Ets1 is required for p53 transcriptional activity in UV-induced apoptosis in embryonic stem cells - PubMed (original) (raw)

Ets1 is required for p53 transcriptional activity in UV-induced apoptosis in embryonic stem cells

Dakang Xu et al. EMBO J. 2002.

Abstract

Embryonic stem (ES) cells contain a p53-dependent apoptosis mechanism to avoid the continued proliferation and differentiation of damaged cells. We show that mouse ES cells lacking Ets1 are deficient in their ability to undergo UV-induced apoptosis, similar to p53 null ES cells. In Ets1(-/-) ES cells, UV induction of the p53 regulated genes mdm2, perp, cyclin G and bax was decreased both at mRNA and protein levels. While p53 protein levels were unaltered in Ets1(-/-) cells, its ability to transactivate genes such as mdm2 and cyclin G was reduced. Furthermore, electrophoretic mobility shift assays and immunoprecipitations demonstrated that the presence of Ets1 was necessary for a CBP/p53 complex to be formed. Chromatin immunoprecipitations demonstrated that Ets1 was required for the formation of a stable p53-DNA complex under physiological conditions and activation of histone acetyltransferase activity. These data demonstrate that Ets1 is an essential component of a UV-responsive p53 transcriptional activation complex in ES cells and suggests that Ets1 may contribute to the specificity of p53-dependent gene transactivation in distinct cellular compartments.

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Figures

Fig. 1. Targeted mutagenesis of the Ets1 gene. (A) The wild-type Ets1 locus and targeting vector showing the targeting strategy and restriction enzyme sites used for screening. The construct was designed such that exons 3–6 and a neomycin cassette were floxed for subsequent excision by CRE recombinase. 5′ and 3′ probes, which were used to discriminate between alleles, are also shown. LoxP sites are indicated by filled triangles. (B) Schematic representation of the wild-type and modified Ets1 locus: (1) wild-type locus; (2) targeted locus with appropriate insertion of loxP sites flanking exons 3–6; (3) double-targeted Ets1 locus generated by High G418 selection; and (4) double-knockout ES cells generated by CRE-mediated excision. (C) _Eco_RV-digested DNA probed with the 1.5 kb genomic fragment external to the targeting construct. WT and clone 270 show the 5.5 kb WT allele only, whereas clone 269 shows both the WT and 3.8 kb targeted band. (D) Confirmation of targeting of clone 269. Genomic DNA from wild-type and Ets1-targeted clone 269 probed with the 1.7 kb 3′ internal genomic fragment. Only clone 269 shows the expected 2.5 and 5.7 kb targeted bands (_Hin_dIII and _Bgl_II digested DNA, respectively) in addition to the expected WT bands. (E) Double-targeted ES cell clones generated by High G418 resistance (17, 2) and subsequent removal of the Ets1 exons 3–6 by CRE recombinase (17–15, 2–34). The CRE spliced and unspliced targeted alleles are indicated by the 13 and 1.7/3.8 kb bands, respectively, using the 5.5 kb 5′ probe. (F) Northern blotting showing absence of Ets1 mRNA expression in Ets1-targeted and spliced ES cell clones using a murine Ets1 cDNA probe. Poly(A)+ mRNA (3 µg) of each was used and reprobed for GAPDH to demonstrate equal loading. These clones were subsequently considered Ets1–/–.

Fig. 2. Reduced expression of p53 mRNA in Ets1–/– ES cells is not due to altered morphology or differentiation status. (A) Northern blotting showing reduced expression of p53 in Ets1–/– ES cells compared with wild-type (WT), double-targeted (Ets1loxP) ES cells. The same blot was used to determine the that relative levels of Oct4 mRNA were unaltered and GAPDH was used to demonstrate equal loading. (B) Photomicrographs of wild-type (1 and 4), Ets1loxP (2 and 5) and Ets1–/– (3 and 6) ES cells. The upper panel is a phase-contrast image whereas the lower panel is labeling with anti-SSEA1–FITC, which is expressed only in undifferentiated ES, indicating that these cultures contain very few differentiated cells. Bar corresponds to 100 µm.

Fig. 3. Ets1–/– ES cells are resistant to UV irradiation-induced apoptosis. (A) Flow cytometric analysis of fixed wild-type, Ets1-targeted and two independent Ets1 null ES cell clones stained with propidium iodide 12 h after UV irradiation (40 J/m2). Percentage of cells with a hypodiploid (sub-2N) DNA content indicative of apoptosis are shown. (B) Graphical representation of the percentage of cells undergoing apoptosis after various doses of UV irradiation. Data is shown as mean ± SD of three independent experiments. (C) Percentage of cells undergoing apoptosis at different time points after irradiation with 40 J/m2 of UV. Data is shown as mean ± SD of three independent experiments. (D–I) Immunofluorescent images of Hoechst 33342-stained WT (D and G), Ets1loxP (E and H) and Ets1–/– (F and I) ES cells, with and without UV irradiation. Significant nuclear condensation and fragmentation is not observed in untreated cells (D–F) or UV-irradiated Ets1 null cells (I). However, a significant number of WT and Ets1-targeted ES cells showed nuclear fragmentation after UV irradiation (G and H). Bar corresponds to 20 µm. (J) Ethidium bromide staining of DNA from WT, Ets1loxP and Ets1–/– ES cells after 40 J/m2 UV irradiation analysed by agarose gel electrophoresis, demonstrating bands of DNA fragmentation in WT and targeted but not Ets1–/– cells. Lane 1 shows pUC19/HpaII marker.

Fig. 4. Addition of exogenous Ets1, but not exogenous p53, restores sensitivity to UV irradiation-induced apoptosis in Ets1–/– ES cells. Western blots of ES cell lysates after transfection with FLAG-Ets1 (A) and exogenous p53 (B), which demonstrate expression of FLAG-Ets1 and increased expression of p53, respectively. (C) FACS analysis of propidium iodide-stained wild-type and Ets1–/– ES cells expressing exogenous CMV-p53 or EF1α-FLAG/Ets1 after UV treatment. These demonstrate characteristic apoptosis after UV treatment of ES cells, which express endogenous or exogenous Ets1, regardless of p53 status.

Fig. 5. The expression of p53 transactivated genes is reduced in cells lacking Ets1 following UV irradiation. (A) Results of a typical northern blot experiment measuring the expression of p53-regulated genes in wild-type (wt), Ets1-targeted (Ets1loxP) and Ets1–/– ES cells before and after UV irradiation (40 J/m2). Cells were analysed and RNA isolated at times indicated after UV treatment. Probes specific for mouse perp, mdm2, cyclin G and bax were used for northern blot analyses with GAPDH as a loading control. Data from five independent experiments of two independent Ets1–/– clones were quantified using a Fuji Image Reader VI.3E and expressed as mean ± SD, relative to a GAPDH loading control. (B) Expression of p53-regulated proteins in wild-type and Ets1–/– ES cells. Cells were isolated at the indicated time points (0, 1, 2, 4 and 8 h) post-UV irradiation (40 J/m2). Antibodies specific for mouse p53, mdm2, Bax, Bcl-2, Bcl-XL and β-tubulin (loading control) were used for western blot analyses. Analysis was performed at least three times on two independent Ets1–/– clones and data from a representative experiment is shown.

Fig. 6. UV enhances p53 transcriptional activity in wild-type but not Ets1–/– ES cells. (A) Wild-type and Ets1–/– ES cells were transfected with wild-type mdm2 promoter-luciferase constructs or those containing mutations in the defined p53- or Ets-binding sites (mdm2, mdm2Δp53I and mdm2ΔEtsA, respectively). Cells were exposed to UV irradiation 24 h after transfection and harvested at 36 h. Luciferase activity was determined relative to a β-gal control. Each assay was performed at least three times in triplicate. Data is shown as the mean of independent experiments ± SD. (B) As above, except the mouse cyclin G promoter and p53 element mutant cyclin GΔp53II promoter-reporter vectors were transfected into wild-type and Ets1–/– ES cells and exposed to UV irradiation. Mean of at least three independent experiments is shown ± SD.

Fig. 7. The UV-induced complex that binds the consensus p53–DNA-binding sequence in ES cells contains p53, Ets1 and CBP. (A) Nuclear extracts were prepared from untreated or UV-irradiated wild-type and Ets1–/– ES cells (40 J/m2) 5 h post-irradiation. The indicated volume of extracts were incubated with [γ-32P]dATP-labeled p53 consensus oligonucleotides with or without a 5-fold excess of unlabeled oligonucleotide competitor and separated on acrylamide gels. The position of the specific p53–DNA complex is indicated. (B) Nuclear extracts of ES cells treated with UV as above were incubated with or without Ets1, p53 or Ets2 antibodies as indicated and analyzed in EMSA using the [γ-32P]dATP-labeled p53 consensus oligonucleotide. Ets1Abs indicates that anti-Ets1 antibody was preabsorbed with recombinant His-tagged Ets1. (C) Nuclear extracts were prepared from UV-irradiated Ets1–/– ES cells transfected with FLAG-Ets1 or FLAG alone, incubated with 3/7 µl antibodies to p53, Ets1 (monoclonal) or FLAG as indicated, and analysed using the [γ-32P]dATP-labeled p53 consensus oligonucleotide. The complex was supershifted by p53, Ets1 and anti-FLAG antibodies only in ES cells containing FLAG-Ets1. (D) EMSA of nuclear extracts from UV-irradiated wild-type and Ets1–/– ES cells with [γ-32P]dATP-labeled p53 consensus oligonucleotides and antibodies to p53 (3 µl) or CBP (3–10 µl) as indicated. The formation of the complex was inhibited by CBP antibody in wild-type ES cells, but not in Ets1–/– ES cells.

Fig. 8. UV induces a complex formation between p53, Ets1 and CBP. Total cell lysates were prepared from wild-type and Ets1–/– ES cells 6 h after exposure to UV irradiation (40 J/m2). These lysates were shown to have similar levels of CBP by western blotting using β-tubulin as a loading control (A). To determine whether complexes containing CBP and p53 were formed, these lysates were immunoprecipitated with CBP (B) or p53 (C) antibodies and the bound complexes were analysed for the presence of p53 and CBP by western blotting (B and C). In each case, similar amounts of CBP (B) or p53 (C) were immunoprecipitated from each ES cell genotype; however, p53 and CBP were co-precipitated after UV irradiation in wild-type but not Ets1–/– ES cells. (D) Similar immunoprecipitations with p53 antibody of wild-type, Ets1–/– (+ FLAG) and Ets1–/– (+ FLAG-Ets1) ES cell lysates. These precipitated complexes were examined for p53, CBP and FLAG-Ets1 by western blotting demonstrating that CBP was co- precipitated with p53 in the presence of exogenous Ets1.

Fig. 9. p53 binds and activates the mdm2 promoter after UV irradiation under physiological conditions in wild-type but not Ets1–/– ES cells. Hybridization of a mdm2 promoter-specific probe to ChIP DNA from wild-type and Ets1–/– ES cells with and without treatment with 40 J/m2 UV irradiation. (A) The top panel shows hybridization to DNA precipitated with p53 antibody and amplified by PCR with mdm2 promoter-specific primers flanking the p53 sites. The second panel is a similar hybridization to input DNA. The specificity of p53 immunoprecipitation is shown by similar PCR amplification of input and p53-precipitated DNA for GAPDH. (B) Precipitation of DNA with antibody to acetylated H3 (IP: AcH3) and input DNA similarly amplified with oligonucleotides to mdm2 or GAPDH, demonstrating specific induction of mdm2 by UV irradiation only in wild-type cells.

Fig. 10. Model for the role of Ets1 in the induction of p53 transcriptional activity after UV irradiation of ES cells. In this model, after exposure to UV, there is a formation of a complex, which includes promoter-bound p53, Ets1 and CBP (where X represents other undefined factors) that interact with the basal transcription machinery leading to strong transcriptional activation. When Ets1 is absent (or other inhibitors present?), the p53–DNA–CBP complex is not stably formed, which leads to only modest transcriptional activation.

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Ets1 is required for p53 transcriptional activity in UV-induced apoptosis in embryonic stem cells - PubMed (original) (raw)