**2.1 Identification of E2F targets involved in DNA damage repair**

Central to understanding the role of the E2F family of transcription factors in DNA repair has been the identification of a large number of putative and demonstrated E2F target genes. Although E2F proteins were originally characterized as important regulators of cell cycle progression, genome-wide screens have demonstrated much broader roles in a variety of primary and immortalized cell types. For example, E2F-1 and E2F-3 bind to the promoters of apurinic/apyrimidinic endonuclease (APE) and other repair enzymes in human primary epidermal keratinocytes, irrespective of their differentiation status (Chang et al., 2006). Similarly, in the GM06990 lymphoblastoid cell line, non-biased genome-wide screening has identified a large number of putative E2F-4 targets involved in responses to DNA damage (Lee et al., 2011). E2F targets important for DNA repair have also been identified in neoplastic cells following therapeutic intervention. For example, treatment of prostate cancer cells with histone deacetylase inhibitors reduces their ability to repair DNA damage induced by radio- and chemotherapy, thus reducing tumour mass (Kachhap et al., 2010). The impaired ability to repair DNA of treated cells was due, at least in part, to decreased recruitment to and activation by E2F-1 to the promoters of key DNA repair genes. Hence, the importance of E2F factors in DNA repair encompasses not only events during carcinogenesis, but also the potential impact of various therapies.

#### **2.2 Role of E2F-1 in responses to DNA damage induced by UV radiation**

UV radiation induces severe DNA damage, which is the principal cause of skin carcinogenesis in humans (Brash et al., 1996). UV-B radiation induces formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6- 4PP), which would result in loss of DNA integrity and genetic instability if left unrepaired. This type of damage to DNA triggers activation of the nucleotide-excision repair pathway, and can occur *via* one or more streams. Such DNA repair streams include (i) global genome repair (GGR), which repairs damage from the entire genome, (ii) transcription-coupled repair (TCR), which generally repairs damage on actively transcribed DNA strands & (iii) transcription domain-associated repair (DAR), which deals with repairing both strands of actively transcribed regions (Nouspikel, 2009).

Normal responses of the epidermis to UV damage are critically dependent on E2F-1 expression. Indeed, increased levels of epidermal apoptosis upon UV-B irradiation have been reported in E2F-1-null mouse epidermis, whereas repair of UV-B-induced DNA photoproducts is more efficient in keratinocytes that overexpress E2F-1 (Berton et al., 2005). UV-induced DNA damage results in stabilization of E2F-1 protein, which stimulates nucleotide excision repair (Berton et al., 2005; Pediconi et al., 2003; Wikonkal et al., 2003). The mechanisms involved include phosphorylation of E2F-1 on Ser31 by ATR and/or ATM kinases (Lin et al., 2001). This modification facilitates E2F-1 recruitment to sites of doublestrand breaks or UV-induced DNA damage. Under these conditions, E2F-1 interacts with two key proteins involved in DNA repair: TopBP1 and GCN5 histone acetyltransferase (Guo et al., 2010a; Guo et al., 2010b). Formation of these E2F-1 complexes is necessary for efficient recruitment of factors involved in nucleotide excision repair. Importantly, the association of E2F-1 with TopBP1 and GCN5 occurs at the expense of the E2F-1-induced expression of proapoptotic p73, thus ensuring that DNA repair, rather than apoptosis, takes place (Berton et al., 2005; Pediconi et al., 2003; Wikonkal et al., 2003). In mouse embryo fibroblasts, UV-C irradiation results in the formation of both CPD and 6-4PP. In these cells, nucleotide excision repair is activated through pathways that involve activation of xeroderma pigmentosum

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

**2.4 Role of E2F-1 in senescence-associated DNA damage** 

and presence of other oncogenic stimuli.

DNA repair genes.

expression.

**2.5 Role of E2F/DP interactions in DNA repair** 

DNA Repair, Cell Cycle Progression and Survival in Development & Disease 165

Senescence is defined as irreversible cell cycle arrest, which occurs both in cultured cells and *in vivo* (Lanigan et al., 2011). Senescence has been recognized as a key mechanism that acts as a barrier to tumour formation and progression. Thus, in spite of any DNA damage that may exist in a long-lived cell, if this cell is senescent it will not undergo clonal expansion to generate daughter cells with altered DNA. A number of molecular mechanisms control cellular senesce, and the E2F/pRb pathway is a key component (Lanigan et al., 2011). Under normal circumstances, the frequency of DNA mutations increases with age. DNA mismatch mutation repair is very efficient in mesenchymal cells from young individuals, as well as in embryonic fibroblasts (Chang et al., 2008). In contrast, these mechanisms are less efficient in senescent cells, in which MSH2 expression is decreased. Associated with these abnormalities is the inhibition of E2F-1 transcriptional activity, which leads to repression of MSH2 gene transcription. Thus, E2F-1 activity is essential to maintain normal capacity of cells to repair mismatch mutations. Whether the reduced activity of E2F-1 also increases the risk of transformation in senescent cells probably depends on cell context, extent of DNA damage,

The interactions between E2F-1 through -6 and their partner DP proteins are essential for normal transcriptional activity, and can also contribute to abnormal regulation of DNA repair factors. Again, depending on the exact context, E2F/DP interactions can positively or negatively modulate DNA repair. For example, following DNA damage by a variety of agents, including doxorubicin, etoposide and UV radiation, the abundance of DP-4 protein is substantially increased, replacing other DP proteins in E2F-1-containing complexes (Ingram et al., 2011). As a result, the capacity of E2F-1 to bind target promoters is strongly reduced, which can result in downregulation of cell cycle regulatory and/or

A positive modulatory role in DNA nucleotide excision repair through inhibition of repressor E2F complexes has been recently attributed to p14Arf (Dominguez-Brauer et al., 2009). Specifically, DNA damage induces p14Arf expression, which directly binds to DP-1, disrupting its interactions with E2F-4. As a result, repressive E2F-4/p130 complexes lose their ability to bind promoters of genes such as XPC, resulting in upregulation of their

To-date, multiple mechanisms that regulate E2F-1 activity at the post-transcriptional level have been identified, although only a handful has been studied in the context of DNA repair. These forms of regulation of E2F-1 activity can have important consequences on its

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-

ability to modulate DNA damage responses, as discussed below.

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

(XPC) gene expression by E2F-1 *via* increased binding to the XPC promoter (Lin et al., 2009). XPC is an essential mediator of DNA damage recognition during global genomic repair, and this phase of repair is actually more efficient in pRB-deficient cells, likely because lack of pRb increases E2F-1 activity.

The importance of E2F in repair of DNA damage induced by UV radiation is further demonstrated by the conservation of this pathway through evolution. For example, in *Arabidopsis* and in maize, MSH2 and MSH6, which are two genes that belong to the mismatch repair system, are targets of E2F transcriptional activation following DNA damage by UV-B radiation (Lario et al., 2011).

#### **2.3 E2F is a key factor to maintain the balance between cell cycle arrest and expression of DNA repair genes following DNA damage**

Given the key roles that pRb family proteins play in the regulation of E2F activity, it is not surprising that they also modulate the function of E2F factors following DNA damage. For example, the zinc finger-containing transcriptional repressor ZBRK1 is an important modulator of GADD45A transcription. The latter is involved in induction of cell cycle arrest in response to DNA damage (Siafakas and Richardson, 2009). E2F-1, but not other E2F proteins, binds to the ZBRK1 promoter, together with pRb, CtIP and CtBP, forming repressor complexes that interfere with ZBRK1 expression (Liao et al., 2010). In pRbdeficient cells, increased susceptibility to DNA damage induced by UV radiation or methylating agents occurs, partly as a result of abnormal cell cycle arrest and DNA repair. In a similar manner, E2F-1 is essential for normal expression of XRCC1 (x-ray repair crosscomplementation group 1), which participates in the repair of single-strand breaks, thus ensuring efficient repair following DNA damage induced by methylating agents (Chen et al., 2008).

In contrast, loss of pRb can improve DNA repair in other circumstances, such as those involving activation of DDB2. Mutations in the *DDB2* gene, which encodes a protein involved in global genomic repair and repair of CPDs, gives rise to xeroderma pigmentosum, a disorder associated with increased risk of cutaneous and ocular tumours (Bennett and Itoh, 2008). DDB2 expression is positively regulated by E2F-1 and E2F-3. Further, deletion of pRb increases DDB2 mRNA and protein levels, together with ability of these cells to repair DNA damage. The latter is associated with more efficient CPD removal relative to that in pRb-expressing cells (Prost et al., 2007).

Solid tumours frequently exhibit hypoxic cores, which contribute to genetic instability within the tumour microenvironment (Bindra et al., 2005). This is partly due to decreased expression of DNA mismatch genes (MLH1 and MSH2), as well as repair genes (RAD51 and BRCA1). E2F factors can also be involved in the downregulation of some of these repair genes, in apparent contrast to their pro-repair roles in other circumstances. Specifically, hypoxic conditions result in the dephosphorylation of the pRb family member p130, which then associates with E2F-4 in the nucleus. This complex can efficiently bind to E2F sites on the RAD51 and BRCA1 promoters, thus interfering with their transcription (Bindra et al., 2005). Thus, E2F factors can positively or negatively regulate DNA repair, depending on cellular context. Given that E2F-4/p130 complexes are also important for cell cycle exit, a balance must exist between these two outcomes, which is essential to avoid increased genetic instability in transformed cells and their clonal expansion.

#### **2.4 Role of E2F-1 in senescence-associated DNA damage**

164 DNA Repair

(XPC) gene expression by E2F-1 *via* increased binding to the XPC promoter (Lin et al., 2009). XPC is an essential mediator of DNA damage recognition during global genomic repair, and this phase of repair is actually more efficient in pRB-deficient cells, likely because lack of

The importance of E2F in repair of DNA damage induced by UV radiation is further demonstrated by the conservation of this pathway through evolution. For example, in *Arabidopsis* and in maize, MSH2 and MSH6, which are two genes that belong to the mismatch repair system, are targets of E2F transcriptional activation following DNA

Given the key roles that pRb family proteins play in the regulation of E2F activity, it is not surprising that they also modulate the function of E2F factors following DNA damage. For example, the zinc finger-containing transcriptional repressor ZBRK1 is an important modulator of GADD45A transcription. The latter is involved in induction of cell cycle arrest in response to DNA damage (Siafakas and Richardson, 2009). E2F-1, but not other E2F proteins, binds to the ZBRK1 promoter, together with pRb, CtIP and CtBP, forming repressor complexes that interfere with ZBRK1 expression (Liao et al., 2010). In pRbdeficient cells, increased susceptibility to DNA damage induced by UV radiation or methylating agents occurs, partly as a result of abnormal cell cycle arrest and DNA repair. In a similar manner, E2F-1 is essential for normal expression of XRCC1 (x-ray repair crosscomplementation group 1), which participates in the repair of single-strand breaks, thus ensuring efficient repair following DNA damage induced by methylating agents (Chen

In contrast, loss of pRb can improve DNA repair in other circumstances, such as those involving activation of DDB2. Mutations in the *DDB2* gene, which encodes a protein involved in global genomic repair and repair of CPDs, gives rise to xeroderma pigmentosum, a disorder associated with increased risk of cutaneous and ocular tumours (Bennett and Itoh, 2008). DDB2 expression is positively regulated by E2F-1 and E2F-3. Further, deletion of pRb increases DDB2 mRNA and protein levels, together with ability of these cells to repair DNA damage. The latter is associated with more efficient CPD removal

Solid tumours frequently exhibit hypoxic cores, which contribute to genetic instability within the tumour microenvironment (Bindra et al., 2005). This is partly due to decreased expression of DNA mismatch genes (MLH1 and MSH2), as well as repair genes (RAD51 and BRCA1). E2F factors can also be involved in the downregulation of some of these repair genes, in apparent contrast to their pro-repair roles in other circumstances. Specifically, hypoxic conditions result in the dephosphorylation of the pRb family member p130, which then associates with E2F-4 in the nucleus. This complex can efficiently bind to E2F sites on the RAD51 and BRCA1 promoters, thus interfering with their transcription (Bindra et al., 2005). Thus, E2F factors can positively or negatively regulate DNA repair, depending on cellular context. Given that E2F-4/p130 complexes are also important for cell cycle exit, a balance must exist between these two outcomes, which is essential to avoid increased genetic instability in transformed cells and their

**2.3 E2F is a key factor to maintain the balance between cell cycle arrest and** 

pRb increases E2F-1 activity.

et al., 2008).

clonal expansion.

damage by UV-B radiation (Lario et al., 2011).

**expression of DNA repair genes following DNA damage** 

relative to that in pRb-expressing cells (Prost et al., 2007).

Senescence is defined as irreversible cell cycle arrest, which occurs both in cultured cells and *in vivo* (Lanigan et al., 2011). Senescence has been recognized as a key mechanism that acts as a barrier to tumour formation and progression. Thus, in spite of any DNA damage that may exist in a long-lived cell, if this cell is senescent it will not undergo clonal expansion to generate daughter cells with altered DNA. A number of molecular mechanisms control cellular senesce, and the E2F/pRb pathway is a key component (Lanigan et al., 2011). Under normal circumstances, the frequency of DNA mutations increases with age. DNA mismatch mutation repair is very efficient in mesenchymal cells from young individuals, as well as in embryonic fibroblasts (Chang et al., 2008). In contrast, these mechanisms are less efficient in senescent cells, in which MSH2 expression is decreased. Associated with these abnormalities is the inhibition of E2F-1 transcriptional activity, which leads to repression of MSH2 gene transcription. Thus, E2F-1 activity is essential to maintain normal capacity of cells to repair mismatch mutations. Whether the reduced activity of E2F-1 also increases the risk of transformation in senescent cells probably depends on cell context, extent of DNA damage, and presence of other oncogenic stimuli.

### **2.5 Role of E2F/DP interactions in DNA repair**

The interactions between E2F-1 through -6 and their partner DP proteins are essential for normal transcriptional activity, and can also contribute to abnormal regulation of DNA repair factors. Again, depending on the exact context, E2F/DP interactions can positively or negatively modulate DNA repair. For example, following DNA damage by a variety of agents, including doxorubicin, etoposide and UV radiation, the abundance of DP-4 protein is substantially increased, replacing other DP proteins in E2F-1-containing complexes (Ingram et al., 2011). As a result, the capacity of E2F-1 to bind target promoters is strongly reduced, which can result in downregulation of cell cycle regulatory and/or DNA repair genes.

A positive modulatory role in DNA nucleotide excision repair through inhibition of repressor E2F complexes has been recently attributed to p14Arf (Dominguez-Brauer et al., 2009). Specifically, DNA damage induces p14Arf expression, which directly binds to DP-1, disrupting its interactions with E2F-4. As a result, repressive E2F-4/p130 complexes lose their ability to bind promoters of genes such as XPC, resulting in upregulation of their expression.

To-date, multiple mechanisms that regulate E2F-1 activity at the post-transcriptional level have been identified, although only a handful has been studied in the context of DNA repair. These forms of regulation of E2F-1 activity can have important consequences on its ability to modulate DNA damage responses, as discussed below.
