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The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells.

Gotoh T, Vila-Caballer M, Santos CS, Liu J, Yang J, Finkielstein CV - Mol. Biol. Cell (2014)

Bottom Line: We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53's negative regulator, Mdm2.Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes.Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

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The hPer2 protein influences the expression of hp53 target genes. (A) HCT116 cells were transfected with either FLAG-hPer2 or siRNA hPer2 and collected at 24 and 48 h posttransfection, respectively. Empty vector (EV) and mock samples were controls. qRT-PCR data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were done by two-tailed unpaired t test and analyses performed using SPSS. NS, not significant; ##p ≤ 0.01. (B) HCT116 cell extracts (20 μg) from the various samples in A were analyzed for hPer2 (top), endogenous hp53 (middle), and tubulin (bottom) by immunoblotting. Bands were quantified using ImageJ, version 1.45, and values normalized to tubulin levels (loading control). Bar graphs indicate the percentage of the remaining hp53 protein. Immunoblot data from a single experiment that was repeated three times with similar results. (C) H1299 cells were transfected with pCS2+FLAG-hPer2, pCS2+FLAG-hp53, empty vector (EV), or a combination of plasmids. Cells were harvested 24 h after transfection and samples prepared for qRT-PCR. Aliquots of cell extracts were analyzed by immunoblotting (right). Asterisks indicate nonspecific signal. Data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were evaluated by ANOVA using Bonferroni or Games–Howell post hoc analyses when needed (SPSS). NS, not significant; #p ≤ 0.05; ##p ≤ 0.01. SFN encodes 14-3-3σ; BAX encodes the proapoptotic factor Bax; MYC encodes the oncogene c-myc; CDKN1a encodes hp21WAF1/CIP1; GADD45α encodes the gadd45α protein.
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Figure 4: The hPer2 protein influences the expression of hp53 target genes. (A) HCT116 cells were transfected with either FLAG-hPer2 or siRNA hPer2 and collected at 24 and 48 h posttransfection, respectively. Empty vector (EV) and mock samples were controls. qRT-PCR data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were done by two-tailed unpaired t test and analyses performed using SPSS. NS, not significant; ##p ≤ 0.01. (B) HCT116 cell extracts (20 μg) from the various samples in A were analyzed for hPer2 (top), endogenous hp53 (middle), and tubulin (bottom) by immunoblotting. Bands were quantified using ImageJ, version 1.45, and values normalized to tubulin levels (loading control). Bar graphs indicate the percentage of the remaining hp53 protein. Immunoblot data from a single experiment that was repeated three times with similar results. (C) H1299 cells were transfected with pCS2+FLAG-hPer2, pCS2+FLAG-hp53, empty vector (EV), or a combination of plasmids. Cells were harvested 24 h after transfection and samples prepared for qRT-PCR. Aliquots of cell extracts were analyzed by immunoblotting (right). Asterisks indicate nonspecific signal. Data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were evaluated by ANOVA using Bonferroni or Games–Howell post hoc analyses when needed (SPSS). NS, not significant; #p ≤ 0.05; ##p ≤ 0.01. SFN encodes 14-3-3σ; BAX encodes the proapoptotic factor Bax; MYC encodes the oncogene c-myc; CDKN1a encodes hp21WAF1/CIP1; GADD45α encodes the gadd45α protein.

Mentions: We then asked whether increased stability of hp53 as a result of hPer2 transfection (Supplemental Figure S3) affects activation of hp53-mediated gene transcription, thus functionally linking hPer2 to the hp53 pathway. First, we monitored the expression of p53-targeted genes in HCT116 cells that were either overexpressing hPer2 (Figure 4A, left, for each set of genes) or silenced for hPer2 expression (Figure 4A, right, for each set of genes). We chose to analyze the expression of those genes because they represent the diversity of cellular pathways controlled by p53 and the various forms of regulation, ranging from transcriptional repression (i.e., MYC) to activation (i.e., SFN [encodes 14-3-3σ ], BAX [encodes Bcl-2–associated X protein (Bax)], CDKN1a [encodes cyclin-dependent kinase inhibitor human p21 (hp21WAF1/CIP1)], and GADD45α [encodes the growth arrest and DNA-damage-inducible protein 45 α (Gadd45α)]). Accordingly, hPer2-transfected cells showed a significant increase in expression of CDKN1a, SFN, GADD45α, and the proapoptotic component BAX, whereas the expression of the MYC oncogene remained invariable (Figure 4A, left, for each set of genes). Specificity of response toward hPer2 was assessed by effectively abrogating its expression using siRNA in HCT116 and monitoring gene transcription in real time (Figure 4A, right, for each set of genes). As shown, down-regulation of hPer2 counteracts the effect of the transcription of p53-target genes triggered by hPer2 overexpression, supporting a role for this circadian modulator in their transcriptional control. Consistent with our previous observations, increased hp53 levels were solely observed in hPer2-transfected samples (Figure 4B), supporting a model in which hPer2 action on hp53-mediated transcription might be the result of its stabilization.


The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells.

Gotoh T, Vila-Caballer M, Santos CS, Liu J, Yang J, Finkielstein CV - Mol. Biol. Cell (2014)

The hPer2 protein influences the expression of hp53 target genes. (A) HCT116 cells were transfected with either FLAG-hPer2 or siRNA hPer2 and collected at 24 and 48 h posttransfection, respectively. Empty vector (EV) and mock samples were controls. qRT-PCR data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were done by two-tailed unpaired t test and analyses performed using SPSS. NS, not significant; ##p ≤ 0.01. (B) HCT116 cell extracts (20 μg) from the various samples in A were analyzed for hPer2 (top), endogenous hp53 (middle), and tubulin (bottom) by immunoblotting. Bands were quantified using ImageJ, version 1.45, and values normalized to tubulin levels (loading control). Bar graphs indicate the percentage of the remaining hp53 protein. Immunoblot data from a single experiment that was repeated three times with similar results. (C) H1299 cells were transfected with pCS2+FLAG-hPer2, pCS2+FLAG-hp53, empty vector (EV), or a combination of plasmids. Cells were harvested 24 h after transfection and samples prepared for qRT-PCR. Aliquots of cell extracts were analyzed by immunoblotting (right). Asterisks indicate nonspecific signal. Data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were evaluated by ANOVA using Bonferroni or Games–Howell post hoc analyses when needed (SPSS). NS, not significant; #p ≤ 0.05; ##p ≤ 0.01. SFN encodes 14-3-3σ; BAX encodes the proapoptotic factor Bax; MYC encodes the oncogene c-myc; CDKN1a encodes hp21WAF1/CIP1; GADD45α encodes the gadd45α protein.
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Figure 4: The hPer2 protein influences the expression of hp53 target genes. (A) HCT116 cells were transfected with either FLAG-hPer2 or siRNA hPer2 and collected at 24 and 48 h posttransfection, respectively. Empty vector (EV) and mock samples were controls. qRT-PCR data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were done by two-tailed unpaired t test and analyses performed using SPSS. NS, not significant; ##p ≤ 0.01. (B) HCT116 cell extracts (20 μg) from the various samples in A were analyzed for hPer2 (top), endogenous hp53 (middle), and tubulin (bottom) by immunoblotting. Bands were quantified using ImageJ, version 1.45, and values normalized to tubulin levels (loading control). Bar graphs indicate the percentage of the remaining hp53 protein. Immunoblot data from a single experiment that was repeated three times with similar results. (C) H1299 cells were transfected with pCS2+FLAG-hPer2, pCS2+FLAG-hp53, empty vector (EV), or a combination of plasmids. Cells were harvested 24 h after transfection and samples prepared for qRT-PCR. Aliquots of cell extracts were analyzed by immunoblotting (right). Asterisks indicate nonspecific signal. Data are presented as the mean ± SEM from three independent experiments performed in triplicate. Statistical comparisons were evaluated by ANOVA using Bonferroni or Games–Howell post hoc analyses when needed (SPSS). NS, not significant; #p ≤ 0.05; ##p ≤ 0.01. SFN encodes 14-3-3σ; BAX encodes the proapoptotic factor Bax; MYC encodes the oncogene c-myc; CDKN1a encodes hp21WAF1/CIP1; GADD45α encodes the gadd45α protein.
Mentions: We then asked whether increased stability of hp53 as a result of hPer2 transfection (Supplemental Figure S3) affects activation of hp53-mediated gene transcription, thus functionally linking hPer2 to the hp53 pathway. First, we monitored the expression of p53-targeted genes in HCT116 cells that were either overexpressing hPer2 (Figure 4A, left, for each set of genes) or silenced for hPer2 expression (Figure 4A, right, for each set of genes). We chose to analyze the expression of those genes because they represent the diversity of cellular pathways controlled by p53 and the various forms of regulation, ranging from transcriptional repression (i.e., MYC) to activation (i.e., SFN [encodes 14-3-3σ ], BAX [encodes Bcl-2–associated X protein (Bax)], CDKN1a [encodes cyclin-dependent kinase inhibitor human p21 (hp21WAF1/CIP1)], and GADD45α [encodes the growth arrest and DNA-damage-inducible protein 45 α (Gadd45α)]). Accordingly, hPer2-transfected cells showed a significant increase in expression of CDKN1a, SFN, GADD45α, and the proapoptotic component BAX, whereas the expression of the MYC oncogene remained invariable (Figure 4A, left, for each set of genes). Specificity of response toward hPer2 was assessed by effectively abrogating its expression using siRNA in HCT116 and monitoring gene transcription in real time (Figure 4A, right, for each set of genes). As shown, down-regulation of hPer2 counteracts the effect of the transcription of p53-target genes triggered by hPer2 overexpression, supporting a role for this circadian modulator in their transcriptional control. Consistent with our previous observations, increased hp53 levels were solely observed in hPer2-transfected samples (Figure 4B), supporting a model in which hPer2 action on hp53-mediated transcription might be the result of its stabilization.

Bottom Line: We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53's negative regulator, Mdm2.Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes.Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

Show MeSH
Related in: MedlinePlus