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: As part of an effort to define novel factors important for hPer2 regulation, we used a bacterial two-hybrid system to screen a human liver cDNA library to search for interacting partners. We chose this library because liver is known to be a peripheral oscillator tissue, and there are comprehensive studies on how circadian components are interlocked and operate in liver tissue (Lamia et al., 2008). Three baits were independently used for screening: full-length hPer2 and two fragments of cDNA encoding the N-terminal (residues 1–821) and C-terminal (residues 822–1255) regions of hPer2. These regions were chosen because of their relevance to Per2 function in various cellular processes and the presence of functional and structural domains known to bind protein counterparts (Griffin et al., 1999; Kume et al., 1999) or small ligand molecules (Yang et al., 2008). The human liver cDNA library (primary size, 6.9 × 106) was screened with the generated pBT recombinant plasmids. Approximately 4 × 106 clones were screened, and 120 were identified as putative positive interactors (67 strong and 53 weak interactors). These clones were maintained in nonselective media containing antibiotics and later patched on selective screening medium containing 5 mM 3-amino-1,2,4-triazole (3-AT) (Figure 1A). All putative clones were then subjected to further screening on a dual selective screening medium (5 mM 3-AT and streptomycin) and confirmed positive. To validate protein–protein interactions, we performed retransformation of the reporter strain using the pTRG-positive clones and recombinant pBT baits. Our results show that 17 clones, which include proteins involved in cellular metabolic processes, RNA binding, regulation of programmed cell death, transcriptional activation, response to stress, and cell cycle progression, reproducibly grow on selective screening medium when cotransformed with the bait plasmid but failed to grow under the same conditions when cotransformed with the empty pBT vector. Among the clones identified in the screening were hp53, the translationally controlled tumor protein (TCTP), and various fragments encoding open reading frame regions of the circadian factor crytochrome (Cry), a known direct interactor of hPer2 (Figure 1A). Remarkably, hp53 was also identified as a positive interactor when screenings were carried out using the sequences encompassing the N- and C-terminal fragments of hPer2, suggesting that more than one interaction site for hp53 might exist within the circadian factor.
Affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.