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Early evidence implicating PML proteins in regulating transcription was uncovered by transfecting C-terminally truncated GAL4-DNA binding/PML fusion protein constructs in yeast and mammalian cells (Ahn et al., 1998). Unexpectedly, in yeast, the full length constructs which comprised the N-terminal Cys-rich region and the coiled coil domain showed no activity using the β-gal assay. Deleting the coiled coil region but keeping the RING finger plus the Cys/His region improved the transactivation activity of PML constructs. Making the GAL4/PML constructs even shorter, which kept only the intact RING finger domain of PML, still showed higher activity than the full length constructs but 6 fold less than those which retained the Cys/His neighboring region. This means that the coiled coil region may block the transactivation function of intact PML. The finger domain itself plays a crucial role in unmasking the transactivation activity of the intact PML, since mutating the Cys residues within this domain significantly reduced the transactivation activity of the C- terminally truncated PML constructs. The transactivation unmasking property was found to apply also in mammalian cells such as Vero cells. Using the same constructs but driven by a different enhancer (SV40 enhancer derived- vector) and expressing them in Vero cells showed about 22 fold increase in transactivation activity in constructs containing the RING domain and Cys/His rich residues. Again mutating the RING finger domain caused a 3 fold-reduction in the transactivation activity. Altogether, there is enough evidence that the N-terminal region has a role in cellular transcription but it is masked by the α- helical coiled coil domain (Ahn et al., 1998).

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1.9.1 PML proteins interact with cellular transcription factors (transcription enhancement)

PML proteins have been discovered to be a positive partner for many cellular transcription factors and thus enhance transcription activity. CBP, a dynamic component of PML-NB (Boisvert et al., 2001) associates with PML-NB through interaction with PML. This association is located between residues 216-331 in the domain common to all PML and residues 331-521 in the N-terminal domain of CBP, PML proteins caused an increase in the transcriptional activity of CBP cofactor (Doucas et al., 1999). Another explanation for the reason for recruitment of CBP to the NB is to keep its level at a steady state inside the cells. Valproic acid, an epilepsy treatment medicine is used to degrade CBP through the ubiquitin proteasome pathway. PML-NB show ubiquitin activity and participate in degrading target proteins to control their levels inside the cells. This might explain why CBP is co- localizing in PML-NB (St-Germain et al., 2008).

PML-NB also show a critical role in tumour suppression after DNA damage by increasing the stability of p53. After DNA damage PML proteins are phosphorylated by ATR and accumulate together with Mdm2 (a negative regulator of p53 levels) in the nucleolus, hence stabilizing p53 by direct interaction between PML and Mdm2 (Bernardi et al., 2004). The transactivation function of p53 is increased with the recruitment of p53 to the nuclear bodies by PML IV in a promoter-dependent manner (Fogal et al., 2000). Another function of PML proteins and in particular PML-IV, is increasing transcription through stabilizing p300 by protecting it from ubiquitin-mediated degradation (Shima et al., 2008).

1.9.2 PML proteins interact with cellular transcription factors (transcription repression)

PML proteins have also been reported to be transcriptional repressors. GAL4/PML fusion proteins inhibited transcription from GAL4-responsive reporter plasmids in HeLa and Cos1 cells (Vallian et al., 1997). Twenty-five years ago, PML protein was depicted as a regulator of the repressive transcriptional function of Daxx. Daxx is an efficient repressor of global cellular transcription: it interacts with Sumo-1-modified PML and is recruited to PML-NB or is recruited to condensed chromatin in PML-

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deficient cells. Movement of Daxx between the condensed chromatin and PML-NB reflects the variable equilibrium between the chromatin and NB and how dynamic is this process which is in the same time needs the accumulation of SUMO-1 modified PML to make the basic structure and framework of NB (Ishov et al., 1999). The Daxx-PML interaction therefore determines the correct of this global repressor, keeping it mainly in the nucleus, despite its locations potential to interact with c-fos although a very tiny proportion of Daxx was reported in the cytoplasmic fraction. In the absence of any inducers of the dissociation of co-repressors, such as all-trans- RA (atRA), Daxx can interact with the PML-RARα complex (Li et al., 2000). This interaction was shown in vivo but could not be confirmed in vitro since it needs highly sumoylated PML, or maybe other cellular factors to associate and facilitate this interaction. Immunofluorescent antibody staining has shown a specific co- localization between Daxx and PML in different tumour cell lines such as HeLa and A549 in addition to normal human fibroblasts. Using reporter gene assays, the ability of PML to inhibit the Daxx repression function in HEK293 cells was measured. Compared to samples which were transfected with GAL4- DNA Binding Domain (DBD) alone, reporter gene expression was greatly reduced when cells were transfected with GAL4-Daxx, and this reduction was rescued in a dose-dependent manner by co-expression of increasing doses of full length PML VI plasmid (Li et al., 2000). This isoform of PML at least is therefore a negative regulator of Daxx repression and its interaction with Daxx may be important for mounting a potent anti-viral response against the invading viruses as it will permit the up-regulation of antiviral response genes.

Many DNA virus proteins, such as Ad5 E4Orf3, disrupt the PML-NB and such disruption is well-correlated with blocking the innate immune response. E4Orf3- deleted viruses do not replicate as well as wild type, but their replication can rescued by a reduction in PML and Daxx using knockdown approaches (Ullman and Hearing, 2008). Another example that reveals the involvement of PML in regulating cellular transcription in eukaryotic cells is the interaction with the specificity protein 1 (SP1). In vitro co-immunoprecipitation assays showed that SP1 and PML interact directly through the C- terminal DNA binding site of Sp1 and the coiled coil regions

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of Sp1 and PML. The physiological significance of such an interaction was demonstrated as an effect of PML on SP1 – dependent gene transcription of genes such as epidermal growth factor receptor (EGFR). In other words, PML represses the transcription of EGFR by inhibiting EGFR’sSp1-dependent activity.PML inhibits transcription from the EGFR promoter through disrupting the DNA binding activity of SP1 and thus PML plays a negative role in regulating the transcriptional activity of the EGFR gene (Vallian et al., 1998a).