Drosophila p53 tumor suppressor directly activates conserved asymmetric stem cell division regulators

Abstract

p53 is the most mutated tumor suppressor gene in human cancers. Besides p53 classical functions inducing cell-cycle arrest and apoptosis in stressed cells, additional p53 non-canonical roles in unstressed cells have emerged over the past years, including the mode of stem cell division regulation. However, the mechanisms by which p53 impacts on this process remain elusive. Here, we show that Drosophila p53 controls asymmetric stem cell division (ASCD), a key process in development, cancer and adult tissue homeostasis, by transcriptionally activating Numb, Brat, and Traf4 ASCD regulators. p53 knockout caused failures in their localization in dividing neural stem cells, as well as a significant decrease in their expression levels. Moreover, p53 directly bound numb, brat, and Traf4 regulatory regions. Remarkably, human and mice genes related to Drosophila brat (TRIM32) and Traf4 (TRAF4) were recently identified in a meta-analysis of transcriptomic and ChIP-seq datasets as predicted conserved p53 targets.

Keywords: Biological sciences; Cell biology; Molecular biology; Stem cells research.

Graphical abstract

Figure 1

p53 is required for proper neuronal lineage formation (A) NB asymmetric division is regulated by an "apical complex" and cell-fate determinants that localize asymmetrically at the apical and basal poles, respectively, of metaphase NBs. NBI asymmetric division renders another NB and a GMC, which receives the determinants and stops self-renewing. The GMC through a terminal asymmetric division generates two different neurons or glial cells: (A) apical, (B) basal. (B) p53 homozygous null mutant viability strongly decays throughout Drosophila life cycle (upper diagram). A significant number of p53 homozygous embryos do not hatch compared to control embryos; no significant (ns) changes in the survival of p53 mutants are observed from L1 to L2 larvae; very significant decay in the survival of p53 mutants is observed again since L3 to the adult eclosion compared to the control. Data are represented as mean ± SD (standard deviation); n = 2 independent experiments (∗∗∗p < 0.001). (C) Ventral views of late stage Drosophila embryos, control, and p53^E8 homozygous null mutants, showing different hemisegments (hs) at each side of the ventral midline (vm). In control embryos, the transcription factor Eve is expressed in a subset of neurons, including one RP2 neuron per hs (blue arrows); in p53^E8 mutants, a significant number (∗∗∗p < 0.001; ∗∗p < 0.01 in the bar graphs) of RP2 duplications (blue arrows in the picture) or losses (blue asterisks) are detected. A diagram of the GMC-1 neuronal lineage is represented. n = number of total hemisegments (hs) or embryos. Scale bar: 20 μm.

Figure 2

Drosophila p53 impacts the localization of the ASCD regulators Numb and Brat in dividing NBs (A) Confocal immunofluorescences showing an embryonic metaphase NB in control or p53^E8 homozygous mutants stained with the apical protein aPKC (in red; arrow); mitotic cells are visualized with PH3 (red), centrosomes are labeled with γ-Tub (green), and membranes are marked by Dlg1 (blue). No significant (ns) defects in the apical localization of aPKC are detected in p53^E8 mutants. (B) Confocal immunofluorescences showing an embryonic metaphase NB in control or p53^E8 homozygous mutants stained with the cell fate determinant Numb (red; arrow); mitotic cells are visualized with PH3 (blue), centrosomes are labeled with γ-Tub (light blue), and membranes are marked by Dlg1 (green). Numb localization is significantly altered (∗∗∗p < 0.001 in the bar graph) in p53^E8 homozygotes. (C) Confocal immunofluorescences showing an embryonic metaphase NB in control or p53^E8 homozygous mutants stained with the apical protein Par-6 (in red; arrow); mitotic cells are visualized with PH3 (blue), and centrosomes are labeled with γ-Tub (green). No significant (ns) defects in the apical localization of Par-6 are detected in p53^E8 homozygous mutants. (D) Confocal immunofluorescences showing an embryonic metaphase NB in control or p53^E8 homozygous mutants stained with the cell fate determinant Brat (red; arrow); mitotic cells are visualized with PH3 (red), and centrosomes are labeled with γ-Tub (blue). Brat localization is significantly altered (∗∗∗p < 0.001 in the bar graph) in p53^E8 homozygote mutants. n = number of metaphase NBs analyzed; scale bar: 5 μm. See also Figures S1 and S3.

Figure 3

Drosophila p53 directly activates the ASCD regulators Numb, Brat, and Traf4 (A) RT-qPCRs reveal a significant decrease in the level of expression of the indicated genes in p53^E8 null homozygotes relative to the control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; data are represented as mean ± SD (standard deviation); n indicates the number of experiments (RT-qPCRs) performed for each gene. (B) Visualization of ChIP-seq data using the Integrative Genomics Viewer (IGV) browser, showing the peaks or regions of interest (ROIs) for p53 binding at the genomes of numb, brat, and Traf4 in about 10 kb (region delimited by red dots) from which the transcription starts (see also Figure S2). Selected peaks or ROIs are highlighted by red open rectangles, and magnification of them are shown indicating, in each case, the primers used to validate those regions. Bar graphs show the quantification of the ChIP-qPCR experiments measuring p53 occupancy at the brat, Traf4, and numb REs. Positive control (corresponding to a p53 RE in the promoter, see Figure S2A) and negative control regions (the p53 3′UTR without any p53 REs) were included. Values in the graphs represent the fold enrichment observed using the p53 Ab for the immunoprecipitation with respect to an unspecific immunoglobulin G (IgG). Data are represented as mean ± SD (standard deviation); n = 3 independent experiments. A t test was used (∗p < 0.05). See also Figure S2.

Figure 4

p53 loss does not induce tumor-like overgrowth in larval brain NB lineages (A) The Drosophila larval central brain (cb) contains type I (NBI) and type II (NBII) NBs. L3, third instar larva; ol, optic lobe; vc, ventral cord; m, medial; l, lateral; d, dorsal; v, ventral; iINP, immature INP; mIPN, mature INP. (B) Confocal immunofluorescences showing an NBII lineage. p53 loss does not induce tumorigenesis or even a significant number of ectopic NBs within NBII lineages. Data are represented as medians within the interquartile range (box) and the maximum and minimum values (whiskers); n = number of NB lineages analyzed. A Mann-Whitney test was used (ns, not significant in the boxplots). (C) Confocal immunofluorescences showing an NBII lineage. p53 downregulation partially suppressed the UAS-Ras^V12scrib² ectopic NB phenotype. Data are represented as medians within the interquartile range (box) and the maximum and minimum values (whiskers); n = number of NB lineages analyzed. A Kruskal-Wallis test was used (ns, not significant; ∗∗p < 0.01, ∗∗∗p < 0.001 in the boxplots). Scale bar: 10 μm. See also Figure S3.