Jongkyeong Chung

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We have reported previously that inducible expression of the p53 gene leads to the cell cycle arrest both at G(1) and G(2)/M in association with induction of p21 and reduction of mitotic cyclins (cyclin A and B) and cdc2 mRNA. In this study, we investigated the mechanism by which p53 regulates transcription of the cdc2 gene. Transient transfection analysis showed that wild type p53 represses whereas various dominant negative mutants of p53 increase cdc2 transcription. The cdc2 promoter activity is not repressed in cells transfected with a transactivation mutant, p53(22/23). An adenovirus oncoprotein, E1B-55K inhibits the p53-mediated repression of the cdc2 promoter, while E1B-19K does not. Since the cdc2 promoter does not contain a TATA sequence, we performed deletion and point mutation analyses and identified the inverted CCAAT sequence located at -76 as a cis-acting element for the p53-mediated regulation. We found that a specific DNA-protein complex is formed at the CCAAT sequence and that this complex contains the NF-Y transcription factor. Consistently, a dominant negative mutant of the NF-YA subunit, NF-YAm29, decreases the cdc2 promoter, and p53 does not further decrease the promoter activity in the presence of NF-YAm29. These results suggest that p53 negatively regulates cdc2 transcription and that the NF-Y transcription factor is required for the p53-mediated regulation.

CRY, PDF and Pyx pathways are required for the near-zero GMF-induced negative geotaxis. (A, C, E) Comparisons of the geotactic positioning of the CRY-, PDF- and Pyx-deficient flies rescued with their coding genes (cry-GAL4/UAS-cry;cry 01 , pdf-GAL4/UAS-pdf;pdf 01 and pyx-GAL4/UAS-pyx;pyx 3 ), respectively. Error bars: SEM. n.s., not significant. **, P < 0.01; ***, P < 0.005 by Student’s t-test. (B, D, F) Comparisons of the geotactic positioning of the flies bearing the GAL4 transgenes only (cry-GAL4/+, pdf-GAL4/+ and pyx-GAL4/+) and the UAS-RNAi transgene only (UAS-cry RNAi/+, UAS-pdf RNAi/+ and UAS-pyx RNAi/+), and the flies with the knockdown of cry, pdf and pyx transcripts (cry-GAL4/UAS-cry RNAi, pdf-GAL4/UAS-pdf RNAi and pyx-GAL4/UAS-pyx RNAi), respectively. Error bars: SEM. n.s., not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.005 by Student’s t-test. For all the data, n = 10 trials. (PDF 19 kb)

CRY and Pyx function in JO for the near-zero GMF-induced negative geotaxis. (A, C, E) Comparisons of the geotactic positioning of CRY-, Pyx-, and PDF-deficient flies in which the expression of CRY, Pyx, and PDF was genetically restored in JO using nan-GAL4, a JO-specific GAL4 driver. Error bars: SEM. n.s., not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.005, ****, P < 0.001 by Student’s t-test. (B, D, F) Comparisons of the geotactic positioning of CRY-, Pyx-, and PDF-deficient flies in which the expression of CRY, Pyx, and PDF was genetically restored using another JO-specific GAL4 driver, iav-GAL4. Error bars: SEM. n.s., not significant. **, P < 0.01; ***, P < 0.005 by Student’s t-test. (G, H) Comparisons of the geotactic positioning by wild-type flies, control flies (GAL4 driver alone, UAS-pdf RNAi alone), and the flies with RNAi knockdown of pdf using cry-GAL4 or pyx-GAL4 driver. Error bars: SEM. n.s.: not significant. **, P < 0.01; ***, P < 0.005; ****, P < 0.001 by Student’s t-test. For all the data, n = 10 trials. (PDF 122 kb)

Near-zero GMF-induced geotactic response under different ZTs and the comparison of vertical choice vs. horizontal choice Y-maze assays. (A) Photos of the test cube (180 × 100 × 140 mm, length × width × height) used for the tube-positioning assay. (B) Comparisons of the geotactic positioning of wild-type flies under the sham and cancellation conditions during ZT0 to ZT2 (n = 10 trials). Error bars: SEM. *, P < 0.05 by Student’s t-test. (C) Photos of the double-layered permalloy (0.5 mm thick) cube (180 × 100 × 140 mm, length × width × height) used for the attenuation of GMF by passive cancellation. (D) Comparisons of the geotactic positioning of wild-type flies under the sham and passive cancellation conditions during ZT5 to ZT8 (n = 10 trials). Error bars: SEM. *, P < 0.05 by Student’s t-test. (E) Comparisons of the exit profiles of wild-type flies making vertical choice versus horizontal choice in the Y-maze assays under the sham condition (n = 12 trials). Error bars: SEM. *, P < 0.05; **, P < 0.01 by Student’s t-test. (PDF 81 kb)