Abstract
RUNX1/ETO (RE), the t(8;21)-derived leukemic transcription factor associated with acute myeloid leukemia (AML) development, deregulates genes involved in differentiation, self-renewal and proliferation. In addition, these cells show differences in cellular adhesion behavior whose molecular basis is not well understood. Here, we demonstrate that RE epigenetically silences the gene encoding P-Selectin Glycoprotein Ligand-1 (PSGL-1) and downregulates PSGL-1 expression in human CD34+ and murine lin− hematopoietic progenitor cells. Levels of PSGL-1 inversely and dose-dependently correlate with RE oncogene levels. However, a DNA-binding defective mutant fails to downregulate PSGL-1. We show by ChIP experiments that the PSGL-1 promoter is a direct target of RE and binding is accompanied by high levels of the repressive chromatin mark histone H3K27me3. In t(8;21)+ Kasumi-1 cells, PSGL-1 expression is completely restored at both the mRNA and cell surface protein levels following RE downregulation with short hairpin RNA (shRNA) or RE inhibition with tetramerization-blocking peptides, and at the promoter H3K27me3 is replaced by the activating chromatin mark H3K9ac as well as by RNA polymerase II. Upregulation of PSGL-1 restores the binding of cells to P- and E-selectin and re-establishes myeloid-specific cellular adhesion while it fails to bind to lymphocyte-specific L-selectin. Overall, our data suggest that the RE oncoprotein epigenetically represses PSGL-1 via binding to its promoter region and thus affects the adhesive behavior of t(8;21)+ AML cells.
Highlights
RUNX1/ETO (RE), the t(8;21)-derived fusion protein, is present in 12% of de novo acute myeloid leukemia (AML) cases and up to 40% of M2 subtype AMLs according to the French– American–British classification.[1]
We show that RE directly interacts with the P-Selectin Glycoprotein Ligand-1 (PSGL-1) promoter region and epigenetically represses PSGL-1 in hematopoietic progenitor cells
As lentivirus-mediated expression of RE-inhibiting peptides induced high mRNA levels of PSGL-1 (Figure 4a), we investigated the connection between RE binding and PSGL-1 expression and examined PSGL-1 genomic sequences for the presence of potential RUNX1-binding motifs (TGT/CGGT) in silico
Summary
RUNX1/ETO (RE), the t(8;21)-derived fusion protein, is present in 12% of de novo acute myeloid leukemia (AML) cases and up to 40% of M2 subtype AMLs according to the French– American–British classification.[1]. PSGL-1 was identified in human primary t(8;21)+ AML patient samples (see Supplementary Figure 4).[31,35] As RE has been shown to induce epigenetic changes to repress its target genes,[31] we examined the RE-binding sites for the presence of several active and repressive histone marks including the repressive H3K27me[3] chromatin mark. This revealed H3K27 trimethylation of the PSGL-1 promoter upstream of exon 1 (Figure 4e). PSGL-1 upregulation occurred within 24 h after shRE-treated Kasumi-1 cells and P-selectin
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