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Abstract
Acute myeloid leukemia 1 (AML1), also denoted Runx1, is a transcription factor essential for hematopoiesis, and the AML1 gene is the most common target of chromosomal translocations in human leukemias. AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. Here we show that both subunits of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1. The regulatory CN subunit binds AML1 with a higher affinity and in addition also interacts with the isolated runt homology domain. The related Runx2 transcription factor, which is essential for bone formation, also interacts with CN. A constitutively active derivative of CN is shown to activate synergistically the GM-CSF promoter/enhancer together with AML1 or Runx2. We also provide evidence that relief of the negative effect of the AML1 sites is important for Ca(2+) activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases this Ca(2+) activation. Both subunits of CN interact with AML1 in coimmunoprecipitation analyses, and confocal microscopy analysis of cells expressing fluorescence-tagged protein derivatives shows that CN can be recruited to the nucleus by AML1 in vivo. Mutant analysis of the GM-CSF promoter shows that the Ets1 binding site of the promoter is essential for the synergy between AML1 and CN in Jurkat T cells. Analysis of the effects of inhibitors of the protein kinase glycogen synthase kinase-3beta and in vitro phosphorylation/dephosphorylation analysis of Ets1 suggest that glycogen synthase kinase-3beta-phosphorylated Ets1 is a target of AML1-recruited CN phosphatase at the GM-CSF promoter.
Highlights
The DNA binding domain of Acute myeloid leukemia 1 (AML1), Runx2, and Runx3 is denoted the runt homology domain (RHD) [10] after the first identified member of this family of transcription factors, the Drosophila protein Runt
AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. We show that both subunits of the Ca2؉/ calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1
We show that relief of the negative effect of the AML1 sites is of importance for Ca2ϩ activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases Ca2ϩ activation
Summary
The DNA binding domain of AML1, Runx2, and Runx3 is denoted the runt homology domain (RHD) [10] after the first identified member of this family of transcription factors, the Drosophila protein Runt. In accordance with these results, mutation of both AML1 sites gave a 4.2 Ϯ 0.4-fold increase in transcription from the GMCSF promoter/enhancer in Jurkat cells (Fig. 1B). Expression of ⌬CN, a C-terminally truncated and thereby Ca2ϩ/calmodulinindependent constitutively active A subunit of CN, activated the wild type GM-CSF reporter ϳ2-fold in Jurkat, whereas it had no significant effect on the AML1 site-mutated reporter (Fig. 1B, cf bars 1, 2, 13, and 14).
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