Characterization of endogenous human promyelocytic leukemia isoforms.

Promyelocytic leukemia (PML) has been implicated in a variety of functions, including control of TP53 function and modulation of cellular senescence. Sumolated PML is the organizer of mature PML bodies, recruiting a variety of proteins onto these nuclear domains. The PML gene is predicted to encode a variety of protein isoforms. Overexpression of only one of them, PML-IV, promotes senescence in human diploid fibroblasts, whereas PML-III was proposed to specifically interact with the centrosome. We show that all PML isoform proteins are expressed in cell lines or primary cells. Unexpectedly, we found that PML-III, PML-IV, and PML-V are quantitatively minor isoforms compared with PML-I/II and could not confirm the centrosomal targeting of PML-III. Stable expression of each isoform, in a pml-null background, yields distinct subcellular localization patterns, suggesting that, like in other RBCC/TRIM proteins, the COOH-terminal domains of PML are involved in interactions with specific cellular components. Only the isoform-specific sequences of PML-I and PML-V are highly conserved between man and mouse. That PML-I contains all conserved exons and is more abundantly expressed than PML-IV suggests that it is a critical contributor to PML function(s).

TRAF interactions with raft-like buoyant complexes, better than TRAF rates of degradation, differentiate signaling by CD40 and EBV latent membrane protein 1.

The CD40 receptor and the Epstein-Barr virus oncoprotein LMP1 are both members of the TNF-receptor family and share several signaling mediators, including TRAF2 and TRAF3. Depending on the cell lineage and stage of maturation, LMP1 and CD40 can have synergistic, antagonist or unrelated effects. Previous publications have suggested that both TRAF2 and TRAF3 move into lipid rafts upon LMP1 expression or CD40 activation, whereas their proteolysis is only enhanced by CD40. However CD40-induced proteolysis of TRAF2 has only been reported in murine cells, and there are conflicting data regarding translocation of TRAF2 into lipid rafts. We therefore investigated TRAF2 and TRAF3 modifications induced by CD40 and LMP1 signaling in a panel of human cell lines of lymphoid and epithelial origins. Upon CD40 stimulation, a marked redistribution of TRAF2 into the buoyant raft fraction was observed in all cell lines and was often associated with a similar redistribution of TRAF3. In contrast, only TRAF3 was redistributed into the raft fraction upon LMP1 expression. Moreover parallel changes in subcellular distribution of TRAF2 and TRAF3 were recorded by electron microscopy. A significant decrease in TRAF2 and TRAF3 concentrations triggered by CD40 ligation was observed in only 1 cell line and there was no evidence that this decrease was required for the negative feed-back on JNK activation. TRAF2 redistribution into raft-like complexes thus appears as the most significant event distinctive of CD40 and LMP1 signaling. On the other hand, the parallel influence of CD40 and LMP1 on TRAF3 redistribution is consistent with functional similarities between the CD40-TRAF3 and LMP1-TRAF3 axes.

Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis.

BCL6 is a POZ/BTB and zinc finger transcription factor that self-interacts and accumulates into discrete nuclear "bodies" of unknown function. We recently reported that BCL6 bodies associate with bromodeoxyuridine (BrdU)-substituted DNA, suggesting their implication in replication. To examine this possibility, we examine here by electron and confocal microscopy the relation between BCL6 bodies and replication foci (RF) using incorporation of various halogenated nucleotides (BrdU, chlorodeoxyuridine, CldU, and iododeoxyuridine, IdU) or PCNA (proliferating cell nuclear antigen) staining. We show that BCL6 bodies are found associated with RF, as revealed by PCNA staining. However, such association is markedly prolonged upon BrdU or CldU incorporation, but less, or not at all, upon IdU incorporation. Pulse-chase and double-labeling experiments indicate that IdU-substituted DNA leaves BCL6 bodies after a few tenths of minutes while BrdU- or CldU-substituted DNA stalls in their vicinity for several hours, thereby giving the characteristic "crowns" of DNA entirely surrounding BCL6 bodies. In all cases, however, the halogenated DNA ends up undergoing a movement from BCL6 bodies toward nucleoplasm and nuclear periphery to reach euchromatin and heterochromatin, respectively. We propose that replicating DNA is prone to be bound by BCL6, while BrdU/CldU incorporation increases this propensity possibly because these two events have synergistic effects on the structure and chromatinisation of the newly synthesized DNA. Finally, despite the known proximity between nuclear sites of transcription and replication, we show via several approaches that BCL6 bodies do not appear to be involved either in RNA synthesis or storage.

Class II histone deacetylases are directly recruited by BCL6 transcriptional repressor.

BCL6 is a member of the POZ/zinc finger (POK) family involved in survival and/or differentiation of a number of cell types and in B cell lymphoma upon chromosomal alteration. Transcriptional repression by BCL6 is thought to be achieved in part by recruiting a repressor complex containing two class I histone deacetylases (HDACs). In this study we investigated whether BCL6 could also target members of class II HDACs. Our results indicate that three related class II deacetylases, HDAC4, HDAC5, and HDAC7 can associate with BCL6 in vivo and in vitro. Using electron microscopy, we found that endogenous BCL6 and class II HDACs partially co-localize in the nucleus. Overexpression experiments showed that BCL6 and HDAC4, -5, or -7 are intermingled onto common nuclear substructures and form stable complexes. A highly conserved domain in the N-terminal region of HDAC5 and HDAC7 as well as the zinc finger region of BCL6 were found necessary for the complex formation in vivo and in vitro. Moreover, our data point to the zinc finger region of BCL6 as a multifunctional domain which, beside its known capacity to bind DNA, is involved in the nuclear targeting of the protein and in the recruitment of the class II HDACs, and hence constitutes an autonomous repressor domain. Since PLZF, a BCL6 relative, could also interact with HDAC4, -5, and 7, we suggest that class II HDACs are largely involved in the control of the POK transcription factors activity.

Adenovirus infection targets the cellular protein kinase CK2 and RNA-activated protein kinase (PKR) into viral inclusions of the cell nucleus.

The effects of the adenovirus infection on the distribution of the cellular protein kinase CK2 and double-stranded RNA-activated protein kinase (PKR) were examined at the ultrastructural level. Immunogold labeling revealed the redistribution of CK2 subunits and PKR to morphologically distinct structures of the cell nucleus. The electron-clear amorphous structures, designated pIX nuclear bodies in our previous work (Rosa-Calatrava et al., 2001), contained CK2 alpha and PKR. The protein crystals, which result from the regular assembly of hexon, penton base, and fiber proteins [Boulanger et al. (1970) J Gen Virol 6:329-332], contained CK2 beta and PKR. Both viral structures were devoid of viral RNA, including the PKR-inhibitor VA1 RNA generated by the RNA polymerase III. Instead, VA1 RNA accumulated in PKR-free viral compact rings in which the viral RNA generated by the RNA polymerase II was excluded.