Nicholas B. La Thangue

Chk2 activates E2F-1 in response to DNA damage

The E2F-1 transcription factor is regulated during cell cycle progression and induced by cellular stress, such as DNA damage. We report that checkpoint kinase 2 (Chk2) regulates E2F-1 activity in response to the DNA-damaging agent etoposide. A Chk2 consensus phosphorylation site in E2F-1 is phosphorylated in response to DNA damage, resulting in protein stabilization, increased half-life, transcriptional activation and localization of phosphorylated E2F-1 to discrete nuclear structures. Expression of a dominant-negative Chk2 mutant blocks induction of E2F-1 and prevents E2F-1-dependent apoptosis. Moreover, E2F-1 is resistant to induction by etoposide in tumour cells expressing mutant chk2. Therefore, Chk2 phosphorylates and activates E2F-1 in response to DNA damage, resulting in apoptosis. These results suggest a role for E2F-1 in checkpoint control and provide a plausible explanation for the tumour suppressor activity of E2F-1.

Acetylation control of the retinoblastoma tumour-suppressor protein

The retinoblastoma tumour-suppressor protein (pRb) and p300/CBP co-activator proteins are important for control of proliferation and in tumour cells these are sequestered by viral oncoproteins such as E1A. pRb is involved in negatively regulating growth, and p300/CBP proteins have histone acetyltransferase (HAT) activity, which influences gene expression. Although it is known that phosphorylation by G1 cyclin-dependent kinases (CDKs) regulates pRb activity, the nature and role of other post-translational modifications is not understood. Here we identify acetylation as a new type of modification and level of control in pRb function. Adenovirus E1A, which binds p300/CBP through an amino-terminal transformation-sensitive domain, stimulates the acetylation of pRb by recruiting p300 and pRb into a multimeric-protein complex. Furthermore, pRb acetylation is under cell-cycle control, and acetylation hinders the phosphorylation of pRb by cyclin-dependent kinases. pRb binds more strongly when acetylated to the MDM2 oncoprotein, which indicates that acetylation may regulate protein–protein interactions in the pRb pathway. The acetylation of pRb defines a new level of cell-cycle control mediated by HAT. Furthermore, our results establish a relationship between p300, pRb and acetylation in which E1A acts to recruit and target a cellular HAT activity to pRb.

A Novel Cofactor for p300 that Regulates the p53 Response

The ability of p53 to function as a transcription factor is instrumental in facilitating the response to cellular stress, and p300/CBP proteins, which act as coactivators for diverse transcription factors, participate in regulating p53 activity. We report a novel cofactor for p300 that facilitates the p53 response by augmenting p53-dependent transcription and apoptosis. JMY and p300 associate in physiological conditions, and, during the cellular stress response, the p300/JMY complex is recruited to activated p53. The bax gene is efficiently activated by JMY, and protein isoforms that arise through alternative splicing alter the functional outcome of the p53 response. The results provide compelling evidence that the p300/JMY coactivator complex plays a central role in facilitating the p53 response.

E2F and cell cycle control: a double-edged sword

The E2F family of transcription factors plays a central role in regulating cellular proliferation by controlling the expression of both the genes required for cell cycle progression, particularly DNA synthesis, and the genes involved with apoptosis. E2F is regulated in a cell cycle-dependent manner, principally through its temporal association with pocket protein family members, the prototype member being the retinoblastoma tumor suppressor protein. Pocket proteins are, in turn, regulated through phosphorylation by cyclin-dependent kinase (cdk). The kinase activity of cyclin/cdk complexes is negatively regulated by cdk inhibitors, and thus both positive and negative growth regulatory signals impinge on E2F activity. Different E2F family members exhibit distinct cell cycle and apoptotic activities. Thus, E2F appears to play a pivotal role in coordinating events connected with proliferation, cell cycle arrest, and apoptosis.

Promoter specificity and stability control of the p53-related protein p73

The p53 family of proteins play instrumental roles in mediating the cellular response to stress. The p53-related gene product, p73, occurs as two distinct protein isoforms, referred to as α and β, which differ in the length of the C-terminal region and arise through alternative splicing of the p73 RNA. Here, we describe an analysis of the transcription properties of p73 and show that although there are certain similarities between transcriptional activation mediated by p73 and p53, such as in their sensitivity to adenovirus E1A and the requirement for p300/CBP co-activator proteins, significant differences are apparent in the response mechanisms. Thus, we find that p73 shows a degree of specificity for the promoters of target genes that is quantitatively distinct from the response mediated by p53. For example, p73 activates the GADD45 gene more efficiently than p53, whereas the reverse situation was apparent for the p21 gene. These effects are, in part, due to the influence of a regulatory domain present in the extended C-terminal of the α isoform. Moreover, we provide evidence that this domain regulates protein abundance by influencing the proteasome-dependent degradation of p73. These data define a novel level of isoform-specific control in regulating p73 activity, and thereby highlight a significant difference between the mechanisms that govern the transcriptional activity of p53 and p73.

Functional interplay between p53 and E2F through co-activator p300

Both E2F and p53 are sequence specific transcription factors that regulate early cell cycle progression. The pathway of control mediated through E2F governs the transition from G1 into S phase whereas p53 in response to genotoxic stress can facilitate cell cycle arrest or apoptosis. The mechanisms which influence the outcome of p53 induction are not clear, although transcription of the p53 target gene, encoding the cdk-inhibitor p21Waf1/Cip1, correlates with p53-mediated cell cycle arrest. Here using a combination of biochemical and functional assays we identify p300 as a co-activator required for p53-dependent transcriptional activation of Waf1/Cip1. Furthermore, we show that the cdk-inhibitor p21Waf1/Cip1 autoregulates in a positive fashion transcription through modulating the activity of the p53/p300 complex, whilst negatively regulating the activity of E2F by preventing cdk-dependent phosphorylation of pRb. Consistent with a role for p21Waf1/Cip1 in the autoregulation of p53-dependent transcription, p300 augments the ability of p53 to cause G1 arrest and, conversely, cells undergoing p53-dependent apoptosis are rescued by p300. Thus, our data suggest that the ability of p300 to interact with p53 influences the physiological consequence of p53 activation. From previous studies it is known that cells expressing aberrant levels of E2F-1 can undergo p53-dependent apoptosis. In addition, we find that E2F-1 can cause apoptosis in p53−/− tumour cells and further p300, which also functions as a co-activator for the E2F/DP heterodimer, enhances the apoptotic activity of E2F-1. In conditions where E2F-1 and p53 co-operate in apoptosis E2F-1 can effectively compete for p300, causing a reduction in p53-dependent transcription. Thus, a functional interaction between p300 and either p53 or E2F-1 has a profound impact on early cell cycle progression, specifically in regulating the contrasting outcomes of cell cycle arrest and apoptosis. These results suggest a critical role for p300 in integrating and co-ordinating the functional interplay between the pathways of growth control mediated by E2F and p53.

The yin and yang of E2F-1: balancing life and death

E2F transcription factors coordinate the timely expression of genes during early cell-cycle progression. In addition, the E2F-1 subunit can induce apoptosis in response to DNA damage. New results reveal an unexpected function for E2F-1 in suppressing apoptosis, which may be important in explaining the contribution of E2F-1 to tumorigenesis.E2F transcription factors coordinate the timely expression of genes during early cell-cycle progression. In addition, the E2F-1 subunit can induce apoptosis in response to DNA damage. New results reveal an unexpected function for E2F-1 in suppressing apoptosis, which may be important in explaining the contribution of E2F-1 to tumorigenesis.

Integration of a growth‐suppressing BTB/POZ domain protein with the DP component of the E2F transcription factor

Transcription factor E2F plays an important role in orchestrating early cell cycle progression through its ability to co‐ordinate and integrate the cell cycle with the transcription apparatus. Physiological E2F arises when members of two distinct families of proteins interact as E2F–DP heterodimers, in which the E2F component mediates transcriptional activation and the physical interaction with pocket proteins, such as the tumour suppressor protein pRb. In contrast, a discrete role for the DP subunit has not been defined. We report the identification and characterization of DIP, a novel mammalian protein that can interact with the DP component of E2F. DIP was found to contain a BTB/POZ domain and shows significant identity with the Drosophila melanogaster germ cell‐less gene product. In mammalian cells, DIP is distributed in a speckled pattern at the nuclear envelope region, and can direct certain DP subunits and the associated heterodimeric E2F partner into a similar pattern. DIP‐dependent growth arrest is modulated by the expression of DP proteins, and mutant derivatives of DIP that are compromised in cell cycle arrest exhibit reduced binding to the DP subunit. Our study defines a new pathway of growth control that is integrated with the E2F pathway through the DP subunit of the heterodimer.

DNA‐damage response control of E2F7 and E2F8

Here, we report that the two recently identified E2F subunits, E2F7 and E2F8, are induced in cells treated with DNA‐damaging agents where they have an important role in dictating the outcome of the DNA‐damage response. The DNA‐damage‐dependent induction coincides with the binding of E2F7 and E2F8 to the promoters of certain E2F‐responsive genes, most notably that of the E2F1 gene, in which E2F7 and E2F8 coexist in a DNA‐binding complex. As a consequence, E2F7 and E2F8 repress E2F target genes, such as E2F1, and reducing the level of each subunit results in an increase in E2F1 expression and activity. Importantly, depletion of either E2F7 or E2F8 prevents the cell‐cycle effects that occur in response to DNA damage. Thus, E2F7 and E2F8 act upstream of E2F1, and influence the ability of cells to undergo a DNA‐damage response. E2F7 and E2F8, therefore, underpin the DNA‐damage response.

Role of LXCXE motif-dependent interactions in the activity of the retinoblastoma protein.

Cell cycle control by pRb requires the integrity of the pocket domain, which is a region necessary for interactions with a variety of proteins, including E2F and LXCXE-motif containing proteins. Through knowledge of the crystal structure of pRb we have prepared a panel of pRb mutant derivatives in which a cluster of lysine residues that demark the LXCXE peptide binding domain were systematically mutated. One of the mutant derivatives, Rb6A, exhibits significantly reduced LXCXE-dependent interactions with HPV E7, cyclinD1 and HDAC2, but retained LXCXE-independent binding to E2F. Consistent with these results, Rb6A could down-regulate E2F-1-dependent activation of different E2F responsive promoters, but was compromised in Rb-dependent repression. Most importantly, Rb6A retained wild-type growth arrest activity, and colony forming activity similar to wild-type pRb. It is compatible with these results that directly targeting HDAC2 to E2F responsive promoters as an E2F/HDAC hybrid protein failed to effect cell cycle arrest. These results suggest that LXCXE-dependent interactions are not essential for pRb to exert growth arrest.