Expression of ZBTB4 mRNA in human tumours. (A) ZBTB4 is expressed in a stage-dependent manner in human neuroblastoma. The graph is a summary of microarray data of ZBTB4 mRNA levels observed in different neuroblastoma tumour stages. The data are taken from the original analysis (Berwanger et al, 2002). Error bars represent standard error of the mean. (B) Confirmation of microarray data. The graph summarizes the RQ–PCR analysis of ZBTB4 expression in an independent set of 98 primary neuroblastoma tumours. Error bars represent standard error of the mean. (C) Expression of ZBTB4 correlates with patient survival. The graph documents the Kaplan–Meier analysis of neuroblastoma patients with ZBTB4 mRNA expression levels above (‘high') and below (‘low') the mean expression level. (D) ZBTB4 is repressed in glioblastoma and in prostate carcinoma relative to normal tissue. The data are taken from the studies summarized in Table 1, and were obtained from www.oncomine.org. The numbers refer to Table 1. In this and the following panel, data are presented as Box and Whisker graph with error bars representing the 5th and 95th percentile. (E) Stage-specific expression of ZBTB4 in breast and prostate carcinoma. The data are taken from the studies summarized in Table 2. The plots are taken from www.oncomine.org (BH, benign hyperplasia; IN, intraepithelial hyperplasia; LPC, localized prostate carcinoma; MPC, prostate carcinoma metastasis).

Depletion of Zbtb4 enables neuroblastoma cells to survive in the presence of vincristine. (A) Colony assays documenting growth of a control clone of SH-EP cells and a clone expressing the indicated shZBTB4 RNA after treatment with the indicated chemotherapeutic agents. Control (DMSO treated) plates were stained at day 7 and experimental plates stained 14 days after plating. Note that the ZBTB4 mRNA expression level of this clone is documented in Supplementary Figure 1A. (B) Cells expressing shZBTB4 have a survival advantage in the presence of vincristine. SH-EP cells were infected with vectors that express GFP and either a control shRNA vector or one of three distinct shZBTB4 RNAs as indicated. At the indicated time points after infection, the percentage of GFP-positive cells was measured by FACScan. All values were normalized to those obtained from a control plate expressing H2B-GFP. At the start of the experiment, approximately 10% of all cells were positive for GFP. (C) Vincristine induces apoptosis in control cells but G1 arrest in Zbtb4-depleted cells. The upper panels document FACS analyses of the DNA content of either a control clone or of a clone expressing shZBTB4 after treatment with DMSO or the indicated concentrations of vincristine for 48 h. The lower panels show immunoblots using antibodies directed against cleaved poly-ADP-ribose-polymerase and β-actin as loading control.

Depletion of Zbtb4 causes resistance to vincristine treatment through upregulation of P21CIP1. (A) Vincristine induces activation of p53. The panels show immunoblots of SH-EP cells infected with either a control vector (left) or a vector expressing a dominant-negative allele of p53 (p53DD; right) probed with the indicated antibodies. (B) Transient depletion of Zbtb4 upregulates P21CIP1 mRNA levels. The panels document the RQ–PCR analysis of relative mRNA expression level of ZBTB4, P21CIP1, 14-3-3σ, BTG2, GADD45, PUMA, BAX and NOXA in SH-EP and SH-SY5Y neuroblastoma cells transfected with either control or siRNA targeting ZBTB4. Cells were harvested 48 h after transfection and expression levels were determined by RQ–PCR. (C) Stable depletion of Zbtb4 upregulates p21Cip1 protein. The panels show immunoblots of p21Cip1 and Cdk2 protein levels in indicated SH-EP cell clones stably infected with either control vectors or vectors expressing shZBTB4. (D) Ectopic expression of Zbtb4 represses P21CIP1. The panels document the RQ–PCR analysis of the relative mRNA expression level of ZBTB4, P21CIP1, PUMA and NOXA in SH-EP cells stably infected with HA-tagged ZBTB4 vector or a control vector. (E) P21CIP1 is required for protection of SH-EP cells from vincristine-induced apoptosis by depletion of Zbtb4. The indicated SH-EP cell clones stably expressing either shZBTB4 or control shRNA were transiently transfected with vectors expressing shP21CIP1 or a scrambled shRNA. At 48 h after transfection, cells were plated in triplicate for vincristine treatment. Cells were treated with either vincristine or DMSO for 3 days; subsequently, the relative cell number was determined. Error bars represent standard error of the mean.

Zbtb4 regulates P21CIP1 expression independently of p53. (A) Expression of a dominant-negative allele of p53 blocks UV- and vincristine-induced upregulation of P21CIP1, NOXA and PUMA. Exponentially growing SH-EP cells were stably infected either with control retroviral vectors or with vectors expressing a carboxyl-terminal fragment of p53 (p53DD). Pools of infected cells were exposed to UVB or treated with 5 nM vincristine or DMSO as a control for 24 h. Cells were harvested and expression of the indicated genes was analysed by the RQ–PCR as before. (B) Regulation of P21CIP1 by Zbtb4 is independent of p53. Control SH-EP cells, SH-EP p53DD-transduced cells, HCT116 cells and HCT116 p53^−/− cells were transfected with siZBTB4 or control (scrambled) siRNA. At 48 h after transfection, cells were harvested and the expression of ZBTB4 and P21CIP1 was determined by the RQ–PCR analysis. Data are plotted as expression relative to an internal standard.

Zbtb4 suppresses cell cycle arrest in response to activation of p53. (A) Zbtb4 represses p21Cip1 expression in response to nutlin treatment. SH-EP cells were stably infected with either control retroviruses or viruses expressing HA-tagged Zbtb4. Pools of infected cells were either treated with DMSO as solvent control or with the indicated concentrations of nutlin for 33 h. The panels show immunoblots of cell lysates probed with the indicated antibodies; β-actin served as a loading control. (B) FACScan analysis showing the response to nutlin. The panel documents the cell cycle distribution of either control cells or SH-EP cells expressing Zbtb4 after treatment with the indicated concentrations of nutlin for 33 h. Error bars represent the standard deviation of triplicates. (C) The upper panels show immunoblots documenting expression of p53, phosphorylated p53 (p53^S15P) and of phosphorylated Chk1 (Chk1^S345P) in SH-EP cells treated with the indicated agents. The lower panels show immunoblots documenting expression of p21Cip1, total Zbtb4 and of exogenous Zbtb4 (detected using a HA antibody) in control SH-EP cells and in SH-EP cells expressing Zbtb4; β-actin was used as a loading control. (D) FACScan analysis of the responses to nutlin, adriamycin and etoposide of control SH-EP cells and of SH-EP cells expressing Zbtb4.

Zbtb4 and Miz1 form a transcriptional repressor complex that represses transcription of P21CIP1. (A) Scheme of the human P21CIP1 gene with indicated p53, Miz1 and putative Zbtb4-binding sites. Also indicated are the positions of primers used for the chromatin immunoprecipitation (ChIP) experiments shown below. Shaded grey areas denote exons. (B) Zbtb4 represses P21CIP1 promoter activity. Shown are reporter assays using P21CIP1 promoter constructs spanning the indicated nucleotides relative to the transcription start site. The graph shows the luciferase activity relative to the standard CMV-βGal after transfection of SH-EP cells with either expression plasmids encoding Zbtb4, Miz1 or both as indicated. The error bars represent the standard deviation of triplicate samples. Controls established that Zbtb4 does not inhibit expression of Miz1 (not shown). (C) ChIP demonstrating binding of Zbtb4 to the core of the P21CIP1 promoter, but not to a control region located in the 3′UTR. Cells were infected with viruses expressing either control shRNA or shZBTB4 as indicated. (D) Activation of P21CIP1 correlates with loss of endogenous Zbtb4 from the P21CIP1 promoter. The panels show ChIP assays documenting binding of p53, Miz1 and Zbtb4 to the P21CIP1 promoter in untreated SH-EP cells and in SH-EP cells exposed to adriamycin. For each protein, the difference between specific antibody and control antibody in percentage DNA bound is plotted. (E) Zbtb4 binds to Miz1 in lysates of co-transfected cells. HEK293 cells were transfected with expression vectors encoding HA-tagged Zbtb4 and/or Miz1 as indicated. Lysates were immunoprecipitated with the indicated antibodies. The left panels show immunoblots of the immunoprecipitates probed with the indicated antibodies. The right panels document an identical experiment except that 0.1 mg/ml ethidium bromide was added to the lysates before immunoprecipitation to exclude that the observed interaction is mediated by binding of both proteins to DNA. Both antibodies IgG mouse and IgG rabbit were used as controls. (F) Endogenous Zbtb4 and Miz1 co-immunoprecipitate in LS174T cells. Rabbit IgG was used as a control. (G) Zbtb4 and Miz1 form a DNA-binding complex. HEK293 cells were transfected with expression plasmids encoding Miz1 and/or Zbtb4 as indicated. Biotinylated oligonucleotides spanning the Zbtb4-binding site, the Miz1-binding site or a control sequence (3′UTR) were added and precipitated using avidin-sepharose. Bound proteins were detected by immunoblotting using the indicated antibodies. (H) Miz1 recruits Zbtb4 to the core P21CIP1 promoter. SH-EP cells were transfected either with scrambled siRNA or siRNA targeting MIZ1. ChIP assays were performed with control and Zbtb4 antibodies and analysed using the indicated primer pairs. The panel shows the difference between the specific signal and the signal obtained with the control antibody.

Zbtb4 recruits Sin3a to repress the P21CIP1 promoter. (A) ChIP assays documenting Zbtb4-induced alterations in histone modification at the P21CIP1 promoter. The assays were performed from either control SH-EP cells or SH-EP cells expressing Zbtb4 using the indicated antibodies. The signals were normalized to precipitations using a H2B antibody. (B) Inhibition of histone deacetylation reduces repression of P21CIP1 by Zbtb4. SH-EP cells were infected with either control retroviruses or retroviruses expressing ectopic Zbtb4. Cell pools were either treated with solvent control or with the histone deacetylase inhibitors trichostatin-A (TSA) or sodium butyrate as indicated. Shown are expression data for P21CIP1 mRNA relative to a control mRNA derived from the RQ–PCR analysis with error bars representing the standard deviation of the mean. The data are plotted as fold repression by Zbtb4 in each experimental condition. The concentration of TSA used was efficient in inducing overall histone acetylation as determined by immunoblot analysis (not shown). (C) Zbtb4 co-immunoprecipitates Sin3a. HEK293 cells were transfected with indicated HA-tagged vectors, and protein lysates were immunoprecipitated with HA antibodies. Associated endogenous Sin3a was visualized by immunoblotting. Here, 2% input of Sin3a is indicated. As a positive control, we used the repressor protein Mnt and a mutated version of Mnt, in which the Sin3a interaction domain had been deleted (MntΔSID) (Hurlin et al, 1997). (D) Endogenous Zbtb4 co-immunoprecipitates Sin3a and Sin3b. HCT116 whole cell extracts were immunoprecipitated with Zbtb4 or pre-immune antibodies; precipitates were probed with antibodies to mSin3b and mSin3a. Here, 2% of input is shown. (E) Association of Zbtb4 with histone deacetylase activity depends on Sds3. HEK293 cells were transfected with constructs expressing Zbtb4 or control vector and simultaneously infected with lentiviruses expressing either control shRNA or shRNA targeting Sds3. At 48 h later, whole cell lysates were prepared and immunoprecipitated with Zbtb4 or normal rabbit serum antibodies. HDAC activity in the immunoprecipitates was analysed. (F) Characterization of lentiviral shRNAs targeting SIN3A and SIN3B. HCT116 cells were infected with lentiviruses expressing the indicated shRNAs. At 48 h after infection, cells were harvested. Immunoblotting was performed with the indicated antibodies. (G) Zbtb4 recruits Sin3a and Sin3b to the core P21CIP1 promoter. Shown are ChIP assays from control SH-EP cells and from SH-EP cells expressing Zbtb4. (H) Zbtb4 shares transcriptional targets with Sin3. Comparison of transcripts regulated in Sin3- and Zbtb4-depleted cells relative to control HCT116 cells. Absolute values of the fold changes are indicated as >2.0-fold. The probability of chance of the overlap between shSIN3 and shZBTB4 as calculated by the hypogeometric distribution is <10⁻⁴.