The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells.
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.
Affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.Show MeSH
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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.
Affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.