Comprehensive Genomic Screens Reveal Multiple Modes of Action of the PLZF-RAR-α Oncoprotein

The t(11;17)(q23;q21) translocation is associated with a retinoic acid-insensitive form of acute promyelocytic leukemia (APL) involving the production of reciprocal fusion proteins PLZF-RARα and RARα-PLZF. These proteins mediate malignant transformation by binding to and dysregulating RARα/RXR and PLZF target genes, respectively. In order to investigate the molecular basis of PLZF-RARα induced leukemia, we performed a genome wide screen for PLZF-RARα direct target genes using a gain of function model in which PLZF-RARα was expressed in human U937 leukemia cells. Chromatin from U937/PLZF-RARα cells was immunoprecipitated using PLZF antibodies, amplified by ligation-mediated PCR and biological triplicates were hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,659 human promoters. We identified 4916 genes directly bound by PLZF-RARα (2/3 biological replicates, FDR <0.2). These genes were highly enriched for ontological categories including immunity and defense (p<10-6), apoptosis (p<2×10-5), cell cycle (p<10-3) and oncogenesis (p<10-2). Gene expression profiling of U937/PLZF-RARα cells revealed that 34% of direct targets were also transcriptionally regulated in response to PLZF-RARα induction. Despite the established role of PLZF-RARα as a transcriptional repressor, 56% of genes bound by PLZF-RARα were upregulated and 44% repressed. Bioinformatic analysis of PLZF-RARα bound sequences using the MATRIXReduce algorithm identified the ‘-AGGTCA-‘ core sequence as the highest ranked position specific affinity matrix (PSAM). Comparison of this matrix with known transcription factor binding sites from the JASPAR core database revealed high similarity to the recognition sequence for the RAR-related orphan receptor A1 (RORA1) (E value: 5.2×10-3), RORA2 (3.5×10-2) and RXRA-VDR (4.4×10-2). This suggests that the natural binding site of PLZF-RARα is similar to that of other nuclear receptors. The ‘GTCA’ core sequence is frequently observed in canonical retinoic acid receptor response elements and this motif was only associated with genes repressed with binding by PLZF-RARα. Together these results are consistent with the idea that PLZF-RARα acts in large part as a dominant negative retinoic acid receptor. A comparison of genes bound directly by PLZF-RARα with gene expression profiles from 22 APL (4 PLZF-RARα, 18 PML-RARα) and 99 acute myeloid leukemias (AML) selected at random from the Erasmus University dataset, using gene set enrichment analysis, revealed that direct targets of PLZF-RARα were differentially repressed in APL when compared to other forms of AML. Overexpression of PLZF-RARα in murine hematopoietic progenitors and human CD34+ cord blood, blocked myeloid differentiation, an effect associated with the repression of C/EBP genes (α, β and ε), which were identified as direct targets of PLZF-RARα by ChIP-chip. Treatment of primary CD34+ cells with ATRA led to an increase in CEBPα and β, but repression of CEBPε was not relieved. Overexpression of PLZF-RARα in primary murine bone marrow led to an increase in the more primitive Sca1+ population, coincident with increased serial replating ability. Overexpression of PLZF-RARα in mouse and human progenitors led to increased proliferation with more cells in the S and G2/M phases of cell cycle. Correlating with this effect, genes with defined roles in hematopoietic stem cell self-renewal including HOXA9 and MPL were bound and activated by the induction of PLZF-RARα in U937 cells. Increased proliferation was also coincident with repressed expression of Cdkn2d (p19) a cyclin dependent kinase inhibitor, also directly bound by PLZF-RARα. PLZF-RARα appears to transform cells through three interlinked modes of action, inhibition of differentiation by direct repression of key myeloid transcription factors, stimulation of proliferation by repression of a cyclin dependent kinase inhibitor and activation of genes critical for self renewal.