**3. Role of miRNAs in E2F regulation of cell growth and DNA repair**

MicroRNAs (miRNAs) are short nucleotide sequences (~21-24nt) that pair with the 3' untranslated regions of target mRNAs. They negatively regulate gene expression by mediating degradation of the target mRNA, or by inhibition of protein translation (Almeida et al., 2011). Small miRNAs regulate many cellular processes, such as apoptosis, differentiation, and proliferation. They are upregulated in many human disorders, including cancer and neurological diseases (Almeida et al., 2011). To-date, approximately 800 miRNAs have been identified in humans. A single miRNA can target multiple mRNAs (Griffiths-

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

**4. Regulation of E2F-1 by post-translational modifications**

genes (Farhana et al., 2002; Martinez-Balbas et al., 2000).

exert either activating or inhibitory effects on E2F-1 transcriptional activity.

E2F-1 (Zhang et al., 2011).

**4.1 Acetylation** 

progression (Kong et al., 2003).

**4.2 Phosphorylation** 

DNA Repair, Cell Cycle Progression and Survival in Development & Disease 167

*7a-d*, *mir-15b-16-2* and *mir-106b-25* during the G1/S transition (Bueno et al., 2010). These miRNAs, in turn, regulate E2F-1 activity. In their absence, E2F-1 induces entry into S phase, but also DNA damage. Indeed, E2F-1 and other oncogenes can induce stalling and collapsing of DNA replication forks, leading to the formation of DNA double-strand breaks (Halazonetis et al., 2008). Thus, *let-7a-d*, *mir-15b-16-2* and *mir-106b-25* play key roles in prevention of DNA damage and replicative stress associated with abnormal regulation of

Another mode of E2F regulation that fine-tunes cell cycle progression and DNA repair occurs at the post-translational level. Post-translational modifications identified in E2F-1 include phosphorylation, acetylation, methylation & ubiquitination. These modifications can

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

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

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

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 progression.

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

#### **3.1 Growth-promoting miRNAs**

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 breaks (Pickering et al., 2009).

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 expression throughout the cell cycle, which would induce apoptosis.

#### **3.2 Tumor suppressor miRNAs**

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

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

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

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-* *7a-d*, *mir-15b-16-2* and *mir-106b-25* during the G1/S transition (Bueno et al., 2010). These miRNAs, in turn, regulate E2F-1 activity. In their absence, E2F-1 induces entry into S phase, but also DNA damage. Indeed, E2F-1 and other oncogenes can induce stalling and collapsing of DNA replication forks, leading to the formation of DNA double-strand breaks (Halazonetis et al., 2008). Thus, *let-7a-d*, *mir-15b-16-2* and *mir-106b-25* play key roles in prevention of DNA damage and replicative stress associated with abnormal regulation of E2F-1 (Zhang et al., 2011).
