p53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation

Conserved elements of apoptosis are also integral components of cellular differentiation. In this regard, p53 is involved in neurogenesis, being required for neurite outgrowth in primary neurons and for axonal regeneration in mice. Interestingly, demethylases regulate p53 activity and its interaction with co-activators by acting on non-histone proteins. In addition, the histone H3 lysine 27-specific demethylase JMJD3 induces ARF expression, thereby stabilizing p53 in mouse embryonic fibroblasts. We hypothesized that p53 interacts with key regulators of neurogenesis to redirect stem cells to differentiation, as an alternative to cell death. Specifically, we investigated the potential cross-talk between p53 and JMJD3 during mouse neural stem cell (NSC) differentiation. Our results demonstrated that JMJD3 mRNA and protein levels were increased early in mouse NSC differentiation, when JMJD3 activity was readily detected. Importantly, modulation of JMJD3 in NSCs resulted in changes of total p53 protein, coincident with increased ARF mRNA and protein expression. ChIP analysis revealed that JMJD3 was present at the promoter and exon 1 regions of ARF during neural differentiation, although without changes in H3K27me3. Immunoprecipitation assays demonstrated a direct interaction between p53 and JMJD3, independent of the C-terminal region of JMJD3, and modulation of p53 methylation by JMJD3-demethylase activity. Finally, transfection of mutant JMJD3 showed that the demethylase activity of JMJD3 was crucial in regulating p53 cellular distribution and function. In conclusion, JMJD3 induces p53 stabilization in mouse NSCs through ARF-dependent mechanisms, directly interacts with p53 and, importantly, causes nuclear accumulation of p53. This suggests that JMJD3 and p53 act in a common pathway during neurogenesis

On the beneficial effect of noise in vertex localization

A theoretical and experimental analysis related to the effect of noise in the task of vertex identication in unknown shapes is presented. Shapes are seen as real functions of their closed boundary. An alternative global perspective of curvature is examined providing insight into the process of noise- enabled vertex localization. The analysis reveals that noise facilitates in the localization of certain vertices. The concept of noising is thus considered and a relevant global method for localizing Global Vertices is investigated in relation to local methods under the presence of increasing noise. Theoretical analysis reveals that induced noise can indeed help localizing certain vertices if combined with global descriptors. Experiments with noise and a comparison to localized methods validate the theoretical results

p53 can promote mitochondria- and caspase-independent apoptosis

International audienceThe tumour suppressor p53 plays a pivotal role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis. Cell cycle arrest is mediated by transcriptional induction of genes whose products inhibit cell cycle progression. Conversely, the molecular events that lead to p53-dependent apoptosis are less clear. Transcriptional activation is commonly implicated but growing evidences show that transrepression and transcription-independent functions can also play a central role in p53-dependent apoptosis. 1 At the cellular level, all studies converge to the crucial role of both the mitochondrial pathway (cytochrome c release, 2 ROS production or/and DCm drop) 3 and caspase activation in p53-induced apopto-sis. 4 In this way, the Bcl-2 antiapoptotic protein as well as caspase inhibition were shown to protect cells from p53-induced apoptosis. We previously showed in rat embryo fibroblasts (e.g. the REtsAF cell line) expressing a temperature-sensitive mutant (tsA58) of the simian virus 40 large tumour antigen (LT) that LT inactivation leads to p53-mediated apoptosis. Moreover, we reported that while bcl-2 overexpression inhibits apopto-sis, caspase inhibition surprisingly accelerates apoptosis and moreover abolishes the protective effect of Bcl-2. 5 These data led us to postulate that caspase inhibition would unmask an alternative route for p53-induced cell death signal, which would lead to a caspase-independent and Bcl-2-insensitive cell death process. In order to ascertain that this new cell death process observed in the presence of ZVAD is dependent on p53 activity, REtsAF cells were transiently transfected with genes encoding temperature-sensitive dominant-negative mutants of p53 (p53 val135 and p53 ala143). 6,7 At restrictive temperature, these mutants are defective in their DNA binding domain and consequently loss their sequence-specific transactivation and transrepression properties. We observed that overexpression of either p53 val135 or p53 ala143 led to a decreased rate of apoptosis in the absence or in the presence of ZVAD (Figure 1a), demonstrating that active p53 is required for these two cell death pathways. Next, we controlled that this unexpected effect of ZVAD on p53-induced cell death could be reproduced in a more physiological model. For this purpose, we induced a p53-dependent apoptosis in primary rat embryo fibroblasts (RE) by addition of 100 mM etoposide, in the absence or in the presence of ZVAD. As observed in REtsAF cells, ZVAD treatment accelerated commitment to death of RE cells. As control, ZVAD reduced staurosporine-induced apoptosis of RE cells, showing its usual protector effect against this p53-independent cell death (Figure 1b). These results argue for a physiological relevance of the proapoptotic effect of ZVAD on p53-mediated apoptosis. In order to characterise the pattern of the p53-induced death of REtsAF cells in the presence of ZVAD, we examined the associated nuclear and cytological alterations. In the absence of ZVAD, the earliest morphological changes observed are the rounding up, the brightening phase, the shrinkage of the cells as well as the blebbing of the plasma membrane (Figure 1c). These events are associated with typical apoptotic chromatin compaction and fragmentation in globular, crescent-shaped figures (stage II chromatin condensation), and they preceded the breaking up of the cells into fragments as well as the fragmentation of DNA, as judged by the flow cytometric analysis of TUNEL-stained cells. In the presence of ZVAD, light microscopy observation showed that some typical morphological features of apoptosis such as loss of adherence and condensed cytoplasm were evident in dying REtsAF cells. Further fluorescence microscopy examination revealed chromatin condensed in lumps (stage I chromatin condensation), and rounded cells without microvilli or protu-sions on the surface (Figure 1c). However, specific events of the final degradation phase such as the nucleus and cell fragmentation do not occur whereas cells completely detached from the substratum. These observations indicate that p53 can promote two cell death pathways showing apoptotic features, one that is caspase-dependent and another that is independent of caspases. Recent data suggest that noncaspase proteases including calpains, cathepsins or serine proteases can also mediate cell death and bring about many of the morphological changes characteristic of apoptosis in a caspase-independent manner. 8 We investigated the possible involvement of these proteases in the novel route by which p53 signal cell death through the use of specific inhibitors: serine protease inhibitors (TPCK, TLCK), calpain protease inhibitors (ALLN, MDL), cysteine protease inhibitors (Lactacystine, Z-FA-fmk). Microscopic studies of cell morphology did not show notable differences in the amount of Z-VAD-dependent cellular death whether the cells were cultured in the presence or in the absence of the drugs. These results suggest that none of the tested proteases are involved in p53-induced apoptosis of REtsAF cells in the presence of ZVAD. Inasmuch as the mitochondrial pathway plays a central role in many models of p53-dependent apoptosis, we investigate