[show abstract][hide abstract] ABSTRACT: We previously showed that in quiescent cells, p300/CBP (CREB-binding protein)family coactivators repress c-myc and prevent premature induction of DNA synthesis. p300/CBP-depleted cells exit G(1) early and continue to accumulate in S phase but do not progress into G(2)/M, and eventually they die of apoptosis. Here, we show that the S-phase arrest in these cells is because of an intra-S-phase block. The inappropriate DNA synthesis that occurs as a result of forced expression of c-myc leads to the activation of the DNA damage response as evidenced by the phosphorylation of several checkpoint related proteins and the formation of foci containing gamma-H2AX. The activation of checkpoint response is related to the induction of c-myc, as the phosphorylation of checkpoint proteins can be reversed when cells are treated with a c-Myc inhibitor or when Myc synthesis is blocked by short hairpin RNA. Using the DNA fiber assay, we show that in p300-depleted cells initiation of replication occurs from multiple replication origins. Chromatin loading of the Cdc45 protein also indicates increased origin activity in p300 knockdown cells. Immunofluorescence experiments indicate that c-Myc colocalizes with replication foci, consistent with the recently reported direct role of c-Myc in the initiation of DNA synthesis. Thus, the inappropriate S-phase entry of p300 down-regulated cells is likely to be because of c-Myc-induced deregulated replication origin activity, which results in replicative stress, activation of a DNA damage response, and S-phase arrest. Our results point to an important role for p300 in maintaining genomic integrity by negatively regulating c-myc.
Journal of Biological Chemistry 04/2009; 284(22):15193-205. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Previously we showed that the E1A binding proteins p300 and CBP negatively regulate c-Myc in quiescent cells and that binding of E1A to p300 results in the induction of c-Myc and thereby induction of S phase. We demonstrated that p300 and HDAC3 cooperate with the transcription factor YY1 at an upstream YY1 binding site and repress the Myc promoter. Here we show that the small E1A protein induces c-Myc by interfering with the protein-protein interaction between p300, YY1, and HDAC3. Wild-type E1A but not the E1A mutants that do not bind to p300 interfered in recruitment of YY1, p300, and HDAC3 to the YY1 binding site. As E1A started to accumulate after infection, it transiently associated with promoter-bound p300. Subsequently, YY1, p300, and HDAC3 began to dissociate from the promoter. Later in infection, E1A dissociated from the promoter as well as p300, YY1, and HDAC3. Removal of HDAC3 from the promoter correlated with increased acetylation of Myc chromatin and induction. In vivo E1A stably associated with p300 and dissociated YY1 and HDAC3 from the trimolecular complex. In vitro protein-protein interaction studies indicated that E1A initially binds to the p300-YY1-HDAC3 complex, briefly associates with it, and then dissociates the complex, recapitulating somewhat the in vivo situation. Thus, E1A binding to the C-terminal region of p300 disrupts the important corepressor function provided by p300 in repressing c-Myc. Our results reveal a novel mechanism by which a viral oncoprotein activates c-Myc in quiescent cells and raise the possibility that the oncoproteins encoded by the small-DNA tumor viruses may use this mechanism to induce c-Myc, which may be critical for cell transformation.
Journal of Virology 04/2009; 83(10):4810-22. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: A mutant Aspergillus carbonarius selected for temperature tolerance after UV treatment, when grown in shake flasks, produced mycelia bearing yellow pigment. Since the mutant was affected in sterol biosynthetic pathway, the pigment was apparently produced to maintain membrane fluidity and rigidity for growth sustenance in low-pH culture broth. Nuclear magnetic resonance analyses characterizing the pigment as a partially saturated canthaxanthin, containing beta-ionone end rings, suggested its application as a retinoid. When tested for this property in retinoic acid receptor expressing prostate cancer cell line, LNCaP, the fungal partially saturated canthaxanthin induced apoptosis. Low apoptosis percentage in DU145 prostrate cancer cells that does not express functional retinoic acid receptor-beta (RAR-beta) suggested binding specificity of the partially saturated canthaxanthin for RAR-beta.
Applied Microbiology and Biotechnology 07/2008; 80(3):467-73. · 3.69 Impact Factor