Antisense-mediated depletion of p300 in human cells leads to premature G1 exit and up-regulation of c-MYC

Abstract

The cAMP-response element-binding protein (CREB)-binding protein and p300 are two highly conserved transcriptional coactivators and histone acetyltransferases that integrate signals from diverse signal transduction pathways in the nucleus and also link chromatin remodeling with transcription. In this report, we have examined the role of p300 in the control of the G(1) phase of the cell cycle in nontransformed immortalized human breast epithelial cells (MCF10A) and fibroblasts (MSU) by using adenovirus vectors expressing p300-specific antisense sequences. Quiescent MCF10A and MSU cells expressing p300-specific antisense sequences synthesized p300 at much reduced levels and exited G(1) phase without serum stimulation. These cells also showed an increase in cyclin A and cyclin A- and E-associated kinase activities characteristic of S phase induction. Further analysis of the p300-depleted quiescent MCF10A cells revealed a 5-fold induction of c-MYC and a 2-fold induction of c-JUN. A direct target of c-MYC, CAD, which is required for DNA synthesis, was also found to be up-regulated, indicating that up-regulation of c-MYC functionally contributed to DNA synthesis. Furthermore, S phase induction in p300-depleted cells was reversed when antisense c-MYC was expressed in these cells, indicating that up-regulation of c-MYC may directly contribute to S phase induction. Adenovirus E1A also induced DNA synthesis and increased the levels of c-MYC and c-JUN in serum-starved MCF10A cells in a p300-dependent manner. Our results suggest an important role of p300 in cell cycle regulation at G(1) and raise the possibility that p300 may negatively regulate early response genes, including c-MYC and c-JUN, thereby preventing DNA synthesis in quiescent cells.

Figure 1

Levels of p300 and CBP and S phase induction in quiescent MCF10A and MSU cells infected with Ad vectors expressing p300-specific AS sequences, AS-1 and AS-2. (A) Schematic representation of the time course used for infection of MCF10A cells with Ad vectors for cell cycle analysis, protein, and RNA quantitation. (B) Western blot showing reduced levels of p300 in AS-1- and AS-2-infected cells. Serum-starved MCF10A cells were infected with Ad vectors at 200 pfu/cell and 18 h after infection cells were lysed in RIPA buffer (14). Fifty micrograms of the protein was Western immunoblotted by using a SDS/7% polyacrylamide gel with an α-p300 polyclonal antibody (sc-584; Santa Cruz Biotechnology). Arrow shows the position of p300. All of the gels shown here and in the following figures were quantitated by using a densitometer (Molecular Dynamics). (C) Western blot showing equal levels of CBP in cells infected with AS-1, AS-2, and Ad-β-gal. Fifty micrograms of protein from the same cell lysate was Western immunoblotted with α-CBP polyclonal antibody (sc-369; Santa Cruz Biotechnology). (D) Induction of DNA synthesis in quiescent MCF10A cells infected with p300 AS vectors. MCF10A cells were infected as shown in Fig. 1A and the cells were harvested at the indicated times. The percentage of cells in G₁, S, and G₂-M were quantitated by FACS analysis as described in Materials and Methods. (E) Western blot shows depletion of p300 in AS p300-infected MSU cell lysates prepared as described above. MSU cells were serum starved for 48 h before infection. (F) Induction of DNA synthesis in serum-starved p300-depleted MSU cells as determined by FACS analysis.

Figure 2

Analysis of cyclin A, cyclin E, CDK2, and CDK2 activity in quiescent MCF10A cells expressing p300 AS sequences. (A) Autoradiogram showing cyclin E/CDK2 activities of the cell lysates prepared at different time periods of infection. Infection protocols were as shown in Fig. 1A. One hundred micrograms of the protein was immunoprecipitated with α-cyclin E antibody, and the immunoprecipitated complexes were incubated with γ [³²P]ATP and histone H1 (19). Phosphorylated histones were analyzed on a SDS/12% PAGE. (B) Autoradiogram showing cyclin A/CDK2 activities of cell lysates used in A. One hundred micrograms of the protein was immunoprecipitated with αcyclin A antibody, and the immunoprecipitated complexes were incubated with γ [³²P]ATP and histone H1. Phosphorylated histones were analyzed on a SDS/12% PAGE. (C) Western blot showing levels of cyclin E in cells infected with Ad vectors at 20, 26, and 32 h after infection. Fifty micrograms of the protein from cell lysates was fractionated on SDS/10% PAGE and probed with an α-cyclin E antibody (sc-247; Santa Cruz Biotechnology). The arrows point to the cyclin E doublet. All Western blot membranes in this report were reprobed with α-actin antibody for loading control (data not shown). (D) Western blot showing levels of cyclin A in cell lysates used in C. Fifty micrograms of the protein from cell lysates was fractionated on SDS/10% PAGE and probed with an α-cyclin A antibody (sc-751; Santa Cruz Biotechnology). (E) Western blot showing CDK2 protein levels in serum-starved MCF10A cells infected with Ad vectors at various time points. Fifty micrograms of the protein from cell lysates was fractionated on a SDS/10% PAGE and immunoblotted by using α-CDK-2 antibody (sc-163; Santa Cruz Biotechnology).

Figure 3

Levels of c-MYC, c-JUN, and p300 in serum-starved MCF10A cells infected with AS p300 vectors. (A) Western immunoblot of c-MYC in quiescent MCF10A cells infected with AS-1 or β-gal vectors. Twenty micrograms of the protein from cell lysates prepared from MCF10A cells at indicated time points was immunoblotted by using an α-c-MYC polyclonal antibody (sc-764; Santa Cruz Biotechnology). (B) Western analysis of c-JUN in serum-starved MCF10A cells infected with Ad vectors. Twenty micrograms of protein from the cell lysates used in A was assayed by Western immunoblotting by using an α-c-JUN antibody (sc-1694; Santa Cruz Biotechnology). (C) Time course of p300 depletion in serum-starved MCF10A cells. Fifty micrograms of the protein lysates at different time periods of infection was analyzed by Western immunoblotting by using an α-p300 antibody (sc-584, Santa Cruz Biotechnology).

Figure 4

Riboprobe analysis of c-MYC, CAD, and p21. (A) RNase protection assay of c-MYC RNA. Twenty micrograms of total RNA extracted from MCF10A cells infected with AS vectors at indicated time points was assayed by RNase protection assay by using a c-MYC specific riboprobe (14). In the Probe lane, 1/100 of the radioactive probe used in RNase protection assays was loaded directly. The smaller arrow indicates the undigested probe, whereas the larger arrow indicates the protected fragment. Details of the probes and the procedures were as described in Materials and Methods. (B) RNase protection assay of CAD mRNA in quiescent MCF10A cells infected with Ad vectors at 30 h after infection. Thirty micrograms of the total RNA was used for the assay by using a CAD riboprobe. In the Probe lane, 1/100 of the probe used in the assay was loaded directly on the gel. As above, the small arrow points to the probe and the large arrow to the protected fragment. The asterisk indicates a nonspecific band. (C) RNase protection assay of p21 mRNA in AS-infected MCF10A cells at 17 and 30 h after infection. Twenty micrograms of total RNA was used in the assay. In the Probe lane, 1/100 of the probe used for assay was loaded directly. The small arrow points to the probe, whereas the large arrow points to the p21-protected fragment. (D) RNase protection assay for GAPDH mRNA samples used in Fig. 4 A, B, and C. Five micrograms of the total RNA was assayed by using a riboprobe specific for GAPDH. Data for the 18-h time point are not shown.

Figure 5

Reversal of p300 depletion-dependent premature S phase induction. (A) Inhibition of premature S phase induction in p300-depleted MCF10A cells by overexpression of MAD. Serum-starved MCF10A cells were coinfected with AS Ad vectors at 200 pfu/cell with or without Ad-MAD (100 pfu/cell), which expresses MAD protein. Ad-β-gal was always used to maintain the multiplicity of infection uniform in all experiments. (B) Reversal of early S phase entry in p300-depleted cells. Serum-starved MCF10A cells were coinfected with either AS p300 sequences and AS-c-Myc or AS-c-Jun. Ad-β-gal was used to maintain the multiplicity of infection. (C) Inhibition of c-MYC protein synthesis in p300 depleted cells. Serum-starved MCF10A cells were coinfected with different adenoviral vectors in combinations as indicated below. In lanes 1, 5, 9, and 13 Ad-β-gal was mixed with AS-c-Myc; in lanes 2, 6, 10, and 14 AS-1 was mixed with AS-c-Myc; in lanes 3, 7, 11, and 15 Ad-β-gal was mixed with Ad -β-gal and in lanes 4, 8, 12, and 16, Ad-β-gal was mixed with AS-1. Protein lysates obtained from these cells at different periods of infection were assayed for c-MYC protein levels by Western blot analysis. Western blot membrane shown (Upper) was reprobed with α-actin antibody.

Figure 6

Up-regulation of c-MYC promoter in p300-depleted cells. (A) Diagram of the TBE1/2 (wild-type MYC) and TBE1 m/2 m in which two TCF-4-binding sites are mutated (20). (B) Time course of promoter–reporter assay. MCF 10A cells were transfected (TX) with appropriate reporter plasmids, serum starved, and infected with Ad vectors before being harvested for luciferase assay as shown. (C) MYC promoter-reporter activity in p300-depleted cells. Cells harvested after transfection and infection as described in B were assayed for luciferase activity. Average values of three experiments with error bars are shown.

Figure 7

S phase induction and c-MYC RNA and c-JUN protein in quiescent MCF10A cells infected at 20 pfu/cell with dl1500 (wild-type E1A) and dl2–36 (mutant E1A that does not bind to p300). (A) Cell cycle analysis of serum-starved cells infected with dl1500 or dl2–36. Cells were serum starved, infected, and harvested at the indicated times, and percent cells in S phase were quantitated by FACS analysis. (B) Levels of c-MYC RNA at the indicated times after infection. Riboprobe analysis was carried out as described in legend to Fig. 4A. The protected fragment is shown by a large arrow, whereas the undigested probe is indicated by a small arrow. (C) RNase protection assay for GAPDH mRNA for the RNA samples used in B. Details for the assay conditions are as shown in Fig. 4D. (D) Western analysis of c-JUN in quiescent MCF10A cells infected with dl1500 and dl2–36. Twenty micrograms of protein was Western immunoblotted by using an α-c-JUN polyclonal antibody (sc-1694; Santa Cruz Biotechnology).