Repression of c-Myc and inhibition of G1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity

Abstract

p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two highly homologous, conserved transcriptional coactivators, and histone acetyltransferases (HATs) that link chromatin remodeling with transcription. Cell transformation by viral oncogene products such as adenovirus E1A and SV40 large T antigen depends on their ability to inactivate p300 and CBP. To investigate the role of p300 in cell-cycle progression, we constructed stable rat cell lines, which conditionally overexpress p300 from a tetracycline-responsive promoter. When p300 was induced in these cells, serum-stimulated S-phase entry was significantly inhibited. The inhibition of S-phase induction was associated with down-regulation of c-Myc, but not of c-Fos or c-Jun. Simultaneous overexpression of c-Myc and p300 before serum stimulation reversed the inhibition of S-phase induction to a significant level, indicating that the inhibition of c-Myc to a large extent is responsible for the p300 inhibition of G1 exit. Similar studies with stable rat cell lines that overexpress a mutant p300, which lacks the HAT activity, showed that the intrinsic HAT activity of p300 is not required for the negative regulation of c-Myc or G1. These findings, and our previously published results (Kolli, S., Buchmann, A. M., Williams, J., Weitzman, S. & Thimmapaya, B. (2001) Proc. Natl. Acad. Sci. USA 98, 4646-4651), establish an important negative regulatory role for p300 in c-Myc expression that may be important in maintaining the cells in the G0/G1 phase of the cell cycle.

Figures

Fig. 3.

(A) Northern blot showing the levels of c-Myc, c-Fos, and c-Jun RNAs in serum-stimulated clone 37 and 48 cells. Serum-starved clone 37 and 48 cells were infected with Ad vectors and stimulated with serum as shown in Fig. 2 A. Poly(A)-containing RNA was isolated at different time points shown and was analyzed by using Northern blot hybridizations. c-Jun and GAPDH in each case were analyzed after reprobing the filters used for c-Myc analysis. One microgram of poly(A)-containing RNA was used in each case. (B) Transcriptional activation activity of Myc in serum-stimulated R12G12 (control) and clone 37 and 48 cells. (Upper) Structure of the Ad vector (AdM4) containing the Myc-responsive promoter-reporter cassette is shown. The inverted terminal repeat (ITR) and 0 and 4 map units of the Ad genome are shown. Serum-starved cells were infected with Ad vectors and were superinfected 2-h later with Ad-M4 or Ad-M4mut at 10 pfu per cell. Sixteen hours later, the cells were serum-stimulated, and at the indicated times the luciferase activity in the lysates was assayed by using 5 μg of protein. The experiments were repeated twice with reproducible values, and the data of one such experiment are shown. For R12G12 cells, only 0- and 3-h time points are shown. Luc, luciferase activity. (C) Determination of the levels of cyclin E/Cdk2 and cyclin A/Cdk2 activities in clone 37 cells. Assay details are as described in Materials and Methods. Phosphorylated histones were resolved on SDS/12% PAGE, dried, and autoradiographed.

Fig. 1.

Induction of p300 in rat cell clones and their growth properties. (A) Immunoprecipitation analysis of p300 (see Materials and Methods for details). Note that p300 levels for clones R12G12, 23, 48, 5, and 35 in lanes 1–10 and clones 37 and 48 cells in lanes 11–14 were assayed in separate experiments. For comparison of p300 between lanes, the levels of p300 in lanes 11–14 were normalized to clone 48 values in lanes 5–6. (B) Growth properties of rat cell clones.

Fig. 2.

Inhibition of the S-phase entry in p300-overexpressing cells. (A) Schematic representation of the time course of serum starvation, vector infection, and harvesting of cells for FACS analysis. (B) Induction of p300 in serum-starved cells. Serum-starved cells were infected 16 h before serum stimulation with Ad vectors, labeled, and harvested at the indicated times, and p300 in the lysates was analyzed by immunoprecipitation. (C) Kinetics of S-phase entry of serum-stimulated cells. Cells were seeded, starved, and infected with Ad vectors as in A. The distribution of cells in G1, S, and G2/M phases after serum stimulation was determined by FACS analysis as described (18). The assays were done in triplicate, and the average number of cells in S phase with SD are shown.

Fig. 4.

Abrogation of p300-mediated S-phase inhibition by Myc. (A) Schematic representation of the time course of vector infection and harvesting of cells for FACS analysis. (B) Serum-starved cells were coinfected with Ad vectors as shown (all at 25 pfu per cell). Sixteen hours later, they were stimulated with serum and were harvested at indicated time points. Distribution of cells in G1, S, and G2/M phase were determined by FACS analysis as described (18). Adβ-gal was used where appropriate to keep the multiplicity of infection constant. Average values obtained in three independent experiments with SD are shown. For statistical validation, the mean cell numbers in lane 7 (cells infected with Adtet plus Adβ-gal) were compared with those of lane 8 (cells infected with Adtet plus Adc-Myc) by independent sample Student's two-tailed t test. P values for lane 7 vs. lane 8 for clone 48, 0.007; for clone 37, 0.011. (C) Determination of cyclin E/Cdk2 and cyclin A/Cdk2 activities in clone 37 cells at 15 h after infection with Ad vectors as shown in B.

Fig. 5.

HAT activity of p300 is not required for the inhibition of Myc- and S-phase induction. (A) Immunoprecipitation of p300 in cell clones AT21, AT41, and 37. (B) Comparison of growth properties of cell clones AT21 and AT41 with R12G12 and 37. (C) Induction of S phase in clones AT21 and AT41 with and without induction of p300. Details of this experiment were as in Fig. 2C. Assays for AT21 and AT41 were done in triplicate, for which the average values and SD are shown. (D) Determination of Myc activity in serum-stimulated clones 37, AT21, and AT41. Details are as in Fig. 3B. Values shown are luciferase activity expressed as percent of Adβ-gal controls. Averages values with SD obtained from three independent experiments are shown. (E) HAT activity of p300 in clone 37 and AT21 with induced and uninduced p300 levels. See Materials and Methods for details. Average values obtained from two experiments are shown. The radioactivity obtained from IgG incubations was subtracted from each matching sample. The values obtained for Adβ-gal infected cells were taken as 1 in calculating the fold induction.