PLZF-RARα Utilizes the Histone Methyl Transferase G9a/GLP and the Histone Demethylase LSD1 to Repress RARα Target Genes and Block Myeloid Differentiation

As a result of the t(11;17) translocation in retinoic acid resistant subtype of acute promyelocytic leukemia (APL), the transcriptional repression domains of the Promyelocytic Leukemia Zinc Finger protein (PLZF) are fused to the ligand binding and DNA binding domains of the Retinoic Acid Receptor α (RARα). The expression of PLZF-RARα as well as the reciprocal RARα-PLZF protein both appear to contribute to leukemogenesis. While the mode of action of PML-RARα has been studied in detail, less is known about transcriptional repression mediated by PLZF-RARα. We and others previously showed an important role of histone deacetylases in PLZF and PLZF-RARα mediated gene repression. We now find that expression of PLZF-RARα also modulates gene expression through changes in the state of histone methylation at target promoters. PLZF-RARα co-precipitated in vivo with endogenous G9a, a histone methyl transferase responsible for the mono and di-methylation of euchromatic histone 3 lysine tail residue 9 (H3K9me1/2), a covalent modification associated with gene repression. Deletion analysis of the PLZF-RARα fusion protein showed that the BTB/POZ domain of PLZF fused to RARα was sufficient to mediate this interaction. PLZF-RARα also bound in vivo to LSD1, a histone demethylase that removes methyl groups from mono or di-methylated Histone 3 lysine 4 (H3K4me1/2), a change generally associated with gene repression. As with G9a the BTB/POZ domain of PLZF was implicated in binding to LSD1. Co-precipitation experiments showed a robust interaction between PLZF-RARα and G9a and LSD1 while RARα, PML-RARα and NPM-RARα bound much more weakly, suggesting that the interaction with these histone modifying enzymes may be a mechanism relatively specific to t(11;17)-associated APL. To identify genes modulated by PLZF-RARα and determine how PLZF-RARα affects the chromatin of such genes we induced expression of PLZF-RARα in a U937 tetracycline-regulated system. PLZF-RARα directly repressed known RARα target genes such as NFE2, PRAM1 and C/EBPε. As a result of PLZF-RARα expression, U937T cells were blocked in differentiation characterized by decreased expression of the myeloid cell surface markers CD11b, CD14 and CD33. Chromatin immunoprecipitation experiments in this cell line showed that PLZF-RARα expression was associated with an increase in H3K9me1/me2 at the NFE2, PRAM1 and C/EBPε promoters. Knockdown of endogenous G9a by shRNA transduction reversed transcriptional repression mediated by the fusion protein on all three promoters. Both results are consistent with the presence of G9a in PLZF-RARα transcriptional complex. By contrast, the H3K4 methylation changes in response to PLZF-RARα were promoter specific and complex: while NFE2 exhibited a decrease in H3K4me1/2, consistent with the recruitment of LSD1 and demethylation, PRAM1 and C/EBPε showed an increase in these two modifications. Inhibition of LSD1 by tranylcypromine treatment as well as knockdown of LSD1 by shRNA only reverted PLZF-RARα repression of NFE2. PLZF-RARα recruitment to all three genes was associated with a decrease in H3K4trimethylation, a modification only accomplished by jumanji-class histone demethylases. Consistent with the biochemical information, knockdown of G9a or its heterodimeric partner GLP, showed a strong biological phenotype, reverting the block in myeloid differentiation caused by PLZF-RARα as measured by the expression of the myeloid cell surface markers CD11b and CD14. Depletion of LSD1 only modestly interfered with the differentiation block mediated by the fusion protein. Gene regulation by PLZF-RARα is associated with a complex set of chromatin changes mediated by a combination of histone deacetylases, methyl transferase and demethylases. All three classes of enzymes may represent therapeutic targets in t(11;17)-APL.