Direct p53 transcriptional repression: in vivo analysis of CCAAT-containing G2/M promoters - PubMed (original) (raw)
Direct p53 transcriptional repression: in vivo analysis of CCAAT-containing G2/M promoters
Carol Imbriano et al. Mol Cell Biol. 2005 May.
Abstract
In response to DNA damage, p53 activates G(1)/S blocking and apoptotic genes through sequence-specific binding. p53 also represses genes with no target site, such as those for Cdc2 and cyclin B, key regulators of the G(2)/M transition. Like most G(2)/M promoters, they rely on multiple CCAAT boxes activated by NF-Y, whose binding to DNA is temporally regulated during the cell cycle. NF-Y associates with p53 in vitro and in vivo through the alphaC helix of NF-YC (a subunit of NF-Y) and a region close to the tetramerization domain of p53. Chromatin immunoprecipitation experiments indicated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in vivo before and after DNA damage, requiring DNA-bound NF-Y. Following DNA damage, p53 is rapidly acetylated at K320 and K373 to K382, histones are deacetylated, and the release of PCAF and p300 correlates with the recruitment of histone deacetylases (HDACs)-HDAC1 before HDAC4 and HDAC5-and promoter repression. HDAC recruitment requires intact NF-Y binding sites. In transfection assays, PCAF represses cyclin B2, and a nonacetylated p53 mutant shows a complete loss of repression potential, despite its abilities to bind NF-Y and to be recruited on G(2)/M promoters. These data (i) detail a strategy of direct p53 repression through associations with multiple NF-Y trimers that is independent of sequence-specific binding of p53 and that requires C-terminal acetylation, (ii) suggest that p53 is a DNA damage sentinel of the G(2)/M transition, and (iii) delineate a new role for PCAF in cell cycle control.
Figures
FIG. 1.
ChIP analysis of NF-Y and p53 on G2/M promoters. (A) ChIP was performed with NIH 3T3 mouse fibroblasts not treated (−) or treated (+) with adriamycin and the indicated antibodies (Ab). (B) (Upper panel) p53−/− mouse embryo fibroblasts (MEF) were used in a similar ChIP analysis with the indicated antibodies. (Lower panel) NIH 3T3 cells arrested in G0 by serum withdrawal were used in a ChIP analysis (6). (C) ChIP with NIH 3T3 cells stably transfected with a reporter construct containing the wild-type cyclin B2 promoter, a mutant lacking two CCAAT boxes (Y1-Y2m), or a mutant lacking all three CCAAT boxes (Y1-Y2-Y3m). Immunoprecipitated DNAs were amplified with oligonucleotides, revealing the luciferase (Luc) transgene as well as the respective endogenous cyclin B2 promoter. Ctl, control.
FIG. 2.
Overexpression of a dominant-negative (DN) NF-YA mutant prevents p53 association with CCAAT promoters. (A) NIH 3T3 cells were infected for 7 h with green fluorescent protein (GFP) control adenovirus (lanes 1 to 4), wild-type NF-YA adenovirus (lanes 5 to 8), or YAm29 adenovirus (lanes 9 to 12). Western blot analysis of overexpressed NF-YA is shown. (B) ChIP analysis with anti-NF-YB antibodies of chromatin derived from cells infected with the three viruses. The indicated bona fide NF-Y targets were PCR amplified. For each promoter, we show two amplifications at different PCR cycles. CTL, control; PLK, Polo-like kinase. (C) Same as panel B, except that wild-type NF-YA chromatin (YA wt) and YAm29 chromatin (YA DN) were analyzed with anti-NF-YB and anti-p53 antibodies (Ab). We included as controls the cyclin A promoter (single CCAAT box) and the p21 promoter (containing a bona fide p53 binding element but not CCAAT boxes).
FIG. 3.
Binding of p53 to NF-Y in solution and on DNA in vitro. (A) Purified recombinant His-tagged NF-Y trimer and HA-p53 were immunoprecipitated with anti-NF-YA (Mab7) and anti-HA antibodies. Load (L), flowthrough (FT), and bound (B) fractions were assayed in Western blots with anti-HA and anti-NF-YB antibodies. In the lowest panel, immunoprecipitation was performed without HA-p53. (B) EMSAs with recombinant NF-Y and p53 and with the cyclin B2 Y1 high-affinity NF-Y binding site (lanes 1 to 5) or a cyclin B2 fragment (−129 to +48) (lanes 6 to 24). p53 was used at 50 ng in lanes 1, 4, 6, 9, 12, 15, and 20 to 24 and at 150 ng in lanes 2, 5, 7, 10, 13, and 16; NF-Y was used at 1 ng in lanes 3 to 5 and 8 to 10, at 2 ng in lanes 19 to 24, and at 5 ng in lanes 11 to 18; TAF11-TAF13 dimer was used at 200 and 600 ng in lanes 15 and 16, respectively. For the supershift EMSAs in lanes 19 to 24, anti-p53 DO1 was incubated with recombinant p53 before (lane 21) or after (lane 22) NF-Y and DNA were added; anti-NF-YB and anti-GST controls were added after NF-Y, p53, and DNA were preincubated. (C) Footprint analysis of NF-Y and p53 on a cyclin B2 fragment (−129 to +48). In the upper rows, 20 ng of NF-Y was incubated alone or with 100 and 300 ng of p53. The lower rows contained unbound DNA and p53 (300 ng) alone. The positions of the Y1, Y2, and proximal Y3 CCAAT boxes are indicated.
FIG. 4.
Binding of p53 to NF-Y in vivo. (A) NF-Y-p53 interactions in vivo in H1299 cells. Immunoprecipitation (IP) with anti-p53 or anti-YA (lanes 4 and 5), and control (Ctl) (lane 3) antibodies (Ab) was followed by Western blot analysis with the indicated antibodies. In lanes 1 and 2, extracts were tested directly in Western blots. NF-Y and p53 were overexpressed in the extracts used in lanes 2, 3, and 5. The different sizes of NF-YA are due to the prevalence of the “short” splicing isoform in lane 1 and overexpression of the “long” isoform in lanes 2, 3, and 5. WCE, whole-cell extracts. (B) Evaluation of endogenous NF-Y-p53 interactions. NIH 3T3 cells were not treated (lanes 1, 3, and 5) or were treated with adriamycin (ADR) for 8 h (lanes 2, 4, and 6). Extracts were analyzed directly in Western blots (lanes 1 and 2) or immunoprecipitated with control (lanes 3 and 4) or anti-p53 (lanes 5 and 6) antibodies. Western blot analysis of eluates with the indicated antibodies is shown in lanes 3 to 6. (C) EMSAs of in vivo-produced NF-Y and p53. The Y1 CCAAT oligonucleotide was used in lanes 1 to 3; a cyclin B2 fragment (−129 to +48) was used in lanes 4 to 8. In lanes 1 and 4, 1 μl of mock-transfected Saos2 cell extracts was used; in lanes 2, 3, and 5 to 8, equivalent amounts of extracts from cells transfected with NF-Y expression vectors were used, together with HA-p53 in lanes 3 and 6 to 8. Supershift with anti-HA antibodies and inhibition with anti-YB antibodies are shown in lanes 7 and 8, respectively.
FIG. 5.
Mapping of the NF-Y-p53 association domains. (A) EMSA of wild-type NF-Y (left panel) and HFM NF-YC-NF-YB dimer with a 56-amino-acid NF-YA mutant (YA9) sufficient for DNA binding (right panel) alone and with increasing doses of p53. (B) Immunoprecipitation (IP) of GST-p53 mutants with the indicated NF-YC mutations and of full-length NF-YB with anti-NF-YB antibodies. Flowthrough (FT) and bound (B) fractions were assayed in Western blots with the indicated antibodies. L, load. (C) Immunoprecipitation of full-length NF-Y trimer with the indicated GST-p53 mutations with antibody Mab7. Western blots are shown.
FIG. 6.
ChIP analysis of p53, H3, and H4 acetylation and HAT association on G2/M promoters. (A) RT-PCR analysis of cyclin B2 and control GAPDH mRNAs following DNA damage by adryamicin for the indicated times. (B) (Left panels) ChIP analysis of NIH 3T3 cells not treated (−) or treated with adriamycin (ADR) for 20 h (+) with the indicated antibodies against nonacetylated p53, Ac-p53 K320, and Ac-p53 K373-382. (Middle and right panels) Anti-YB antibodies used as a positive control, together with anti-Ac-H3, anti-Ac-H4, and anti-PCAF antibodies. Titration of the input DNAs is shown. (C) NIH 3T3 cells were treated with adriamycin for the indicated times (in hours), and ChIP analysis of the cyclin B and Cdc2 promoters was performed with the indicated antibodies as described above. (Left panels) PCAF and control antibodies. Titration of the input DNAs is shown. (Right panels) Representative cycles of semiquantitative PCR. (D) (Left panels) ChIP analysis of the Bax and Mdm2 promoters. (Right panels) Time course for PCR amplification of the Mdm2 and Bax promoters.
FIG. 7.
HDAC recruitment to G2/M promoters upon DNA damage. (A) NIH 3T3 cells were treated for the indicated times with adriamycin (ADR), and ChIP analysis was performed with antibodies against the indicated HDACs. Two sets of representative PCR cycles are shown for each time point. Ctl, control. (B) Chromatin from the Y1-Y2m-containing stable clones (Fig. 1) was used for ChIP analysis with the indicated antibodies. Immunoprecipitated DNAs were PCR amplified to show endogenous cyclin B2 (left panels) and the luciferase reporter construct mutated in two of the three CCAAT boxes (right panels).
FIG. 8.
Repression of cyclin B2 by p53 and PCAF. (A) PCAF (100 and 300 ng) was cotransfected in COS cells with 100 ng of p53, with wild-type cyclin B2, and with Y1-Y2-Y3m. Error bars indicate standard deviations. (B) Same as panel A, except that PCAF (100 and 300 ng) was transfected alone or with p53, together with the Mdm2 reporter. (C) Transcriptional analysis of Mdm2 and cyclin B2 reporters in p53 −/− Saos2 cells with 100 ng of wild-type (wt) p53 and the indicated mutants of p53. (D) Representative levels of the p53 proteins in panel C were checked in Western blots. (E) Same as panel A, except that wild-type p53 or p53 K320R (100 ng) was transfected with PCAF and assayed on the cyclin B2 promoter in NIH 3T3 cells.
FIG. 9.
Effect of a nonacetylated p53 mutant on transcriptional repression. (A) Dose-response analysis of wild-type p53 (p53wt) and mutant p53-9KR in Saos2 cells with the cyclin B2 and Mdm2 reporters. Error bars indicate standard deviations. (B) Levels of expression of wild-type p53 and p53-9KR in panel A are shown in Western blots. (C) Immunoprecipitation of overexpressed wild-type p53 and mutant p53-9KR from transfected H1299 cells. Western blot analysis was performed with anti-p53 and anti-YA antibodies. WCE, whole-cell extracts; Ctl, control. (D) ChIP analysis of NF-Y and p53 in NIH 3T3 cells transfected with wild-type p53, mutant p53-9KR, and control vectors, together with the cyclin B2-luciferase construct, before and after adriamycin (ADR) treatment for 8 h. PCRs with the transfected templates are shown in the right panels, and those with the control endogenous cyclin B2 gene are shown in the left panels. Two sets of PCRs are shown. Note that fewer PCR cycles are required for the transfected templates.
FIG. 10.
Short-term events on G2/M promoters following DNA damage.
References
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