PML Colocalizes with and Stabilizes the DNA Damage Response Protein TopBP1

The function of the PML tumor suppressor gene, which encodes a multifunctional protein, is consistently disrupted by t(15;17) in acute promyelocytic leukemia (APL) (26). Promyelocytic leukemia protein (PML) suppresses cell growth (31) and plays an essential role in multiple pathways of apoptosis (37, 49). PML-deficient thymocytes are resistant to programmed cell death induced by many apoptosis-inducing agents (49). PML overexpression induces G1/G0 cell cycle arrest (21) and lengthens the G1 phase of the cell cycle (30). PML-induced G1 cell cycle arrest is associated with increased expression of p53 and p21 and an inhibitor of cell cycle checkpoint kinases and results in hypophosphorylation of Rb (21, 30). PML induces apoptosis through both p53-dependent and p53-independent pathways (11, 35). Recent study showed that PML inhibits A20 protein expression induced by tumor necrosis factor alpha through the NF-κB site (52). That study suggests that PML induces apoptosis by sensitizing the tumor necrosis factor death receptor pathway. PML is normally modified in vivo by the small ubiquitin-like modifier protein SUMO-1 at three different sites (K65, K160, and K490) (15, 60). This modification is essential for the organization of PML nuclear bodies (NBs) and for PML's role in transcription regulation and apoptosis (14, 42). Transfection of the PML SUMO-1 mutant into a normal human cell line forms normal PML NBs (23) but not in PML-deficient cells (10, 11), indicating that SUMO-1 modification of PML is essential for the formation of PML NBs (14, 60). SUMO-1 modification is essential for many of PML functions, including interaction with p53, Sp100, and Daxx (14, 22, 23, 60). Substantial evidence demonstrates that PML plays a role in the regulation of gene expression (24, 59). PML interacts with transcription coactivator CREB-binding protein in vivo and activates the trancription of target genes (3, 6, 19). Documented evidence also shows that PML is associated with p53 in vivo and activates the transcription of p53 target genes (8, 9, 34). Activation of transcription of other genes by PML has also been reported (22, 46). Interestingly, evidence that PML plays a role in transcriptional repression has also been documented. When PML is fused downstream to the GAL4 DNA-binding domain, it represses GAL4-mediated transcription (45), possibly through a mechanism involving recruitment of the transcriptional corepressor histone deacetylases (51). PML interacts and associates in vivo with histone deacetylases and represses transactivation by deacetylation of the target promoter. The in vivo association of PML with other transcriptional corepressors has also been reported (16). Another mechanism by which PML represses transactivation is the interaction of PML with transcription factors, which are then prevented from binding to the target sites. PML sequesters and inhibits transcription mediated by Daxx (22, 23). PML interacts with Sp1 and inhibits Sp1-mediated transactivation of the epidermal growth factor receptor promoter (47). In addition, PML interacts with Nur77 (53) and NF-κB (52) and represses transcription mediated by these transcription factors in a promoter-specific manner. Recent studies suggest that PML may play a role in the maintenance of genome stability (58) and DNA repair (4, 58). PML colocalizes in vivo with BLM, a RecQ DNA helicase deficient in patients with Bloom syndrome (2, 58). Deficiency of BLM results in genome instability (7, 50). PML was also shown to colocalize with the alternative lengthening of telemeres (ALT), suggesting that PML plays a role in maintaining the stability of the telomere ends (10, 56). PML is associated in vivo with several DNA repair proteins, including Mre11, Rad51, and H2AX, and is localized to the single-stranded DNA (ssDNA) repair foci in response to ionizing radiation (IR) (4, 27, 36). These studies suggest that PML plays a role in the organization of the double-strand break (DSB) DNA repair complex. TopBP1, a topoisomerase IIβ-binding protein, was initially identified by yeast two-hybrid screening as the human homologue of the fission yeast Rad4/Cut5 protein, which involves in cellular responses to DNA damage and replication checkpoint controls (40, 41). TopBP1 is a DNA damage response gene, containing multiple copies of the Brca1 carboxyl-terminal motif, which has been shown to bind DNA (54). TopBP1 is a substrate of ATM kinase and is phosphorylated rapidly after exposure of the cells to IR (55). Specific antisense morpholino oligomers inhibit TopBP1 expression and induce cell death by apoptosis, indicating that TopBP1 function may be required for cell survival against IR (55). There is also evidence that TopBP1 functions in a manner similar to its fission yeast counterpart and likely involve a role in repair of DSB DNA damage and the replication checkpoint in mammalian cells (13, 25). In response to IR exposure TopBP1 forms IR-induced foci, a nuclear speckled staining pattern similar to that of the PML NBs in vivo (13, 55). Since TopBP1 plays a role in DNA damage response and replication checkpoint controls, we investigated whether PML NBs and TopBP1 foci are functionally associated in vivo. Our study demonstrated that PML colocalized with TopBP1 at the endogenous levels in response to IR. TopBP1 and PML colocalized with the IR-induced bromodeoxyuridine (BrdU) ssDNA foci and several proteins critical for DSB DNA repairs. PML and TopBP1 were coinduced by IR and interferon (IFN), and overexpression of PML significantly induced the expression of TopBP1 via stabilization of the protein. Our study demonstrated a PML-dependent induction of TopBP1 expression. The results presented here suggest that PML plays a role in regulating TopBP1 functions by association and stabilization of the protein in response to IR-induced DNA damage.