tumor incidence

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5195 of E2F 1À/À mice can be further increased by removing precursor cells that can give rise to tumors (Spike et al., E2F 2 (Zhu et al., 2001), but not E2F 3, suggesting that 2004). In other cases, the loss of Rb appears to disrupt tumor suppression is a characteristic only of E2Fs 1 and the coordination between cell cycle exit and differentia- 2 (Cloud et al., 2002). tion. In RbÀ/À skin for example, suprabasal keratino- The relative importance of loss of Rb-mediated cytes continued to divide even though they lost repression versus activation by E2Fs is still not clear expression of basal keratins and induced expression and may vary with the cell type or apoptotic stimulus. In of more differentiated keratin (Ruiz et al., 2004). In human fibroblasts, overexpression of an E2F DNA- sensory hair cells of the ear, Rb is normally upregulated binding domain construct can induce cell death regard- during terminal differentiation, but when Rb is removed less of the phase of the cell cycle, indicating that Rb’s there is increased proliferation not only of the proli- active repression of target genes is important to inhibit ferating progenitors but also of the normally post- apoptosis (Young and Longmore, 2004). In contrast to mitotic, differentiated cells (Sage et al., 2005). Although E2F-dependent cell cycle genes, Rb was found at the these mature cells showed abnormal orientation, they promoter of E2F apoptotic targets at all stages of the were functional even while expressing proliferation cell cycle in human fibroblasts. This was distinct from markers such as PCNA, and acute loss of Rb in these mouse cells, where Rb was never detected at E2F mature cells was sufficient for them to re-enter the cell apoptotic targets, although the repressive E2F 4 and cycle. These examples demonstrate that, at least in some other pocket proteins were present. These results may tissues, Rb plays a critical role in coordinating cell also reflect the fact that human fibroblasts showed cycle exit with differentiation and in maintaining the significantly higher levels of nuclear Rb than did mouse quiescent state of the differentiated cells, and loss of fibroblasts (Young and Longmore, 2004). In addition, Rb does not prevent differentiation even when cell cycle different pocket proteins appear to be important in exit is blocked. inhibiting apoptosis in different tissues. In post-mitotic Recent studies showed that Rb and E2F proteins may neurons, p130 is a key player in suppressing expression have a more direct role in differentiation. The impor- of E2F target genes by binding to E2F 4. In fact, tance of E2F and pocket protein activity has been shown overexpression of p130 is sufficient to protect neurons in confluent (non-dividing) MEFs that are induced to from apoptotic stimuli, and removal of p130 with differentiate into adipocytes. Under these conditions, siRNA is sufficient to induce apoptosis. This suppressive removal of E2F 4 results in spontaneous differentiation activity of p130 requires its interaction with corepressors even though the cell cycle is not affected, suggesting that SUV39H1 and histone deacetylases (HDACs) (Liu E2F 4 suppresses differentiation independently of proli- et al., 2005). feration (Landsberg et al., 2003). Recently, lung-specific A role for Rb and E2F in regulating apoptosis was deletion of Rb has been reported. Not unexpectedly, also observed in Drosophila, where overexpression of these mice showed both increased proliferation and dE2F1 or removal of RBF all led to increased apoptosis increased apoptosis in the lung. More interest- (Asano et al., 1996; Du et al., 1996b; Du and Dyson, ingly, these lungs demonstrated a specific effect on the 1999). dE2F1 was shown to be required for UV neuroendocrine cells, which are normally scattered at irradiation-induced expression of the pro-apoptotic gene airway branch points. Not only were these clusters hac-1 (the Drosophila homolog of APAF-1) (Zhou and comprised of more cells when Rb was ablated, but there Steller, 2003). Although this pro-apoptotic role of was also an increased number of individual aggregates, activating E2Fs has been firmly established in both suggesting that Rb normally prevents cells from adopt- Drosophila and mammalian systems, recent evidence ing the neuroendocrine fate (Wikenheiser-Brokamp, indicates that dE2F1 can also play a protective role in 2004). Although this ability to restrict differentiation inhibiting UV induced apoptosis. Specifically, develop- has not been genetically separated from the ability ing wing discs show UV-dependent apoptosis in the to restrict proliferation, it is potentially of particular intervein area but not at the dorsal/ventral boundary. interest in tumorigenesis since small cell lung cancer, Wing discs lacking dDP or deficient in dE2F1 show a which is derived from neuroendocrine cells, is specifi- reversal of this phenotype, with decreased apoptosis in cally associated with loss of Rb function. the intervein area and increased apoptosis at the D/V In addition to regulating E2F, Rb has been shown boundary. These apoptotic effects are apparently due to to interact with numerous other proteins. The best a repression of the apoptotic regulator hid by RBF and characterized of these are involved in differentiation and the activating E2F dE2F1 (Moon et al., 2005). include basic helix–loop–helix transcription factors such as Id and MyoD. The muscle defect of RbÀ/À embryos appears to be due to an inability of muscle cells to The role of Rb in differentiation complete differentiation (Rb family-mediated muscle Similar to indirect models of apoptosis, loss of Rb could differentiation is reviewed by De Falco and Simone in indirectly prevent differentiation if cells unable to exit this issue). Muscle cells lacking Rb can growth arrest, the cell cycle could not differentiate. In some hemato- but they show delayed upregulation of late differentia- poetic cell types, Rb null cells cannot reach the final tion markers and are capable of re-entering the cell cycle stage of differentiation, and in mice these cells may when stimulated with mitogens. Rb can cooperate with contribute to myeloproliferative disorders. It has been the transcription factor MyoD to induce transcription of speculated that this is due to the increase in the pool of muscle target genes, and MyoD was shown to bind to

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5196 Rb (Gu et al., 1993), although the physiological –cycle-dependent manner (Ren et al., 2002) or a library significance of these interactions is unclear. An alter- of CpG islands (Wells et al., 2003), as GC rich regions native model suggests that Rb may promote differentia- are often found at promoters and origins of replication. tion indirectly by sequestering inhibitors of MyoD The ChIP on CHIP screen with the cell–cycle-regulated (Nguyen and McCance, 2005). Recently, specific post- promoter array found that in quiescent cells, antibodies translational modifications have been demonstrated to against the repressive E2F 4 precipitated the promoters affect the ability of Rb to mediate differentiation. of genes involved not only in the cell cycle but also in Differentiated myotubes showed an increase in acety- DNA replication, DNA damage and repair, chromo- lated Rb, and an acetylation site mutant of Rb, although some condensation and separation, the G2/M check- able to acutely arrest the cell cycle, was not able to point, and mitosis. Many components of multimeric cooperate with MyoD to induce differentiation and complexes appeared in the screen, suggesting that they maintain permanent growth arrest (Nguyen et al., 2004). are transcriptionally co-regulated. The importance of One model for Rb activation of MyoD involves its pocket protein repression in controlling these genes was ability to sequester inhibitors such as HDACS or E1a- confirmed by the finding that in p107À/À;p130À/À like inhibitor of differentiation (EID-1) (MacLellan MEFs many of these genes were deregulated, as expec- et al., 2000; Miyake et al., 2000). Non-actetylated Rb ted if repression by E2F 4 complexes controlled their appears less able to bind MDM2 and therefore has a expression. In S-phase cells, the activating E2F 1 decreased ability to target EID-1 for degradation precipitated primarily genes involved in DNA replication (Nguyen et al., 2004). Verification of this role of Rb in and repair, suggesting that these genes require not only differentiation in vivo will require further use of mouse de-repression but also activation. In the ChIP on CHIP models. screen of CpG islands, targets other than cell cycle- regulated genes were found, including genes involved in embryogenesis, differentiation and development. As the Transcriptional targets of Rb and E2Fproteins binding of E2F or Rb to a given promoter does not necessary mean that E2F or Rb will be the rate-limiting Since Rb and E2F proteins are transcriptional regula- determinant of expression for that gene, combining the tors, identification of their targets is critical to our microarray gene expression data and the CHIP on CHIP understanding of their biological functions. Rb and E2F data should allow a better identification of Rb/E2F proteins were originally known for their ability to target genes. In addition, these newly identified genes can regulate the G1/S transition, so E2F target genes were be studied to determine whether they are important to initially identified by examining the promoters of genes the biological roles of Rb/E2F described above. known to be turned on at the G1/S transition for E2F- The relative simplicity of the Drosophila Rb and E2F binding sites. These early studies identified E2F target proteins has made it an ideal system for the genome- genes that are either cell cycle regulators (such as cyclins wide identification of Rb/E2F targets. Specifically, E, A and B) or DNA replication factors (such as PCNA, RNAi has been used to deplete the activating E2F dihydrofolate reductase (DHFR) and thymidine kinase). (dE2F1), the inhibitory E2F (dE2F2), dDP, RBF or More recently, microarray technology has allowed RBF2 from Drosophila cell lines (Dimova et al., 2003) identification of E2F target genes at the genome level. or specific tissues (Stevaux et al., 2005). Although it Several different approaches have been carried out. is possible that secondary changes in gene expression Initial microarray screens focused on the overexpression could have occurred, the screen did detect actual of E2F or Rb family members (Muller et al., 2001; changes in expression, suggesting that the E2F/Rb inter- Polager et al., 2002). While these studies identified large actions were rate limiting under these conditions. arrays of altered genes, they cannot distinguish genes A surprising result of this screen was that very few directly regulated by Rb or E2F from genes that genes showed both a decrease in expression in the changed expression due to secondary effects of Rb or absence of dE2F1 and an increase in expression in the E2F expression (such as alterations in the cell cycle). absence of dE2F2, suggesting that most genes are more Furthermore, overexpression of E2Fs or Rb might also influenced either by repression or by activation rather result in non-physiological binding and activation or than by both activities equally (Dimova et al., 2003). repression. Most of the genes found to be regulated largely by To identify potential direct targets of Rb and E2F dE2F1 were involved in processes similar to those found proteins, a second generation of screens used the so in the ChIP on CHIP screens, including the cell cycle, called chromosomal immunoprecipitation (ChIP) on DNA replication and repair, mitosis, chromosome CHIP approach to identify genes which have promoters segregation, and checkpoints. However, the genes bound to endogenous E2F/Rb (Ren et al., 2002). regulated primarily by dE2F2 were not involved in Specifically, small DNA fragments that are bound by DNA repair or S-phase processes. While the functions the endogenous Rb and E2F proteins are enriched by of many genes in this category are unknown, a surpri- Chromosomal IP and are then amplified and hybridized sing number appear to be unrelated to the cell cycle and to a promoter array. However, as the complete human instead are involved in development, including male and promoter microarray is not currently available, the female specific genes. The importance of dE2F2 repres- current ChIP on CHIP screens have used either a sion was confirmed by finding that expression of many promoter library of genes known to be regulated in a cell of these genes was deregulated in de2f2 null flies.

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5197 Further examination of repression of these differen- recruited to E2F sites to repress transcription. Rb that tiation genes has highlighted the complexity of Rb/E2F has been phosphorylated by cyclinD/cdk4 cannot bind control of transcription and the importance of interac- to HDACs, allowing the expression of targets such as tions with other factors. In comparing the requirement cyclin E (Zhang et al., 2000). This mode of repression by for dE2F2 and RBF2 to repress expression in Drosophila Rb is in direct contrast to activation by E2F, which can cell lines, ovaries and embryos, microarray data showed bind to the histone acetylases (HATs) p300/CBP and that different genes were deregulated in each tissue with P/CAF, making the DNA more accessible to transcrip- surprisingly little overlap, suggesting cell-type specific tion factors. RB/E2F-binding status, acetylation of requirements for expression (Stevaux et al., 2005). E2F-dependent promoters and induction of transcrip- Comparison of dE2F2 and RBF2 binding to the tion have been shown to be coordinated in a number of promoters of these genes showed that in some cases studies examining the proteins localized to the promo- the differences in expression correlated with dE2F2/ ters of E2F-dependent genes at different stages of the RBF2 occupancy of the promoter, suggesting that cell cell cycle (Takahashi et al., 2000; Ferreira et al., 2001; type-specific factors determine dE2F2/RBF2 distribu- Rayman et al., 2002; Caretti et al., 2003; Taubert et al., tion. In contrast, at other promoters dE2F2 and RBF2 2004). As compared to quiescent cells, in late G1 there is were always present, suggesting that the functional a loss of the pocket proteins and repressive E2Fs (E2F relevance of promoter occupancy also varies with cell 4/p130), which are replaced by activating E2Fs. At the type. These findings emphasize the need to further same time, there is a switch from HDACs to HATs. This understand the mechanisms determining Rb and E2F is accompanied by hyperacetylation of histones H3 and functions in specific contexts. H4 and the initiation of E2F-dependent gene expression (Frolov and Dyson, 2004). Rb may also repress transcription through its ability Mechanisms of transcriptional control to recruit histone methylase activity. Endogenous Rb associates with SUV39H1, an enzyme which methylates E2F and Rb control transcription through a variety of lysine 9 on histone H3. Additionally, HP1, a protein mechanisms. While E2F alone can activate transcrip- which binds methylated lysine 9 and is associated with tion, binding by Rb not only blocks transcriptional transcriptionally silent regions of chromatin, can bind activation but also leads to active repression. Inhibition Rb (Nielsen et al., 2001). Furthermore, Rb interacts with of Rb function would be predicted to both remove the DNA methyltransferase enzyme DNMT1, which active repression and to allow transactivation by E2F. cooperates to repress reporter genes (Robertson et al., The relative importance of de-repression versus activa- 2000); however, DNA methylation was not detected at tion may depend on the specific target. For example, the repressed promoters, suggesting that the effect of mutation of the E2F-binding site upstream of B-myb DNMT1 may not be through its enzymatic activity but and cyclin E results in premature expression, reflecting a instead might help recruit other co-repressors. requirement for active suppression. In contrast, similar While general mechanisms of repression and activa- mutations result in loss of appropriate upregulation of tion have been well studied, more recent efforts have DHFRand thymidine kinase, indicating a requirement been directed toward elucidating the mechanisms by for activation (Mundle and Saberwal, 2003). which various E2F and Rb family members carry out A role for chromatin remodeling by Rb as a part of its their distinct functions. Since the crystallization of repressive activity was first identified by the interaction an E2F 4/DP DNA-bound heterodimer suggested of Rb with BRG1, a component of the human SWI/ that all the DNA-contacting residues are conserved SNF complex. BRG1 interacts with the unphosphory- among E2F family members (Zheng et al., 1999), it is lated pocket domain, and in BRG1-deficient cells its re- unlikely that specificity is conferred by variations in the expression is capable of conferring a growth arrested, DNA sequence of the E2F-binding sites. An alternative flat cell phenotype (Dunaief et al., 1994). However, it hypothesis is that specificity of E2F family members is has since been shown that the Rb-binding site of BRG1 determined by their interaction with other transcription is not conserved in the Drosophila BRG homolog, and factors (Figure 4). The marked box domain of E2F mutation of human BRG1 so that it could no longer family members may play a role in this specificity. bind Rb did not affect its ability to arrest cells in G1, This domain, directly downstream of the DBD, was suggesting that the genetic interaction between BRG1 first found to be important in the interaction of E2F 1 and Rb does not reflect a direct biochemical interaction. with the adenoviral protein E4, and it allowed for a Instead, it appears that BRG1 induces expression of a synergistic effect of these two proteins on the activation cyclinE/cdk2 inhibitor, resulting in decreased phosphor- of the E2 promoter, leading to the hypothesis that ylation of Rb, thus allowing it to remain in an active this region might also bind to cellular transcription state (Kang et al., 2004). factors (Jost et al., 1996). This was confirmed by studies One mechanism of Rb repression is through recruit- examining the specificity of E2F 1 induced apoptosis. ment of co-repressors. Rb was first shown to interact Construction of chimaeric proteins containing E2Fs 1 with HDAC 1 through its pocket domain (Brehm and 3 sequences indicated that the marked box domain et al., 1998). Although HDAC and E2F both bind to and adjacent regions of E2F 1 were critically involved Rb’s pocket domain, they can simultaneously interact in the ability of activating E2Fs to induce apoptosis with hypophosphorylated Rb, allowing HDAC to be (Hallstrom and Nevins, 2003).

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5198 the promoter by their specific interaction with other transcription factors (Figure 4). Post-translational modifications of E2F 1 may also regulate its ability to associate with different E2F promoters, although specific co-factors have not been defined. In response to DNA damage, E2F 1 was shown to re-localize from cell cycle promoters to some apoptotic targets, such as p73. E2F 1 was acetylated in Figure 4 Combinatorial control of gene expression by Rb/E2F response to DNA damage, and expression from the with other transcription factors. Owing to specific interactions with p73 promoter but not the DHFRpromoter depended other transcription factors, individual members of the Rb and E2F families can differentially regulate specific target genes. As shown both on E2F 1 acetylation and on the acetyltransferase in the repression model, specific Rb/E2F complexes may be p300/CBP-associated factor (PCAF). This model sug- preferentially recruited to an E2F-binding site based on the ability gests that acetylation of E2F 1 may regulate its ability of Rb and/or E2F to bind to another transcription factor with an to interact with other transcription factors, adapting its adjacent binding site. Similarly, in the activation model, the ability localization under different circumstances. Further, of individual E2F family members to bind to other transcription factors may preferentially recruit both activators to promoters these other factors may specifically require the recruit- containing adjacent binding sites, synergistically activating tran- ment of PCAF rather than other acetyltransferases such scription. as p300 that can also activate E2F-dependent transcription (Pediconi et al., 2003). Finally, recent work in Drosophila has also contri- buted to further understanding of the differences bet- Indeed, the ubiquitous E box-binding transcription ween activating and repressive E2Fs. In an attempt to factor TFE-3 was identified in a yeast two-hybrid screen understand the specific function of dE2F2, the repressive using the marked box domain of E2F 3 as bait to E2F, native complexes were purified from embryo identify interacting proteins (Giangrande et al., 2003, extracts (Korenjak et al., 2004). In addition to either 2004). Although this marked box domain shows 55% RBF or RBF2, these complexes contained components homology between E2Fs 1 and 3, TEF-3 specifically of the dMyb complex, a known transcriptional reg- bound only to E2F 3. E2F 3 and TFE-3 could syner- ulator. The dMyb complex showed no co-staining with gistically activate p68, a polymerase a subunit with both actively transcribed regions of chromatin, consistent an E-box and E2F sites proximal to the promoter, and with its association with repressive E2Fs. Depletion of they could directly bind to one another through the E2F these dMyb subunits by RNAi resulted in upregulation 3 marked box domain. In fact, the synergistic activation of genes normally controlled exclusively by dE2F2 required this interaction, since an E2F 3 chimaera with repression, demonstrating that dMyb is required for the E2F 1 marked box domain could not activate dE2F2/RB repression. Rather than playing a role in the transcription, and in the absence of E2F 3, TFE-3 was cell cycle, these E2F target genes are involved in unable to bind to the p68 promoter. These findings differentiation, often in expression of sex-specific genes. suggest that the specificity of transcriptional activation Interestingly, the Myb/Rb/E2F interaction also appears by individual E2F proteins is determined at least in part to be evolutionarily conserved: Rb and components of by the transcription factors that bind synergistically the Myb complex form part of the synMuv class of with the E2F transcription factor (Figure 4). genes in Caenorhabditis elegans that control vulval Similarly, the repressive E2Fs, E2Fs 4 and 5 were development, and, although less studied, the human found to specifically interact with the SMAD transcrip- Myb homologs also appear to bind to Rb directly. tion factors (Chen et al., 2002). In response to transforming growth factor-b (TGF-b) signaling, SMAD2 and 3 are phosphorylated, allowing them to Concluding remarks bind to SMAD4, and this complex can either activate or repress transcription of target genes. The TGF-b In summary, regulation of specific target genes by response element of c-myc contains a consensus E2F Rb/E2F depends not only on the presence of specific Rb site adjacent to the SMAD-binding site, and mutation of and E2F family members but also on their post-trans- either site led to decreased SMAD binding and loss of lational modifications and other interacting factors. TGF-b responsiveness. Chromatin immunoprecipitation Although Rb has been extensively studied since its experiments demonstrated that SMADs 2–4, as well as identification as the first tumor suppressor gene, much E2Fs 4 and 5 and p107 were found at the TGF-b remains to be discovered about both Rb and E2F’s response element, but that the other RB family members physiological roles and the specific mechanisms of and activating E2Fs were absent. In fact, SMAD3 could action that account for their roles. Recent molecular directly bind to both E2Fs 4 and 5 as well as p107. discoveries have begun to provide a mechanistic expla- This interaction was required for repression of c-myc nation for the observation that some target genes are in response to TGF-b since c-myc expression did controlled by specific E2F and Rb family members, and not decrease in E2F 4À/À;E2F 5À/À or p107À/À cells. further understanding of the specific interactions bet- This repressive ability of E2Fs 4 and 5 may therefore, ween E2F/Rb family members and other transcription in some cases, be due to their ability to be recruited to factors may also explain complex patterns of timing and

Oncogene Retinoblastoma family genes W Du and J Pogoriler 5199 levels of induction. The mechanisms and targets found Acknowledgements in biochemical and microarray experiments will need to be examined in vivo to determine which interactions and We thank Dr Kay Macleod for reading of this paper. This targets are crucial for determining the physiological work is supported by grants from NIH and ACS to WD. outcome. WD is a Scholar of the Leukemia and Lymphoma Society.

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