p53 requires the stress sensor USF1 to direct appropriate cell fate decision

Genomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1-deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway

Cell cycle arrest regulation in response to UV exposure : implications of USF1 transcription factor in the control of p53 availability

L'exposition aux ultraviolets solaires constitue un facteur de risque majeur dans le développement de cancers cutanés. L'initiation de ces cancers est cependant pondérée par des mécanismes cellulaires de protection qui contrecarrent l'instabilité génomique éventuellement promue par les UV. Dans ces mécanismes, le suppresseur de tumeur p53 joue un rôle fondamental en régulant l'expression de nombreux gènes permettant de bloquer le cycle cellulaire et de réparer l'ADN ou, si les dommages cellulaires sont trop importants, d'activer l'apoptose. Les régulateurs de la stabilité de la protéine p53 en réponse au stress UV sont ainsi capitaux pour assurer la stabilité du génome. En réponse au stress UV in vivo et in vitro, nous mettons en évidence que le facteur de transcription USF1 est primordial à l'activation du programme génétique contrôlé par la protéine p53. Nos données convergent vers un modèle dans lequel USF1 agit sur la voie p53 par deux moyens. D'une part, USF1, assure par interaction physique la stabilité de p53 en contrecarrant de manière mutuellement exclusive l'association du suppresseur de tumeur à MDM2 son inhibiteur physiologique. D'autre part, USF1 agit synergiquement avec le suppresseur de tumeur pour transcrire certains gènes cibles de p53 comme le régulateur du cycle cellulaire CDKN1A (p21). Ces deux niveaux de régulation dépendent étroitement du niveau de stress et permettent d'assurer un contrôle optimal de l'arrêt du cycle cellulaire en réponse à l'exposition UV. Collectivement, nos données montrent qu'USF1, par le contrôle de la voie p53, est un facteur essentiel contre l'instabilité génomique induite par les UV.Ultraviolets (UV) solar exposure is a critical risk factor in skin cancers development. Initiation of these cancers is however lowered by cellular protective mechanisms that counteract the genomic instability potentially promoted by UV. In these mechanisms p53 protein is critical in regulating a large number of genes that blocks the cell cycle to allow DNA repair or, if damages are beyond repair, to activate apoptosis. The regulators of p53 stability in response to UV are thus crucial to ensure genomic stability. In response to UV stress, we found by in vivo and in vitro studies that USF1 is essential in the activation of p53 genetic program. Our data converge to a model whereby USF1 acts on p53 pathway by two means. On one hand, USF1 stabilizes p53 from MDM2 mediated degradation by a physical association to the tumor suppressor in a MDM2 mutually exclusive manner. On the other hand USF1 synergizes with the tumor suppressor in the transcription of several targets of p53 protein and particularly the CDKN1A inhibitor of the cell cycle. These two levels of regulation are closely dependent in the stress level and ensure an optimal control of the cell cycle progression in response to UV. Collectively, our results show that USF1, by controlling p53 pathway, is a critical factor against the genomic instability promoted by UV

Régulation de l’arrêt du cycle cellulaire en réponse à l’exposition UV : implications du facteur de transcription USF1 dans le contrôle de la disponibilité de la protéine p53

Ultraviolets (UV) solar exposure is a critical risk factor in skin cancers development. Initiation of these cancers is however lowered by cellular protective mechanisms that counteract the genomic instability potentially promoted by UV. In these mechanisms p53 protein is critical in regulating a large number of genes that blocks the cell cycle to allow DNA repair or, if damages are beyond repair, to activate apoptosis. The regulators of p53 stability in response to UV are thus crucial to ensure genomic stability. In response to UV stress, we found by in vivo and in vitro studies that USF1 is essential in the activation of p53 genetic program. Our data converge to a model whereby USF1 acts on p53 pathway by two means. On one hand, USF1 stabilizes p53 from MDM2 mediated degradation by a physical association to the tumor suppressor in a MDM2 mutually exclusive manner. On the other hand USF1 synergizes with the tumor suppressor in the transcription of several targets of p53 protein and particularly the CDKN1A inhibitor of the cell cycle. These two levels of regulation are closely dependent in the stress level and ensure an optimal control of the cell cycle progression in response to UV. Collectively, our results show that USF1, by controlling p53 pathway, is a critical factor against the genomic instability promoted by UV.L'exposition aux ultraviolets solaires constitue un facteur de risque majeur dans le développement de cancers cutanés. L'initiation de ces cancers est cependant pondérée par des mécanismes cellulaires de protection qui contrecarrent l'instabilité génomique éventuellement promue par les UV. Dans ces mécanismes, le suppresseur de tumeur p53 joue un rôle fondamental en régulant l'expression de nombreux gènes permettant de bloquer le cycle cellulaire et de réparer l'ADN ou, si les dommages cellulaires sont trop importants, d'activer l'apoptose. Les régulateurs de la stabilité de la protéine p53 en réponse au stress UV sont ainsi capitaux pour assurer la stabilité du génome. En réponse au stress UV in vivo et in vitro, nous mettons en évidence que le facteur de transcription USF1 est primordial à l'activation du programme génétique contrôlé par la protéine p53. Nos données convergent vers un modèle dans lequel USF1 agit sur la voie p53 par deux moyens. D'une part, USF1, assure par interaction physique la stabilité de p53 en contrecarrant de manière mutuellement exclusive l'association du suppresseur de tumeur à MDM2 son inhibiteur physiologique. D'autre part, USF1 agit synergiquement avec le suppresseur de tumeur pour transcrire certains gènes cibles de p53 comme le régulateur du cycle cellulaire CDKN1A (p21). Ces deux niveaux de régulation dépendent étroitement du niveau de stress et permettent d'assurer un contrôle optimal de l'arrêt du cycle cellulaire en réponse à l'exposition UV. Collectivement, nos données montrent qu'USF1, par le contrôle de la voie p53, est un facteur essentiel contre l'instabilité génomique induite par les UV

USF1 counteracts MDM2-mediated p53 degradation upon cellular stress.

<p>p53 protein-protein interactions and MDM2 mediated p53 degradation were studied in B16 melanoma cells knocked down for <i>Usf1</i> (sh-<i>Usf1</i>) and their controls (sh-<i>CT</i>). (A–B) sh-<i>CT</i> and sh-<i>Usf1</i> cells were treated with 10 µM MG132 for 3 hours then irradiated or not irradiated with UVB. Western blot analysis of proteins immunoprecipitated from cell lysates (A) Immunoprecipitation analysis to assay ubiquitinated flag-tagged p53 after transfection of sh-<i>CT</i> or sh-<i>Usf1</i> with the corresponding cDNA. Cells were treated with MG132, were or were not irradiated with UVB and analyzed 3 hours later. The values reported indicate the level of p53 ubiquitination (normalized to the total amount of flag-tagged protein recovered). p53 expressing sh-<i>Usf1</i> cells treated with MG132 has been arbitrary chosen as the reference (100%) since it is the condition where normalized-level of p53-ubiquitinated protein is the highest. (B) sh-<i>CT</i> and sh-<i>Usf1</i> cells were treated with 10 µM MG132 for 3 hours then irradiated or not irradiated with UVB. Western blot analysis of proteins immunoprecipitated from cell lysates with a mix of two MDM2 antibodies (SMP14 and 3G9) and blotted with p53 antibody (1C12). (C) Western blot analysis showing basal levels of USF1, MDM2 and HSC70 (loading control) in sh-<i>CT</i> and sh-<i>Usf1</i> cell lysates. (D) Western blot showing the effect of Nutlin-3 (10 µM, 6 h) treatment on the levels of flag-tagged p53 and GFP proteins in sh-<i>CT</i> and sh-<i>Usf1</i> cells; antibodies to USF1, p53, GFP and HSC70 (loading control) were used. (E) Western blot analysis of p53, MDM2 and HSC70 (loading control) in sh-<i>CT</i> and sh-<i>Usf1</i> cells over-expressing either p53 or p53 plus MDM2. (F) Same experiment as in D but in sh-<i>Usf1</i> KD cells over-expressing either GFP or USF1. (G) Immunofluorescence analysis of p53 expression and localization in sh-<i>Usf1</i> KD cells treated as in D and stimulated with vehicle (DMSO) or Nutlin-3 (10 µM) for 6 hours. Experiments have been done in triplicate and 15 to 20 microscopic fields analyzed per condition. (H) Quantification of the level of p53 and USF1 interaction in B16 melanoma cells using Thermo Scientific Cellomics HCS Solution (fluorescent microscopy) using Duolink PLA technology. Quantification of p53-USF1 interaction level using specific primary antibodies and Duolink PLA technology in B16 melanoma sh-<i>CT</i> cells over-expressing either p53 or p53 plus MDM2 (left panel). The graph represents the cumulative level of fluorescence observed in B16 cells under specific spotted form. p53 plus GFP is used as control condition. Quantification of p53-USF1 interaction level in B16 melanoma sh-<i>Usf1</i> cells over-expressing p53 plus MDM2 and or not different forms of USF1 (wild type or negative dominant (AUSF)) (right panel). p53 plus MDM2 is used as control condition.</p

<i>Usf1</i> KO mice present defective induction of p53 protein.

<p>The back of <i>Usf1</i> KO mice (<i>Usf1^-/-</i>) and WT mice (<i>Usf1^+/+</i>) were irradiated or not irradiated with an UVB dose corresponding to the mice MED (5 kJ/m²) and the skin was analyzed 5 h later. (A) RT-qPCR analysis of <i>Trp53</i> and <i>Usf1</i> mRNA relative level (expressed as a ratio to the value for the <i>Hprt</i> transcript) in skin extracts from protected (-) and UV-exposed (+) areas. Error bars: SD, n>9. (B) Western blot showing USF1, p53, γH2AX and HSC70 (loading control) immunoreactivity 5 h after skin irradiated or not irradiated with UVB. The graph reports the mean ratio between the p53 signal (normalized to that for HSC70) in skin-exposed areas versus non-irradiated areas (controls). Error bars: SD, n = 8 for each condition. (C) <i>Usf1^+/+</i> (Usf1 WT) and <i>Usf1^-/-</i> (<i>Usf1</i> KO) skins were or were not irradiated with UVB (5 kJ/m²) and analyzed for the induction of transcripts <i>in vivo.</i> RT-qPCR analysis of <i>CDKN1a</i> (p21), <i>SFN</i> (14-3-3σ) and <i>PCNA</i> transcripts in UVB-irradiated skin and non-exposed controls; values reported were normalized to those for the <i>Hprt</i> transcript. Transcripts were assayed <i>in vivo</i> 5 hours after irradiation. Error bars: SD, n = 4 <i>in vivo</i> (D) Immunohistochemical labeling of cyclobutane pyrimidine dimers (CPD) showing their localization and abundance in skin areas (x100) exposed or not exposed to UVB. Dashed lines indicate the boundary between the dermis (d) and the epidermis (e), and arrows indicate positive nuclei. (E) The level of CPDs in total DNA extracts from skin was quantified by ELISA. The graph shows the mean difference in the CPD absorbance values between for exposed and protected skin areas. Error bars: SD, n = 4. (F) Immunofluorescence staining with the Ki-67 antibody of inter-follicular cycling cells in skin areas (x100) exposed or not exposed to UVB. (G) The graph shows the mean percentage of cycling cells (calculated as Ki-67-positive cells/total Dapi-stained cells) in protected and UV-exposed skin areas. Error bars: SD, n = 3. Student's <i>t</i> test was used to test the significance of differences (*, <i>p</i> <0.05, **, p<0.01, ***, p<0.001).</p

Model of regulation of p53 stabilization by USF1 in response to stress.

<p>USF1 prevents MDM2-mediated p53 degradation under stress conditions, thereby ensuring the stability and tumor suppressor activity of the p53 protein. Left Panel, in the absence of stress, p53 is targeted to proteasomal degradation after binding to MDM2, maintaining cell proliferation. Right Panel, under DNA-damage context, USF1 counteracts MDM2 function by interacting with p53 thereby increasing its transcriptional activity to control transient cell cycle arrest and DNA repair processes. In the absence of USF1, p53 stabilization is abolished abrogating cell cycle control in response to DNA damage and thereby favoring genomic instability.</p

USF1 is required to stabilize p53 protein following genotoxic stress.

<p>B16 melanoma cells knocked down for <i>Usf1</i> (sh-<i>Usf1</i>) and their controls (sh-<i>CT</i>) were analyzed for post-translational regulation of p53. (A) Western blot analysis of the effect of USF1 re-expression on p53 protein levels in sh-<i>Usf1</i> cells irradiated or not irradiated with UVB and tested 6 h after irradiation. Cells were transfected with the cDNA indicated (as described in the materials and methods) and analyzed for USF1, p53 and HSC70 (loading control). (B) Western blot showing USF1, p53 and HSC70 immunoreactivity in sh-<i>CT</i> and sh-<i>Usf1</i> cells at the indicated time following treatment with MG132 (10 µM). (C–D) Time course of p53 accumulation and Ser15-phosphorylation in sh-<i>CT</i> and sh-<i>Usf1</i> cells treated with vehicle (DMSO) in C or MG132 (10 µM) plus UVB (0.3 kJ/m²) irradiation in D. (E–F) p53 degradation in sh-<i>CT</i> and sh-<i>Usf1</i> cells pretreated for 3 h with MG132 (10 µM) and then with cycloheximide (CHX 20 µM) (E). Cells were analyzed at the time points indicated after addition of CHX. The graphs show the results of densitometric analysis of p53 immunoreactive bands (normalized to the loading controls H2AX or HSC70). (G) Western blot showing flag-tagged p53 and GFP proteins in sh-<i>CT</i> or sh-<i>Usf1</i> cells after co-transfection of the corresponding cDNA. (H, upper panel) Immunoprecipitation analysis to assay flag-tagged p53 after transfection of sh-<i>CT</i> or sh-<i>Usf1</i> with the corresponding cDNA. Cells were treated with MG132, were or were not irradiated with UVB and analyzed 3 hours later. (H, lower panel) sh-<i>CT</i> and sh-<i>Usf1</i> cells were treated with 10 µM MG132 for 3 hours then irradiated or not irradiated with UVB. Western blot analysis of proteins immunoprecipitated from cell lysates with USF1 antibody and blotted with p53 (1C12) and USF1 antibodies.</p

Loss of ARHGEF1 causes a human primary antibody deficiency

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Mutations in the adaptor-binding domain and associated linker region of p110δ cause Activated PI3K-δ Syndrome 1 (APDS1)

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