The Acute Promyelocytic Leukemia (APL)-Associated Fusion Proteins PML/RAR and PLZF/RAR Directly Bind to and Inhibit GATA-1.

Abstract

The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation. The APL is a well characterized subtype of AML, which is related in the 95% of the cases by the presence of the t(15;17) and in 2% by the presence of t(11;17). In several cell models the resulting PML/RAR and PLZF/RAR fusion proteins (X-RAR) recapitulate the leukemic phenotype by inducing a state of refractoriness to various inducers of myeloid differentiation. Accordingly, expression in animal models of both PML/RAR and PLZF/RAR leads to the development of leukemia. The treatment with all trans retinoic acid (t-RA) is able to overcome the block of differentiation of PML/RARα- but not that of PLZF/RAR-positive-blasts. These fusion proteins block differentiation through several mechanisms such as aberrant chromatin modeling by aberrant recruitment of histone deacetylase activity or the deregulation of differentiation-relevant transcription factors such as PU.1, VDR or C/EBPalpha. The deregulated function of these transcription factors can be due to their transcriptional down-regulation or to a sequester by direct interaction. Nothing is known about how the X-RAR block erythroid and megacaryocytic differentiation. Therefore we investigated whether and how the X-RAR interfere with the functionality of the differentiation-relevant transcription factor GATA-1. It has been recently reported that the lack of GATA-1 severely impairs erythroid differentiation and contributes to the accumulation of immature megakaryocytic blasts. He we report thatGATA-1 directly interacts with X-RAR in vivo as revealed by co-immunoprecipitation and mammalian two hybrid assays;GATA-1 expression was not transcriptionally deregulated by the X-RAR;the GATA-1 binding capacity to the H2S beta-globin locus was severely inhibited by the presence of the X-RAR as revealed by ChIP experiments in K562, whereas the transactivation of the GATA-target promotor alpha-IIb was not impaired by the the X-RAR in classical transient promoter studies;treatment with t-RA restored GATA-1 binding to the H2S locus of the beta-globin gene;the overexpression of GATA-1 in the presence of EPO reduced the the colony forming units of PLZF/RAR-positive Sca1+/lin− hematopoietic stem cells (HSC) and diminished the replating efficiency of PML/RAR-positive HSC, but did not increase erythroid differentiation monitored by TER 119 expression. Taken together our data demonstrate that the X-RAR interfere with the functionality of GATA-1 by direct interaction with GATA-1. It remains to definitively clarify whether the X-RAR inhibit the access of GATA-1 to its target promoters or whether they interfere with the accessibility of the GATA-1 for transcriptional co-activators. The fact that the overexpression of GATA-1 did not increase differentiation of HSC and the X-RAR were unable to inhibit the transactivation of a GATA-1 target promoter strongly suggests that the X-RAR interfere with the GATA-1 functionality in the context of the chromatin.