Cyclin A Is a Mediator of p120^E4F-Dependent Cell Cycle Arrest in G₁

Abstract

E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120 E4F expression induces cell cycle arrest in G 1 . Although p21 WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120 E4F can induce a G 1 block in p21 ؊/؊ cells, suggesting that other proteins are essential for the p120 E4F -dependent block in G 1 . We show here that cyclin A promoter activity can be repressed by p120 E4F and that this repression correlates with p120 E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120 E4F-arrested cells from the G 1 block. These data identify the cyclin A gene as a cellular target for p120 E4F and suggest a mechanism for p120 E4F -dependent cell cycle regulation.E4F is a ubiquitously expressed GLI-Krüppel-related mammalian transcription factor which was first identified as a cellular factor binding to regulatory regions of the adenovirus E4 promoter and responsible for E4 regulation of expression in the course of adenovirus infection (13,35,36). The adenovirus E4 promoter contains two ATF binding sites which are also targets for E4F, as observed in extracts from adenovirus-infected cells. E4F DNA binding specificity differs from that of CREB-ATF protein family members, as only subsets of ATF sites are recognized by E4F. For example, E4F binds to two out of the four ATF binding sites found in the E4 promoter but recognizes none of the ATF sites found in the E2 or E3 promoter (37). Divergence in binding specificities between E4F and ATF proteins was further demonstrated by point mutagenesis of their DNA recognition sequence and by methylation interference assays (37). E4F is synthesized as a 120-kDa protein (p120 E4F ) that upon proteolytical cleavage gives rise to p50 E4F , a 50-kDa amino-terminal fragment (11). Interestingly the murine homolog of E4F, termed AP3, was independently identified as the cellular factor responsible for the repression of the E1A promoter in mouse fibroblasts (13). Although p50 E4F and p120 E4F recognize the same DNA motifs in vitro, they differentially regulate gene expression in vivo. p50 E4Ftransactivates expression of the adenoviral E4 gene in a E1A-dependent fashion (34, 35). p120 E4F, on the other hand, is likely to play a key role in mammalian cell cycle control. Indeed, overexpression of p120 E4F in NIH 3T3 fibroblasts inhibits progression from G 1 to S phase (12). Moreover, it was recently reported that p120 E4F interacts directly with the key cell cycle regulators pRB (retinoblastoma tumor suppressor protein) and p53 (10, 39). One proposed mechanism to explain the cell cycle arrest mediated by p120E4F is the stabilization of p21 WAF1 through a post transcriptional mechanism (12). Howev...

The LIM-only protein FHL2 is a negative regulator of E4F1

“…As previously described, ectopically expressed E4F1 localized almost exclusively in the nucleus of exponentially growing U2OS cells (Figure 3a) (Fajas et al, 2000(Fajas et al, , 2001.…”

“…We and others had previously demonstrated that E4F1 overexpression leads to a strong reduction of DNA synthesis, which results from a cell cycle arrest in G1 (Fernandes et al, 1998;Fajas et al, 2000Fajas et al, , 2001Sandy et al, 2000). To evaluate the impact of FHL2 on E4F-mediated growth arrest, we monitored DNA synthesis in asynchronously growing U2OS cells transiently cotransfected with various combinations of E4F1 and Flag-tagged FHL2, together with GFP expressing plasmids.…”

“…Co-immunoprecipitation experiments were performed with an anti-FHL2 antibody or corresponding preimmune serum on nuclear extracts prepared from U2OS cells (Figure 6a), which expresses FHL2 and E4F1 (Fajas et al, 2000(Fajas et al, , 2001Amaar et al, 2002). As a negative control, we performed the same experiment in RDE cells that express similar levels of E4F1 (Figure 6b) but almost undetectable levels of FHL2 (Genini et al, 1997;Scholl et al, 2000).…”

“…p50 E4F1 transactivates expression of the adenoviral E4 gene through multiple CRE-like elements in an E1A-dependent fashion (Fernandes et al, 1997). On the contrary, p120 E4F1 , at least when overexpressed, is likely to play a role of repressor for the adenoviral E4 and E1A promoters (Fognani et al, 1993;Fernandes et al, 1997) and for the cellular cyclin A2 gene (Fajas et al, 2001;Tessari et al, 2003;Ahmed-Choudhury et al, 2005), a property that might rely on its direct interaction with histone deacetylase 1 (Colombo et al, 2003).…”

“…Indeed, embryos lacking E4F1 die at the peri-implantation stage, while in vitro cultured E4F À/À blastocysts exhibit severe defects in mitotic progression, chromosomal missegregation and increased apoptosis (Le Cam et al, 2004). Conversely, enforced expression of p120 E4F1 in various cell lines inhibits progression from G1 to S phase (Fernandes et al, 1998;Fajas et al, 2000Fajas et al, , 2001Sandy et al, 2000;Rizos et al, 2003;Fenton et al, 2004) and that of p50 E4F1 enhances cell death in E1A/ras-transformed cells (Fernandes et al, 1999). Interestingly, the E4F1-induced G1 arrest appears to depend, at least in part, on pRB and p53 pathways (Fajas et al, 2000;Sandy et al, 2000;Rizos et al, 2003).…”