Comprehensive genomic screens identify a role for PLZF-RARalpha as a positive regulator of cell proliferation via direct regulation of c-MYC

Abstract

The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RARalpha) and RARalpha-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARalpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARalpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARalpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARalpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARalpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARalpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARalpha.

Figure 1

Identification of PLZF-RARα direct target genes by ChIP-chip and gene expression arrays. (A) U937T:PLZF-RARα cells were withdrawn from tetracycline for 48 hours and PLZF-RARα expression was assessed by Western blot of whole-cell lysate using PLZF monoclonal antibody (2A9). (B) Loci-specific qPCR using unamplified ChIP samples confirmed PLZF-RARα binding to 100% of putative target promoters compared with uninduced controls (fold enrichment in brackets), a negative control exon region (—). Data are expressed as mean ± SEM of 3 independent experiments (**P < .01). (C) Comparison of PLZF-RARα ChIP dataset (FDR < 0.2, 2/3 replicates; 4916 genes) with gene expression dataset (> 1.5-fold, P < .05; 4472 genes) identified 1672 genes bound and transcriptionally regulated by PLZF-RARα.

Figure 2

PLZF-RARα direct transcriptional target genes are associated with an APL-specific phenotype. Gene expression profiles of 22 APL (PLZF-RARα [n = 4] and PML-RARα [n = 18]) and 99 AML patients were normalized by GC-RMA, and GSEA was used to generate a ranked list of genes distinguishing these phenotypes (red = up-regulated in APL vs AML blue = down-regulated in APL vs AML). The distribution of PLZF-RARα direct targets (1672 genes) within this ranked gene list was calculated and expressed as number of genes/decile. PLZF-RARα–bound and regulated genes are highly enriched in deciles 8 to 10, representing genes down-regulated in APL versus AML (●, P < .001), whereas genes from a randomly selected dataset were not significantly enriched in these quartiles (○, .01 < P < .1).

Figure 3

PLZF-RARα binds an extended repertoire of RAREs. (A) The frequency of consensus RAREs (PuG(G/T)TCA)(0-20 bp)(PuG(G/T)TCA) in PLZF-RARα–bound promoters (4916 genes) was calculated. The total number of RAREs and genes containing RAREs are shown to the far right. The frequency of spacer lengths corresponding to each RARE was also calculated. (B) The ECHS1 promoter peak contains multiple RAREs (underlined). (C) MATRIXReduce was used to identify consensus motifs in PLZF-RARα–enriched peak sequences (2/3 biologic replicates, FDR < 0.2, differentially expressed > 1.5-fold, P < .05; 1672 genes). The top-scoring motif identified in PLZF-RARα–bound promoters was most similar to nuclear hormone receptor binding sites containing the RARE (A/G)GGTCA half site.

Figure 4

PLZF-RARα differentiation block in U937 cells is responsive to retinoic acid. (A) U937T:PLZF-RARα cells were induced by tetracycline withdrawal for 5 days and the cell-surface expression of myeloid/monocytic differentiation markers (CD11b, CD33, CD14) was assessed by flow cytometry. Expression was calculated as the fold change in mean fluorescence intensity between PLZF-RARα–expressing and nonexpressing cells. In the absence of ligand (), PLZF-RARα inhibited the expression of CD11b, CD33, and CD14; however, in the presence of 10nM ATRA (■), PLZF-RARα enhances the expression of these markers. (B) Transcriptional response of PLZF-RARα target genes to low-dose ATRA (10nM). Data are expressed as mean ± SEM of 3 independent experiments.

Figure 5

PLZF-RARα increases the proliferative capacity of hematopoietic progenitors. (A) PLATE 293T retroviral producer cells were transfected with MPG, MPG-PLZF, and MPG-PLZF-RARα retroviral vectors, and whole-cell lysates were Western blotted with PLZF (2A9) antibody. (B) Murine lin⁻ hematopoietic progenitors were infected with MPG or MPG-PLZF-RARα retrovirus and plated in methylcellulose under myeloid-promoting conditions. Cell-cycle profiles of day-8 colonies were assessed after a 3-hour BrdU pulse. Cells were stained with anti–BrdU-APC antibody and 7-amino-actinomycin D and analyzed by flow cytometry. (C) Human lineage-depleted (Lin⁻) CD34⁺ progenitors from cord blood were infected with MPG or MPG-PLZF-RARα retrovirus and maintained in liquid culture. Cell counts were performed over a period of 40 days. (D) PLZF-RARα directly binds to the promoters of several key regulators of G1/S transition. RNA was extracted from MPG or MPG-PLZF-RARα–expressing murine colonies at day 8 and the expression of PLZF-RARα proliferation target genes (c-Myc, Dusp6, Cdkn2d, and Cdkn1b) was analyzed by qRT-PCR. Data are expressed as mean ± SEM of 3 independent experiments (**P < .05).

Figure 6

Sustained c-Myc expression is associated with survival of retinoic acid–treated PLZF-RARα–transformed murine progenitors. (A) Murine lin⁻ hematopoietic progenitors were infected with MPG or MPG-PLZF-RARα retrovirus and plated in methylcellulose under myeloid-promoting conditions for 7 days. Granulocytic differentiation was assessed by flow cytometry using CD11b-Pe-Cy7 and Gr-1-APC antibodies and gene expression was assessed by qRT-PCR. (B) Day-7 colonies were disaggregated and replated in the presence of 1μM ATRA (or dimethyl sulfoxide control). After 24 or 48 hours, several colonies were picked and disaggregated and differentiation was measured by flow cytometry as in panel A and changes in gene expression were measured by qRT-PCR. (C) Replated colonies treated with ATRA or control were harvested after 9 days of ATRA treatment, and differentiation was measured by flow cytometry as in panel A. Changes in gene expression were measured by qRT-PCR. Data are expressed as mean; error bars represent the maximum deviation of duplicate experiments. (D) PLZF-RARα blocks differentiation and activates proliferation by directly affecting genes involved in granulocyte differentiation and cell-cycle control. RA induces differentiation and transient growth arrest. The persistence of c-Myc expression and down-regulation of Dusp6 is associated with the continued survival of RA-treated PLZF-RARα–expressing cells.

Figure 7

PLZF-RARα functionally antagonizes PLZF-mediated repression at the c-MYC promoter. (A). PLZF and PLZF-RARα bind to an overlapping region of the c-MYC promoter, highlighted by the dotted box. This region lies upstream of the P0 promoter, approximately 1.5-kB upstream of the major transcriptional start site initiated from promoter P1. PLZF endogenous ChIP-chip was performed in the KG1a cell line and compared with PLZF-RARα ChIP-chip target genes identified in the U937:PLZF-RARα–inducible cell line. The major c-MYC transcripts initiated from this promoter region are indicated by solid black lines. Sequence analysis of the enriched peak using PATCH Public 1.0 identified putative RXR and RAR binding sites (underline) that may bind PLZF-RARα, overlapping with a previously characterized PLZF binding site (bold, enlarged font). (B) The pXP2-MYC2.5-kB luciferase reporter vector contains a 2.5-kB region of the c-MYC promoter harboring the PLZF-RARα–enriched peak. The c-Myc reporter (200 ng) was cotransfected with the indicated PLZF, PLZF-RARα, or PLZF-RARα mutant plasmids (800 ng) in 293 cells and lysates were harvested at 48 hours for luciferase activity. Data are expressed as mean ± SEM of 3 independent experiments (**P < .05).