**4.1 Acetylation**

166 DNA Repair

Jones, 2004). Consistent with their role in cancer, miRNAs control cell proliferation by regulating E2F factors and, thereby, expression of genes that are important for cell cycle

The E2F signalling pathway is regulated by many different types of miRNA clusters, including *miRNA-17-92, miRNA-106b-25, miRNA-34, miRNA330-3p, miRNA-128, miRNA-195,* 

O' Donnell et al. were the first to provide evidence that E2F is a target for miRNAs (O'Donnell et al., 2005). They showed that miRNA-17 and miRNA-20a decrease E2F-1 translation efficiency. This type of regulation prevents uncontrolled activation of E2F-1 during normal cell cycle progression. Disruption of miRNA-17 and miRNA-20a leads to improperly timed expression of E2F-1, resulting in the accumulation of DNA double strand

An auto-regulatory loop between E2F-1 and E2F-3 and the miRNA-17-92 clusters has been demonstrated. E2F-1 and E2F-3 bind to and upregulate the transcription of the miRNA-17- 92 cluster. In turn, the miRNA-17-92 cluster downregulates expression of these two transcription factors (Sylvestre et al., 2007; Woods et al., 2007). This negative feedback loop is important to prevent the accumulation of E2F-1 and E2F-3, thereby allowing proper progression of the cell cycle, preventing apoptosis. Another negative feedback loop has been observed between the miRNA-106b-25 clusters and E2F-1 (Petrocca et al., 2008). miRNA106b and miRNA93 downregulate E2F-1 expression. Reciprocally, transcription of these miRNAs is activated by E2F-1. In this manner, properly timed expression of E2F-1 during the G1/S transition is maintained, as the presence of these miRNAs prevents continuous E2F-1

The E2F signalling pathway is also regulated by the miRNA-34 family of clusters (Tazawa et al., 2007). miRNA-34b decreases E2F-1 and E2F-3 transcript levels in a p53-dependent manner, inhibiting cell proliferation and inducing senescence in tumour cells. This demonstrates that miRNAs can function as tumor suppressors. A similar role has been suggested for miRNA-195 (Xu et al., 2009), miRNA-128 (Cui et al., 2010), miRNA-330-3p

Overexpression of miRNA-195 causes cell cycle arrest at the G1/S boundary, by interfering with the expression of cell cycle regulatory proteins, such E2F-3, Cyclin D1 and cyclindependent kinase 6 (CDK6). As a result, pRb remains hypophosphorylated, allowing activation of E2F-dependent target genes (Xu et al., 2009). Exogenous expression of miRNA-127 in glioma cells represses E2F-3a translation, thereby decreasing cell proliferation (Cui et al., 2010). Similarly, in oral squamous cell carcinoma, miRNA193a significantly represses cell

Several miRNA clusters, including *mir17-92, mir-106a-92* and *mir106b-25,* are downregulated by p53 via E2F-dependent mechanisms. This leads to decreased proliferation and/or promotes senescence in normal and transformed cells (Brosh et al., 2008). In addition, in response to mitogenic stimulation, E2F-1 activates transcription of the miRNA clusters *let-*

expression throughout the cell cycle, which would induce apoptosis.

growth and down-regulates E2F-6 translation (Kozaki et al., 2008).

**3.3 Role of miRNAs in modulation of DNA repair by E2F-1** 

(Lee et al., 2009) and miRNA193a (Kozaki et al., 2008).

progression.

*miRNA-37 and miRNA-193a*, as described below.

**3.1 Growth-promoting miRNAs** 

breaks (Pickering et al., 2009).

**3.2 Tumor suppressor miRNAs** 

E2F-1 is acetylated at three highly conserved lysine residues (K117, K120 and K125) by the p300/CREB-binding protein (CBP) or by p300/CBP-associated factor (P/CAF) acetyltransferase (Martinez-Balbas et al., 2000; Marzio et al., 2000). P/CAF directly interacts with E2F-1 through its adenosine deaminase 2 (ADA2) binding domain (Martinez-Balbas et al., 2000). Acetylation of E2F-1 allows for marked stabilization and significant increase in E2F-1 protein levels. This leads to an increase in transcriptional activation of E2F-1 target genes (Farhana et al., 2002; Martinez-Balbas et al., 2000).

Increases in E2F-1 protein levels upon DNA damage are partly due to cell type-specific acetylation (Blattner et al., 1999; Meng et al., 1999; Zhu et al., 1999). For example, adriamycin-mediated treatment induces E2F-1 acetylation in human glioblastoma T98G cells (Pediconi et al., 2003) , but not in HeLa cells (Ozaki et al., 2009). In response to DNA damage, E2F-1 switches to activate pro-apoptotic gene expression, rather than cell cycle progression. This change requires E2F-1 acetylation and recruitment to promoters of proapoptotic target genes, such as p73 (Pediconi et al., 2003). P/CAF, but not p300, is required for E2F-1 stabilization upon DNA damage by doxorubicin (Ianari et al., 2004). On the other hand, overexpression of p300 can be sufficient for acetylation and stabilization of E2F-1 in cells treated with camptothecin, a drug that causes double strand break during DNA replication (Galbiati et al., 2005). The distinct actions of these two acetyltransferase can thus determine the outcome of cellular responses by modulating cellular DNA damage checkpoints (p300) or apoptotic events (P/CAF). The stabilization of E2F-1 by acetylation could also allow it to directly interact with activating signal cointegrator-2 (ASC-2), a mitogenic transcription factor co-activator that regulates cellular proliferation and cell cycle progression (Kong et al., 2003).

#### **4.2 Phosphorylation**

E2F-1 is phosphorylated on several residues, giving rise to modifications that can alter different functional aspects. E2F-1 was first identified as a substrate for phosphorylation in a cell-free system (Bagchi et al., 1989). This post-translational modification interfered with E2F-1 DNA binding activity. Consistent with these observations, E2F-1 and E2F-3 showed decreased DNA binding capacity upon phosphorylation by cyclin A-activated cyclindependent kinase 2 (cdk2) (Dynlacht et al., 1997; Krek et al., 1995). Complexes containing

Post-Transcriptional Regulation of E2F Transcription Factors: Fine-Tuning

and Dagnino, 2007; Ivanova et al., 2009).

and context dependent (Ivanova et al., 2009).

**4.3 Methylation** 

discrepancies are not clear.

**4.4 Ubiquitination** 

DNA Repair, Cell Cycle Progression and Survival in Development & Disease 169

In epidermal keratinocytes, E2F-1 is tightly regulated during normal proliferation and differentiation (Ivanova and Dagnino, 2007; Ivanova et al., 2009; Wong et al., 2003). E2F-1 is localized in the nucleus in undifferentiated keratinocytes, but differentiation induces its export to the cytoplasm, where it is degraded. The signaling pathways involved in E2F-1 turnover in differentiating keratinocytes involve activation by Ca2+ of protein kinase C eta and delta, followed by activation of p38β. The latter appears to phosphorylate E2F-1 at Ser403 and Thr433. Once E2F-1 is phosphorylated, it is exported from the nucleus in a CRM1-dependent fashion, and degraded in the proteasome. This sequence of events involving E2F-1 phosphorylation, ubiquitination, nuclear export and subsequent degradation is required for proper keratinocyte differentiation (Ivanova et al., 2006; Ivanova

E2F-1 degradation subsequent to phosphorylation also occurs in HeLa cells. Specifically, phosphorylation of E2F-1 at Ser403 and Thr433 by TFIIH-cdk7 targets E2F-1 for degradation during S phase (Vandel and Kouzarides, 1999). Phosphorylation at Ser403 is also induced upon DNA damage (Real et al., 2010). Ser403 and Thr433 in E2F-1 are also phosphorylated by glycogen synthase kinase-3β (GSK3β) in HEK293T cells (Garcia-Alvarez et al., 2007). In U2OS osteosarcoma cells treated with doxorubicin, Ser403 is phosphorylated, but is not a substrate of either p38 MAP or GSK3β kinases (Real et al., 2010). Under these conditions, phosphorylation of Ser403 results in changes in E2F-1 target selectivity. Thus, the mechanisms and consequences of E2F-1 phosphorylation on Ser403 appear to be cell-type

Lysine methylation plays critical regulatory roles for histones and non-histone proteins (Huang et al., 2008). The consequences of methylation on E2F-1 activity are controversial at present. E2F-1 is methylated by Set9, a histone H3 methyltransferase, at Lys185, both *in vitro* and in cultured cells (Kontaki et al., 2010, Xie et al., 2011). It has been reported that DNA damage in p53-deficent H1299 lung carcinoma cells is associated with loss of E2F-1 methylation by the lysine-specific demethylase 1(LSD1). Demethylation stabilizes E2F-1, allowing its upregulation of p73. Importantly, methylation of E2F-1 at Lys185 impairs its acetylation and phosphorylation on Ser364, targeting E2F-1 for ubiquitination and degradation in doxorubicin-treated cells (Kontaki et al., 2010). In stark contrast, methylation of E2F-1 at Lys185 by Set9 in U2OS and HCT116 cells treated with adriamycin resulted in E2F-1 stabilization and cell apoptosis (Xie et al., 2011). The reasons for these pronounced

Many studies have shown that the expression of E2F-1 is regulated by the ubiquitin proteasome pathway, and that E2F-1 is protected from degradation by binding to pRb (Campanero and Flemington, 1997; Hateboer et al., 1996; Hofmann et al., 1996). In mammalian and plant cells, E2F-1 is regulated at the S/G2 phases of the cell cycle through ubiquitination by the SCFSKP2-dependent pathway (del Pozo et al., 2002; Marti et al., 1999). *In vitro*, ROC-cullin ligase ubiquitinates E2F-1 in a Skp2-independent manner. Further, phosphorylation of E2F-1 by cyclin A/cdk complexes does not affect E2F-1 ubiquitination (Ohta and Xiong, 2001). Another E3 ubiquitin ligase complex, the anaphase-promoting complex or cyclosome (ACPC/C), also regulates E2F-1 stability during late S phase (Peart et

cyclin A, cdk2, E2F-1, and DP-1 are formed during Late S-phase to terminate E2F-dependent DNA binding and transcription, and enable orderly S-phase progression (Krek et al., 1995). In the absence of cyclin A-cdk2 activity, there is decreased E2F-1 phosphorylation and increased DNA binding activity (Li et al., 1997). This results in S-phase delay and/or arrest, by mechanisms that involve transcriptional activation of E2F-dependent cell cycle checkpoint genes. Together, these data demonstrate that E2F-1 phosphorylation is essential for timely activation of E2F-1 function and orderly cell cycle progression and survival. A second proline-directed kinase, c-Jun N-terminal protein kinase (JNK1), can phosphorylate E2F-1 in response to stress stimuli mediated by tumor necrosis factor-alpha, decreasing its ability to bind DNA and activate target gene transcription (Kishore et al., 2003).

Following DNA damage, Chk2 and ATM phosphorylate E2F-1 on Ser364 and Ser31, respectively (Lin et al., 2001). E2F-1 phosphorylated on Ser31 subsequently interacts with 14- 3-3τ (Wang et al., 2004). This interaction prevents E2F-1 association with the **S**KP1-**C**ullin-**F**box/ S-phase kinase-associated protein 2 (SCFSkp2) ubiquitin ligase. As a result, E2F-1 is not ubiquitinated and is protected from degradation. The net result of phosphorylation of E2F-1 at Ser31 and Ser364 after DNA damage is activation of the pro-apoptotic gene p73, as well as accumulation of p53 through upregulation of p19ARF expression. The latter protein inhibits ubiquitination and degradation of p53, inducing apoptosis (Weber et al., 1999). In additon, the ATM and Chk2 promoters are activated by E2F-1, thereby forming a positive feedback pathway that promotes apoptosis (Berkovich and Ginsberg, 2003) .

The phosphorylation of E2F can also affect its ability to interact with other proteins. In *Drosophila melanogaster*, phosphorylation of E2F-1 and E2F-2 enhances their ability to interact with the SCFslmb ubiquitin ligase complex, targetting it for degradation during S phase (Heriche et al., 2003). *In vitro*, E2F-1 is phosphorylated at Ser337 by complexes containing p34cdc2 and cyclin B (Dynlacht et al., 1997). The significance of this finding is not clear, as E2F-1 phosphorylation on these residues occurs during late G1 phase, and is mediated by cyclin D-cdk4 complexes (Mann and Jones, 1996). Phosphorylation of E2F-1 at Ser332 and Ser337 enhances E2F-1 interactions with the adenovirus E4 protein, simultaneously attenuating its ability to bind pRb (Fagan et al., 1994). Upon adenovirus infection, the enhanced interaction between E2F-1 and E4 increases the efficiency of E2A transcription, which is required for viral DNA replication (Hardy et al., 1989).

Changes in phosphorylation status also modulate the activity and subcellular localization of E2F-4 and E2F-5, although these changes are unlikely to be mediated by cyclin A-dependent cdk activity (Dynlacht et al., 1997). Regulation of E2F-4 and E2F-5 by phosphorylation is important during entry into quiescence associated with cell differentiation, but varies depending on the cell type. For example, hypophosphorylated forms of E3F-4 efficiently associate with p130 in the nucleus, forming transcriptional repressor complexes associated with growth arrest in muscle cells (Shin et al., 1995). In contrast, in human intestinal crypt cells, hypophosphorylated E2F-4 is imported into the nucleus in response to mitogenic stimuli or inhibition of p38 MAP kinase, where it activate genes necessary for S phase entry (Deschenes et al., 2004).

E2F-5 is phosphorylated by cyclin E/cdk2 complexes on Thr251 in the transcriptional activation domain, stimulating cell cycle progression (Morris et al., 2000). This modification stabilizes E2F-5 interaction with the co-activator p300/CBP, resulting in transcription of genes required for DNA synthesis. Significantly, phosphorylation of E2F-5 at Thr251 does not affect its DNA binding activity, intracellular localization or ability to interact with pRb family proteins.

In epidermal keratinocytes, E2F-1 is tightly regulated during normal proliferation and differentiation (Ivanova and Dagnino, 2007; Ivanova et al., 2009; Wong et al., 2003). E2F-1 is localized in the nucleus in undifferentiated keratinocytes, but differentiation induces its export to the cytoplasm, where it is degraded. The signaling pathways involved in E2F-1 turnover in differentiating keratinocytes involve activation by Ca2+ of protein kinase C eta and delta, followed by activation of p38β. The latter appears to phosphorylate E2F-1 at Ser403 and Thr433. Once E2F-1 is phosphorylated, it is exported from the nucleus in a CRM1-dependent fashion, and degraded in the proteasome. This sequence of events involving E2F-1 phosphorylation, ubiquitination, nuclear export and subsequent degradation is required for proper keratinocyte differentiation (Ivanova et al., 2006; Ivanova and Dagnino, 2007; Ivanova et al., 2009).

E2F-1 degradation subsequent to phosphorylation also occurs in HeLa cells. Specifically, phosphorylation of E2F-1 at Ser403 and Thr433 by TFIIH-cdk7 targets E2F-1 for degradation during S phase (Vandel and Kouzarides, 1999). Phosphorylation at Ser403 is also induced upon DNA damage (Real et al., 2010). Ser403 and Thr433 in E2F-1 are also phosphorylated by glycogen synthase kinase-3β (GSK3β) in HEK293T cells (Garcia-Alvarez et al., 2007). In U2OS osteosarcoma cells treated with doxorubicin, Ser403 is phosphorylated, but is not a substrate of either p38 MAP or GSK3β kinases (Real et al., 2010). Under these conditions, phosphorylation of Ser403 results in changes in E2F-1 target selectivity. Thus, the mechanisms and consequences of E2F-1 phosphorylation on Ser403 appear to be cell-type and context dependent (Ivanova et al., 2009).

#### **4.3 Methylation**

168 DNA Repair

cyclin A, cdk2, E2F-1, and DP-1 are formed during Late S-phase to terminate E2F-dependent DNA binding and transcription, and enable orderly S-phase progression (Krek et al., 1995). In the absence of cyclin A-cdk2 activity, there is decreased E2F-1 phosphorylation and increased DNA binding activity (Li et al., 1997). This results in S-phase delay and/or arrest, by mechanisms that involve transcriptional activation of E2F-dependent cell cycle checkpoint genes. Together, these data demonstrate that E2F-1 phosphorylation is essential for timely activation of E2F-1 function and orderly cell cycle progression and survival. A second proline-directed kinase, c-Jun N-terminal protein kinase (JNK1), can phosphorylate E2F-1 in response to stress stimuli mediated by tumor necrosis factor-alpha, decreasing its

Following DNA damage, Chk2 and ATM phosphorylate E2F-1 on Ser364 and Ser31, respectively (Lin et al., 2001). E2F-1 phosphorylated on Ser31 subsequently interacts with 14- 3-3τ (Wang et al., 2004). This interaction prevents E2F-1 association with the **S**KP1-**C**ullin-**F**box/ S-phase kinase-associated protein 2 (SCFSkp2) ubiquitin ligase. As a result, E2F-1 is not ubiquitinated and is protected from degradation. The net result of phosphorylation of E2F-1 at Ser31 and Ser364 after DNA damage is activation of the pro-apoptotic gene p73, as well as accumulation of p53 through upregulation of p19ARF expression. The latter protein inhibits ubiquitination and degradation of p53, inducing apoptosis (Weber et al., 1999). In additon, the ATM and Chk2 promoters are activated by E2F-1, thereby forming a positive feedback

The phosphorylation of E2F can also affect its ability to interact with other proteins. In *Drosophila melanogaster*, phosphorylation of E2F-1 and E2F-2 enhances their ability to interact with the SCFslmb ubiquitin ligase complex, targetting it for degradation during S phase (Heriche et al., 2003). *In vitro*, E2F-1 is phosphorylated at Ser337 by complexes containing p34cdc2 and cyclin B (Dynlacht et al., 1997). The significance of this finding is not clear, as E2F-1 phosphorylation on these residues occurs during late G1 phase, and is mediated by cyclin D-cdk4 complexes (Mann and Jones, 1996). Phosphorylation of E2F-1 at Ser332 and Ser337 enhances E2F-1 interactions with the adenovirus E4 protein, simultaneously attenuating its ability to bind pRb (Fagan et al., 1994). Upon adenovirus infection, the enhanced interaction between E2F-1 and E4 increases the efficiency of E2A transcription,

Changes in phosphorylation status also modulate the activity and subcellular localization of E2F-4 and E2F-5, although these changes are unlikely to be mediated by cyclin A-dependent cdk activity (Dynlacht et al., 1997). Regulation of E2F-4 and E2F-5 by phosphorylation is important during entry into quiescence associated with cell differentiation, but varies depending on the cell type. For example, hypophosphorylated forms of E3F-4 efficiently associate with p130 in the nucleus, forming transcriptional repressor complexes associated with growth arrest in muscle cells (Shin et al., 1995). In contrast, in human intestinal crypt cells, hypophosphorylated E2F-4 is imported into the nucleus in response to mitogenic stimuli or inhibition of p38 MAP kinase, where it activate genes necessary for S phase entry

E2F-5 is phosphorylated by cyclin E/cdk2 complexes on Thr251 in the transcriptional activation domain, stimulating cell cycle progression (Morris et al., 2000). This modification stabilizes E2F-5 interaction with the co-activator p300/CBP, resulting in transcription of genes required for DNA synthesis. Significantly, phosphorylation of E2F-5 at Thr251 does not affect its DNA binding activity, intracellular localization or ability to interact with pRb

ability to bind DNA and activate target gene transcription (Kishore et al., 2003).

pathway that promotes apoptosis (Berkovich and Ginsberg, 2003) .

which is required for viral DNA replication (Hardy et al., 1989).

(Deschenes et al., 2004).

family proteins.

Lysine methylation plays critical regulatory roles for histones and non-histone proteins (Huang et al., 2008). The consequences of methylation on E2F-1 activity are controversial at present. E2F-1 is methylated by Set9, a histone H3 methyltransferase, at Lys185, both *in vitro* and in cultured cells (Kontaki et al., 2010, Xie et al., 2011). It has been reported that DNA damage in p53-deficent H1299 lung carcinoma cells is associated with loss of E2F-1 methylation by the lysine-specific demethylase 1(LSD1). Demethylation stabilizes E2F-1, allowing its upregulation of p73. Importantly, methylation of E2F-1 at Lys185 impairs its acetylation and phosphorylation on Ser364, targeting E2F-1 for ubiquitination and degradation in doxorubicin-treated cells (Kontaki et al., 2010). In stark contrast, methylation of E2F-1 at Lys185 by Set9 in U2OS and HCT116 cells treated with adriamycin resulted in E2F-1 stabilization and cell apoptosis (Xie et al., 2011). The reasons for these pronounced discrepancies are not clear.

#### **4.4 Ubiquitination**

Many studies have shown that the expression of E2F-1 is regulated by the ubiquitin proteasome pathway, and that E2F-1 is protected from degradation by binding to pRb (Campanero and Flemington, 1997; Hateboer et al., 1996; Hofmann et al., 1996). In mammalian and plant cells, E2F-1 is regulated at the S/G2 phases of the cell cycle through ubiquitination by the SCFSKP2-dependent pathway (del Pozo et al., 2002; Marti et al., 1999). *In vitro*, ROC-cullin ligase ubiquitinates E2F-1 in a Skp2-independent manner. Further, phosphorylation of E2F-1 by cyclin A/cdk complexes does not affect E2F-1 ubiquitination (Ohta and Xiong, 2001). Another E3 ubiquitin ligase complex, the anaphase-promoting complex or cyclosome (ACPC/C), also regulates E2F-1 stability during late S phase (Peart et

Post-Transcriptional Regulation of E2F Transcription Factors: Fine-Tuning

requires the presence of DP-1 or DP-2 (Zaragoza et al., 2010).

Zaragoza et al., 2010).

transcription of E2F target genes.

complexes (Masuhiro et al., 2008).

differentiation (Timchenko et al., 1999).

**5.3 C/EBP** 

al., 2003).

**5.4 SOCS3** 

DNA Repair, Cell Cycle Progression and Survival in Development & Disease 171

and DP-4 (Helin and Harlow, 1994; Milton et al., 2006; Ormondroyd et al., 1995). Different DP proteins have distinct modulatory effects on E2F. For example, DP-1β can mediate E2F translocation to the nucleus, whereas DP-1α, which shows reduced affinity for E2F, participates in E2F nuclear export and translocation to the cytoplasm. In this manner, DP-1α indirectly represses the ability of E2F-1 to activate transcription (Ishida et al., 2005). DP-4 can mediate transcriptional repression as well (Milton et al., 2006). Furthermore, a growing body of evidence shows that other proteins that interact with DP factors, such as C/EBP, TRIP-Br and SOCS3, can modulate E2F activation of gene transcription (Masuhiro et al., 2008;

CCAAT/Enhancer Binding Protein (C/EBP) factors are generally characterized as effectors of cellular growth arrest. Within the C/EBP family, C/EBPα has been shown to associate with and repress E2F-1 (Wang et al., 2007). This interaction has been demonstrated through co-immunoprecipitation assays and is independent of pRb family proteins. Rather, it

The effect of C/EBP repression on E2F activity has been demonstrated in multiple tissues. In primary murine keratinocytes, C/EBPα and β are upregulated as these cells differentiate and move from the basal to the suprabasal layers of the epidermis. Further, the repression of E2F target genes via the action C/EBP is necessary for proper differentiation (Lopez et al., 2009). Interactions between C/EBP and E2F also play important roles during senescence. Indeed, C/EBPα and HDAC1 are recruited to hepatic DNA from older, but not young, mice (Wang et al., 2008). Recruitment of these two factors is accompanied by decreased

In mouse 3T3-L1 preadipocytes, C/EBPα, but not C/EBPβ, disrupts E2F-p107 and induces E2F-p130 complexes, leading to decreased proliferation, likely involved in preadipocyte

In mouse hepatocytes devoid of C/EBPβ, E2F target genes are repressed and DNA synthesis is severely impaired. In these cells, C/EBP β interacts with E2F-1, facilitating recruitment of CBP and p300 to E2F target genes. The recruitment of these multiprotein complexes results in upregulation of E2F targets involved in cell proliferation (Wang et al., 2007). C/EBPβ is also required for expression of E2F-3 and S-phase progression in uterine epithelial cells (Ramathal et al., 2010). In primary epidermal keratinocytes, C/EBPα interferes with DNA synthesis in response to DNA damage (Johnson, 2005). However, the mechanisms involved are not fully undertood. It has been proposed that C/EBPα functions with E2F/pRb complexes to repress transcription of S-phase genes. In neuroblastoma cells, C/EBP is involved in induction of apoptotic gene transcription by E2F-1 (Marabese et

The Suppressor of Cytokine Signaling (SOCS) family of proteins act as negative feedback regulators of the JAK-STAT pathway. Recently, SOCS factors have also been shown to associate with DP-1 and DP-3. SOCS3 inhibits transcriptional activation of E2F target genes and cell cycle progression. The mechanisms involved in this repression include SOCS3 inhibition of E2F/DP dimerization, thus preventing the formation of the E2F DNA-binding

al., 2010). The presence of multiple E3 ligases that interact with and mediate degradation of E2F-1 enables orderly control of E2F-1 expression under multiple circumstances.

### **5. Regulation of E2F activity by protein-protein interactions**

The first type of protein-protein interactions shown to modulate E2F transcriptional activity included association with the retinoblastoma family of proteins (pRB, p107 and p130). pRB is a key regulator of E2F-1, -2 and -3 activity and G1/S-phase transition (Weintraub et al., 1995). The importance of pRb regulation of E2F is evidenced by the fact that a majority of human tumours exhibit inactivating alterations in the pRb pathway (Nevins, 2001). Subsequent studies have revealed thet E2F forms complexes with a multitude of additional proteins, underlining the levels of complexity of E2F regulation.

Protein-protein interactions also appear to assist or provide target specificity to E2F under certain conditions. This effects appear to involve cooperative interactions between E2F and other transcription factors, mediated by binding to neighbouring consensus sites on target promoters. Consensus binding sites for various transcription factors have been identified in the promoters of a subset of E2F target genes. These sites are generally adjacent to the E2F binding sites, and include recognition sequences for YY1, TFE3, and C/EBPα (Schlisio et al., 2002; van Ginkel et al., 1997). These sites possess biological significance, and assist E2F in binding to its consensus sequence. This determines the specific phase of the cell cycle in which E2F activates such promoters. In addition, as these other transcription factors do not interact equally well with all E2F members, they constitute a mechanism of activation of individual E2F factors (Giangrande et al., 2003; Schlisio et al., 2002).

#### **5.1 Retinoblastoma family proteins**

pRb binds predominantly to E2F-1, E2F-2, and E2F-3, blocking their transactivation domains (Flemington et al., 1993; Xiao et al., 2003). Under certain circumstances, such as during responses to transforming growth factor-beta in certain cell lines, pRb also binds E2F-4 and represses transcription (Yang, et al. 2008). The pRb family of proteins can also repress transcription of E2F target genes by recruiting other factors, such as histone deacetylases, thus creating transcriptional repressor complexes (Dick, 2007; Morrison et al., 2002; Herrera et al., 1996). pRb is, in turn, regulated by cyclin and cyclin-dependent kinases (Cdk),which deactivate pRb through phosphorylation. Specifically, Cyclin D/Cdk4 and Cyclin E/Cdk2 complexes phosphorylate pRb in the G1 phase of the cell cycle, allowing E2F-1, E2F-2 and E2F-3 to activate target genes (Connell-Crowley et al., 1997; Smith et al., 1996). The other pRb family proteins, p107 and p130, generally bind to E2F-4 and E2F-5, and function to modulate their nucleocytoplasmic shuttling during different periods of the cell cycle. Specifically, E2F-4 and E2F-5 translocate into the nucleus outside of the G1 and S-phases, and act as transcriptional repressors in complexes containing p107 and p130 (Ginsberg et al., 1994; Moberg et al., 1996) (Hijmans et al., 1995) (Guo et al., 2009).

#### **5.2 DP proteins**

Optimal binding of E2F to DNA requires cooperative interactions with a member of the other subfamily of E2F proteins, the DP (Dimerization Partner) family. In fact, with the exception of E2F-7 and -8, all functional E2F complexes identified contain a member of the E2F family associated with a DP protein. The DP family is composed of three known members, DP-1 (with isoforms DP-1α and DP-1β), DP-2 (and its mouse orthologue DP-3), and DP-4 (Helin and Harlow, 1994; Milton et al., 2006; Ormondroyd et al., 1995). Different DP proteins have distinct modulatory effects on E2F. For example, DP-1β can mediate E2F translocation to the nucleus, whereas DP-1α, which shows reduced affinity for E2F, participates in E2F nuclear export and translocation to the cytoplasm. In this manner, DP-1α indirectly represses the ability of E2F-1 to activate transcription (Ishida et al., 2005). DP-4 can mediate transcriptional repression as well (Milton et al., 2006). Furthermore, a growing body of evidence shows that other proteins that interact with DP factors, such as C/EBP, TRIP-Br and SOCS3, can modulate E2F activation of gene transcription (Masuhiro et al., 2008; Zaragoza et al., 2010).
