Growth suppression by acute promyelocytic leukemia-associated protein PLZF is mediated by repression of c-myc expression - PubMed (original) (raw)

Growth suppression by acute promyelocytic leukemia-associated protein PLZF is mediated by repression of c-myc expression

Melanie J McConnell et al. Mol Cell Biol. 2003 Dec.

Abstract

The transcriptional repressor PLZF was identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (APL). Ectopic expression of PLZF leads to cell cycle arrest and growth suppression, while disruption of normal PLZF function is implicated in the development of APL. To clarify the function of PLZF in cell growth and survival, we used an inducible PLZF cell line in a microarray analysis to identify the target genes repressed by PLZF. One prominent gene identified was c-myc. The array analysis demonstrated that repression of c-myc by PLZF led to a reduction in c-myc-activated transcripts and an increase in c-myc-repressed transcripts. Regulation of c-myc by PLZF was shown to be both direct and reversible. An interaction between PLZF and the c-myc promoter could be detected both in vitro and in vivo. PLZF repressed the wild-type c-myc promoter in a reporter assay, dependent on the integrity of the binding site identified in vitro. PLZF binding in vivo was coincident with a decrease in RNA polymerase occupation of the c-myc promoter, indicating that repression occurred via a reduction in the initiation of transcription. Finally, expression of c-myc reversed the cell cycle arrest induced by PLZF. These data suggest that PLZF expression maintains a cell in a quiescent state by repressing c-myc expression and preventing cell cycle progression. Loss of this repression through the translocation that occurs in t(11;17) would have serious consequences for cell growth control.

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Figures

FIG. 1.

FIG. 1.

PLZF expression in U937T cells induces cell death. (A) At the indicated hour post tetracycline withdrawal, 106 U937T:PLZF45 cells were collected and blotted for PLZF expression with monoclonal antibody 2A9. (B) PLZF45 cells without or with PLZF expression were seeded at 5 × 104/ml, and 100 μl of each culture was assayed in triplicate with a CellTitre 96 Aq kit (Promega) at the time points indicated. The data shown are averages of three independent experiments, and the error bars represent standard deviations. (C) Induction of apoptosis was measured by annexin V positivity. Cells were grown in tetracycline-containing (no PLZF) or tetracycline-free (plus PLZF) medium for 6 days. At the time points indicated, 106 cells were withdrawn from each culture, incubated with fluorescein isothiocyanate-conjugated annexin V antibody and PI, and then analyzed by flow cytometry. The cells were separated by annexin V labeling, and the annexin V-positive, PI-negative population was quantified.

FIG. 2.

FIG. 2.

PLZF suppresses expression of c-myc. PLZF45 and Neo1 cells were withdrawn from tetracycline, and mRNA was harvested at 12, 24, and 48 h, labeled with Cy3 or Cy5, and hybridized against Lymphochip. (A) Expression data for PLZF, c-myc, and c-myc target genes from the Lymphochip microarray analysis. Change in gene expression is taken from the average signal of at least three hybridization spots of the cDNA on the array and is expressed relative to the level at the zero time point for each message. (B) Total RNA was purified at the time points indicated either before or after tetracycline withdrawal and used in a Northern blot assay. c-myc and GAPDH expression in PLZF45 (left), RARα-PLZF (center), and Neo1 (right) cells was assessed in each case. M, marker. The values at the top are numbers of days in culture.

FIG. 3.

FIG. 3.

PLZF interacts specifically with a sequence from the c-myc regulatory region. (A) Schematic of the c-myc promoter showing the location of EMSA probes Site1 (black box 1) through Site5 (black box 5) and the EMSA probes created by PCR and restriction digestion. H, _Hin_dIII site; B, _Bgl_I site; K, _Kpn_I site; N, _Not_I site; X, _Xho_I site; long, full-length PCR probe. Locations of the P0, P1, and P2 promoters are indicated by arrows. Scale bar, 200 bases. (B) Nuclear extracts were used in an EMSA on Site1 through Site5 and the probes spanning the proximal promoter region, long. The extracts used are indicated as follows: 0, probe only; −, PLZF45 nuclear extract with no PLZF; +, PLZF45 nuclear extract with PLZF; H, HEL cell nuclear extract; A, anti-PLZF monoclonal antibody added. IL-3R, PLZF binding site from the IL-3Rα promoter. (C) The fourth base of the PLZF consensus site in Site2 was mutated from C to A (Site2Mut4) and used in EMSAs with nuclear extracts from PLZF45 cells with and without PLZF expression. 0, probe only; −, PLZF45 nuclear extract with no PLZF; +, PLZF45 nuclear extract with PLZF; A, anti-PLZF monoclonal antibody added. (D) The interaction between PLZF and Site2 (lane 3) was competed with increasing concentrations of unlabeled Site2 probe (lanes 4 and 5) but not by unlabeled noninteracting probe Site3 (lanes 6 and 7). Black asterisk, PLZF shift; grey asterisk, PLZF supershift.

FIG. 4.

FIG. 4.

ChIP of PLZF on the c-myc promoter. (A) Lysates were made 12 and 24 h after tetracycline withdrawal as indicated, and chromatin was immunoprecipitated with antibodies to PLZF and FLAG M2 as a nonspecific control or no antibody. This was then purified and used as a template for PCR. PCR was carried out with primers to the c-myc 5′ regulatory region. −, no PLZF; +, PLZF expressing; PLZF, anti-PLZF monoclonal antibody; FLAG, anti-FLAG M2 epitope antibody; noAb, no antibody added; input, 10% of the immunoprecipitation input; g, genomic DNA; NTC, no-template control PCR. (B) Lysates from cells induced for 24 h were immunoprecipitated (IP) with either an anti-PLZF monoclonal antibody (PLZF), the RNA polymerase II large subunit (PolII), or preimmune rabbit immunoglobulin G (IgG), and the chromatin was used for PCR of the c-myc 5′ regulatory region. (C) Lysates from a 24-h induction were immunoprecipitated with either the anti-PLZF monoclonal antibody (PLZF) or the FLAG control, and the purified chromatin was queried with primers to the cyclin A2 promoter. (D) Lysates from a 24-h induction were immunoprecipitated with either anti-PLZF monoclonal antibody 2A9 or a FLAG control, and the purified chromatin was queried with primers to the bcl-6 promoter. The sequences of all of the primers used are listed in Table 1.

FIG. 5.

FIG. 5.

PLZF represses a c-myc promoter reporter construct. 293T cells were seeded into 24-well dishes and transfected with 100 ng of reporter plasmid and 400 ng of effector plasmid. All luciferase activity is expressed relative to the expression of an internal Renilla luciferase promoter, and each bar represents the combined data from at least three experiments, each in triplicate. (A) The promoter constructs shown on the left, empty pXP2, two fragments of the c-myc promoter cloned into pXP2—full length (cmyc2.5) and 142 bp (cmyc0.14)—a minimal tk promoter (tkluc), or four PLZF binding sites upstream of the minimal tk promoter (PLZF-tkluc), were transfected with either empty expression vector SG5 (white bar), an SG5 vector expressing PLZF (black bar), or RARα-PLZF (grey bar). The white boxes represent the c-myc promoter fragments, the grey boxes represent the tk promoter, and the black bars represent PLZF binding sites. The effect of each plasmid on the activity of the promoter fragments is shown on the right. (B) A mutation in the PLZF binding site (black bar) was introduced into the 2.5-kb c-myc construct to create cmyc2.5ΔPLZF (grey bar). The effect of the mutation on PLZF-mediated repression is shown. Repression is expressed relative to the level of activity with the empty SG5 vector, which was normalized to 100%. (C) The effect of PLZF on the series of minimal promoter mutant constructs depicted was expressed relative to the activity of the construct in the presence of the empty vector SG5. White box, c-myc minimal promoter; grey bar, mutation. The grey box represents the tk promoter, and the black bars represent PLZF binding sites.

FIG. 6.

FIG. 6.

PLZF-mediated c-myc repression is reversible. (A) Replacement of tetracycline into inducible cell medium turned off PLZF expression. PLZF45 cells were grown in tetracycline (lane 1) or withdrawn from tetracycline for 24 (lanes 2 and 3), 48 (lanes 4 and 5), or 72 (lanes 6 and 7) h. Tetracycline was added back to each culture for 48 (lanes 3 and 7) or 72 (lane 5) h, and then 106 cells were collected from each condition, lysed, and blotted with PLZF monoclonal antibody 2A9. The lower half of the blot was probed separately with a monoclonal antibody against GAPDH. Lane 8, control for PLZF expression. (B) mRNA was purified from the cultures in panel A and used in a real-time PCR. PLZF, c-myc, and GAPDH messages in each condition were quantified, and the Ct for each message in the absenceof PLZF was set as 1. For each message, the change in Ct (ΔCt) between samples was determined in triplicate and averaged and expressed relative to the change in GAPDH to convert into the relative change in expression.

FIG. 7.

FIG. 7.

c-myc expression rescued PLZF-mediated cell cycle arrest. (A) Schematic (top) of the bicistronic c-mycER:GFP vector that was transfected into 293T cells. The lysates were blotted with a monoclonal antibody to c-myc. The black arrow indicates the band resulting from the bicistronic c-myc-ER fusion construct, and the grey arrow indicates endogenous c-myc. (B) PLZF45 cells were split between −PLZF and +PLZF (by tetracycline withdrawal), pulsed with BrdU, and grown in the absence (black bar) or presence (grey bar) of 200 nM 4OHT. The proportion of BrdU-labeled cells in each condition was determined by fluorescence-activated cell sorter analysis with a Phoenix Red-conjugated anti-BrdU monoclonal antibody. (C) PLZF45 cells were electroporated with c-mycER:GFP or the GFP vector and then plated into +tet or −tet medium. Cultures were then split in two again and c-myc was activated by addition of 4OHT to one-half of each culture. After 4 days, cells were pulsed with BrdU and labeled with an anti-BrdU monoclonal antibody conjugated to Phoenix Red. Cells were analyzed by flow cytometry; those expressing GFP were gated, and the extent of BrdU incorporation was assessed in the GFP-positive cells. The percentage of BrdU-labeled cells for −PLZF (white bars) and +PLZF (grey bars) under each condition is expressed relative to the −PLZF value under each condition, which was designated 100. Vector, MIGR1 transfected; c-mycER, c-mycER:GFP transfected. The data presented are from a representative experiment, and the same result was observed in three repetitions. (D) Cells were electroporated and treated with 4OHT as in the experiment whose results are shown in panel C. Three days postelectroporation, GFP-positive cells were separated by flow cytometry and RNA was extracted and transcribed into cDNA. PLZF (left panel) and hTERT (right panel) transcript levels under each condition were assessed by quantitative reverse transcription-PCR, and each change was expressed relative to GAPDH. The experiment was performed in triplicate, and the error bars represent the standard error of the mean. The data shown are from a representative experiment; the same effect was observed in three repetitions.

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