Figure 1.
Dose-dependent repression of the RLIM promoter by p53.
(A) H1299 cells or (B) Hep3B cells were cotransfected with 100 ng of RLIM promoter-luciferase reporter construct NP500-Luc, 30 ng pRL-SV40 and increasing amounts (0, 1, 2.5 and 10 ng) of pCMV-HA-p53 plasmids. The total amount of plasmid in each transfection was adjusted to be the same using the empty vector. Cells were harvested 30 h after transfection and lysed for measuring luciferase activity. Data represent the mean of three independent experiments normalized to pRL-SV40 activity and are presented as fold of induction; bars, ± S.D. (C) Various p53 expression constructs (wild type and mutants with mutations in codons 175, 248, 273 and 282) or pCMV-vector plasmid were cotransfected with 100 ng of p53-Luc luciferase reporter gene (Stratagene). Cells were harvested 30 h after transfection and lysed for luciferase assays. (D) Effects of p53 and various mutants on RLIM promoter activity. The indicated p53 expression plasmids or pCMV-vector plasmid were transfected with 100 ng of RLIM promoter-luciferase plasmid NP500-Luc. Cells were harvested 30 h after transfection and lysed for luciferase assays.
Figure 2.
p53 represses RLIM at both mRNA and protein levels.
(A) U2-OS cells were treated with 20 µM etoposide for the indicated period (0, 6 and 12 h) to induce the expression of endogenous p53. Cells were harvested for total RNA isolation followed by qPCR using primers specific for RLIM, p21, p53 and β-actin mRNA, respectively. (B) U2-OS cells were transfected with 20 µM p53 siRNA or 20 µM NS (non-specific) siRNA as control. RNA isolation and qPCR were performed as described above. (C) U2-OS or Hep3B cells were transfected with increasing amounts of p53 and harvested 24 h after transfection. Cells were harvested for total RNA isolation followed by qPCR using primers specific for RLIM and β-actin respectively. (D) U2-OS cells were treated with 20 µM etoposide for the indicated period (0, 6 and 12 h) to induce the expression of endogenous p53. Cells lysates were immunoblotted with antibodies to p53, RLIM and β-actin. (E) U2-OS or (F) Hep3B cells were transfected with different amount of plasmids encoding p53. Cells were harvested 40 h after transfection and lysates were immunoblotted with antibodies to p53, RLIM and β-actin.
Figure 3.
Sp1 Binds to the RLIM Promoter both in Vitro and in Vivo.
(A) Schematic representation of the RLIM promoter region in NP500-Luc. The four putative Sp1 binding sites S1 to S4 (denoted as the solid bar⧫) are shown. The arrow indicates the transcription start site. The triangles mark the flanking sites of the PCR fragment amplified in the ChIP assays (–200 bp to +50 bp). (B) ChIP analysis for the binding of endogenous Sp1 to the RLIM promoter. Chromatin immunoprecipitations (ChIP) were performed with antibodies against either IgG as control or Sp1. DNA recovered from immunocomplexes were subjected to PCR using primers flanking Sp1 binding region, or primers corresponding to irrelevant upstream chromatin region. PCR was performed without chromatin as negative control (B, primers corresponding to binding region. C, primers corresponding to irrelevant upstream chromatin region). (C) EMSA analysis of the binding of Sp1 to the RLIM promoter in vitro. EMSA was performed using DIG-labeled PCR fragments of the RLIM promoter containing each putative Sp1 binding site as indicated. DIG labeled probes were incubated in binding reactions in the presence or absence of 150 ng rhSp1 protein (Promega). Lanes 1, 4 and 7: probes only. Lanes 2, 5 and 8: rhSp1+ probes. Lanes 3, 6 and 9: rhSp1+ probes +50-fold excess cold unlabeled competitor DNA. Bands for the Sp1/DNA complexes and free probes are indicated. (D) EMSA analysis of the binding of Sp1 or p53 to the RLIM promoter region using the LightShift Chemiluminescent EMSA Kit. Lanes 1, 4 : Biotin-labeled probes only. Lane 2: rhSp1+ Biotin-labeled probes. Lane 3: rhSp1+ Biotin-labeled probes +50-fold excess cold unlabeled competitor DNA. Lane 5: rhp53+ biotin-labeled probes. Lane 6: rhp53+ biotin-labeled probes +50-fold excess cold unlabeled competitor DNA.
Figure 4.
p53 inhibits Sp1-mediated activation of the RLIM promoter.
(A) Sp1 activated the RLIM promoter activity. H1299 cells were cotransfected with 100 ng of NP500-Luc with either 160 ng pCMV empty vector as control or increasing amounts (0, 10, 20, 40, 80 and 160 ng) of pCMV-Myc-Sp1 plasmid. The total amount of plasmid in each transfection was adjusted to be the same using the empty vector. Cells were harvested 30 h after transfection and lysed for measuring luciferase activity. (B) p53 inhibits Sp1-stimulated activity of the RLIM luciferase reporter. H1299 cells were cotransfected with 100 ng of NP500-Luc and increasing amounts (0, 1, 2.5, 5 and 10 ng) of pCMV-HA-p53 plasmid in the presence of a fixed amount (160 ng) of pCMV-Myc-Sp1 plasmid. H1299 cells were transfected with 100 ng NP500-Luc alone as control. Cell lysates were used for luciferase assays as described above. (C) p53 mutants can not inhibit Sp1-stimulated activity of the RLIM luciferase reporter. The indicated p53 wild type or mutant constructs were cotransfected with 100 ng of RLIM promoter reporter construct NP500-Luc and 160 ng pCMV-Myc-Sp1 plasmid. Cells were harvested 30 h after transfection and the cell lysates were prepared and used for luciferase assays as described above.
Figure 5.
Identification of the essential RLIM promoter regions required for p53 mediated transrepression.
(A) Schematic representation of the RLIM promoter luciferase reporter NP500-Luc (−500/+100) and various 3′ serial deletions luciferase reporter constructs of the RLIM promoter (NP500-DN 100 to NP500-DN250). The four putative Sp1 binding sites S1 to S4 are highlighted (denoted as the ). The broken line (…) indicates the deleted region of individual construct. The arrow indicates the transcription start site. (B) Various RLIM promoter luciferase constructs described in Figure. 5A or control pGL3 vector were transfected into cells for luciferase reporter assays. For p53, 10 ng of pCMV-HA-p53 plasmid was used. For vector, 10 ng of pCMV-vector plasmid was used. Aliquots of cell extracts were assayed for luciferase activity as described above.
Figure 6.
Sp1-binding sites mutations abrogate the repression of RLIM promoter by p53.
(A) Schematic representation of the RLIM promoter luciferase reporter construct NP500-Luc and various mutated constructs with the Sp1 binding sites mutations (NP500-M1 to NP500-M1234). The four Sp1 binding sites S1 to S4 (wild type denoted as □ and mutations denoted a ▪) are shown. The sequences of the Sp1 binding sites are shown in the lower panel with the mutated sites underlined. (B–D) Cotransfection was performed as described in Figure. 5B using NP500-Luc and various mutated constructs with indicated Sp1-binding sites mutations, respectively. Luciferase assays were performed as described above. (E) EMSA analysis of the binding of Sp1 to RLIM promoter with different Sp1 binding site mutations (M1–M4). EMSA was performed as described in Figure. 3D. Lane 1: rhSp1+Biotin-labeled probes containing wild type Sp1-1 binding site +50-fold excess cold unlabeled competitor DNA. Lane 2: rhSp1+Biotin-labeled probes containing wild type Sp1-1 binding site. Lane 3: biotin-labeled probes containing wild type Sp1-1 binding site only. Lanes 4, 6, 8: rhSp1+Biotin-labeled probes containing mutant Sp1 binding site. Lanes 5, 7, 9: Biotin-labeled probes containing mutant Sp1 binding site only.
Figure 7.
p53 interacts with Sp1 and inhibits its binding to the RLIM promoter.
(A) H1299 cells were transfected with pCMMV-HA-p53, pCMV-Myc-Sp1 alone or both as indicated. Whole-cell extracts were immunoprecipitated with anti-HA antibody and subjected to Western blot analysis with anti-Myc antibody. As control, 10% of the cell lysates were used as input. (B) Effect of p53 on Sp1 binding to the RLIM promoter. Purified recombinant Sp1 and p53 proteins were used for EMSA. The DIG-labeled probe spanning the –100/+50 region of the RLIM promoter containing three putative Sp1 binding sites (S2, S3 and S4) was incubated in binding reactions with either rhSp1 protein alone (Lane1) or together with increasing amounts of p53 protein (Lanes 2 and Lane 3). EMSA was performed as described above. Bands for the Sp1-DNA complex and free probes are indicated. (C) ChIP analysis for the binding of Sp1 to the RLIM promoter. Chromatin immunoprecipitations (ChIP) were performed with antibodies against either IgG as control or Myc. DNA recovered from immunocomplexes were subjected to PCR using primers flanking Sp1 binding region. (D) p53 prevents the binding of Sp1 to the RLIM promoter in vivo. The H1299 cells transfected with either control vector plasmid (C, lanes 1, 3 and 5) or pCMV-Myc-Sp1 together with pCMV-HA-p53 plasmids (S+P, lanes 2, 4 and 6) were subjected to ChIP assays using anti-Myc, anti-HA or IgG antibodies as indicated. DNA purified from input chromatin (Input) or immunocomplexes were subjected to PCR analysis using primers flanking the –200/+50 region of the RLIM promoter or p21 promoter as a positive control.
Figure 8.
Mechanism of inhibition of RLIM by p53.
(A) p53 directly interacts with and sequesters Sp1 from the RLIM promoter, thereby inhibiting the transcriptional activation of RLIM by Sp1. (B) Dual mechanism of inhibition of RLIM by p53. p53 inhibits the transcriptional activation of RLIM by Sp1. p53 also activates the expression of RLIM E3 ubiquitin ligase Siah-1, leading to the degradation of RLIM protein.