**5.3 C/EBP**

170 DNA Repair

al., 2010). The presence of multiple E3 ligases that interact with and mediate degradation of

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

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

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.,

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),

E2F-1 enables orderly control of E2F-1 expression under multiple circumstances.

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

proteins, underlining the levels of complexity of E2F regulation.

individual E2F factors (Giangrande et al., 2003; Schlisio et al., 2002).

1994; Moberg et al., 1996) (Hijmans et al., 1995) (Guo et al., 2009).

**5.1 Retinoblastoma family proteins** 

**5.2 DP proteins** 

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

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 transcription of E2F target genes.

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 differentiation (Timchenko et al., 1999).

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 al., 2003).

#### **5.4 SOCS3**

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 complexes (Masuhiro et al., 2008).

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

**6.2 SV40 large-T antigen** 

type of chaperone protein Hsc70 and ATP.

E2F (Nemethova et al., 2004).

**6.4 Human parvovirus NS1** 

**6.3 Adenovirus E1A** 

DNA Repair, Cell Cycle Progression and Survival in Development & Disease 173

pathways that act as fail-safe mechanisms for E2F activity, such as p53-mediated apoptosis (Moody and Laimins, 2010). The key HPV viral protein involved in activating E2Fs is E7. This protein carries an LXCXE domain characteristic of proteins that associate with pRb family proteins (Lee et al., 1998). In this manner, E7 proteins bind to pRb, p107 and p130, dissociating them from E2F factors. The mechanisms involved in this effect include blockade by E7 of the pRb-E2F binding domain (Lee et al., 1998). As a result, E2F species bind to and activate target genes without the possibility of repression. E7 also induces pRb proteasomal degradation, by increasing its ubiquitination (Moody and Laimins). Furthermore, there is evidence to indicate that E7 also binds to p300/CBP, allowing this acetyltransferase to facilitate and rapidly increase the transcription of E2F target genes (Bernat et al., 2003).

The simian virus 40 (SV40) genome encodes a protein that shares some characteristics with HPV E7, termed large T-antigen. Similar to HPV E7, large-T antigen has a LXCXE domain, which can bind all three pRb family proteins, leading to release of free E2F and expression of its target genes (DeCaprio, 2009). In addition, large-T antigen binds preferentially to the hypophosphorylated form of pRb, present during the G1-phase of the cell cycle (Ludlow et al., 1989). The characterization of the interactions between large-T antigen and complexes containing p130 or p107 and E2F-4 has been central to understanding the mechanisms involved in deactivation of pRb family proteins by this viral factor (Sullivan et al., 2000). Dissociation of p107 or p130 from E2F also requires Large-T antigen interactions with the J

Similar to HPV E7, large-T antigen binds to p300/CBP through its C-terminus (Eckner et al., 1996). This interaction is likely involved in histone acetylation and transcriptional activation of E2F target genes. Significantly, mutations in the C-termimus of large-T antigen impair its ability to bind p300/CBP, but are without effect on its capacity to disrupt pRb binding to

Adenovirus protein E1A functions in a similar manner to HPV E7 and SV40 large-T antigen. E1A interacts with multiple cellular proteins, including the pRb family and p300/CBP (Raychaudhuri, 1991; Liu, 2007). X-ray crystallographic characterization of E1A has revealed that its N-terminal domain competes with the transactivation domain of E2F for binding to pRb. This induces a decrease in E2F binding to pRb by competition (Liu, 2007). Similar to other viral oncoproteins, E1A also has an LXCXE domain that binds to pRb, p107 and p130 (Dyson, 1992). E1A also binds the 400-kDa protein p400, which mediates further interactions with TRRAP/PAF400, along with the DNA helicase TAP54α/β). Together, these proteins form a chromatin remodeling complex, which contributes to cell transformation and

Human parvovirus B19 (B19V) is the only pathogenic human parvovirus, and it targets cells of the erythroid lineage, expecially erythroid progenitors (Wan et al., 2010). The B19V protein NS1 (nonstructural protein 1) interacts with E2F-4 and E2F-5, inducing their nuclear accumulation and G2 arrest, necessary for viral replication (Wan et al., 2010). Simultaneously, NS1 also decreases expression of E2F-1, E2F-2, and E2F-3, resulting in

activation of E2F target genes that mediate viral DNA replication (Liu, 2007).
