Abstract
AbstractAlteration of lineage-specific transcriptional programs for hematopoiesis causes differentiation block and promotes leukemia development. Here, we show that AML1/ETO, the most common translocation fusion product in acute myeloid leukemia (AML), counteracts the activity of retinoic acid (RA), a transcriptional regulator of myelopoiesis. AML1/ETO participates in a protein complex with the RA receptor alpha (RARα) at RA regulatory regions on RARβ2, which is a key RA target gene mediating RA activity/resistance in cells. At these sites, AML1/ETO recruits histone deacetylase, DNA methyltransferase, and DNA-methyl-CpG binding activities that promote a repressed chromatin conformation. The link among AML1/ETO, heterochromatic RARβ2 repression, RA resistance, and myeloid differentiation block is indicated by the ability of either siRNA-AML1/ETO or the DNA methylation inhibitor 5-azacytidine to revert these epigenetic alterations and to restore RA differentiation response in AML1/ETO blasts. Finally, RARβ2 is commonly silenced by hypermethylation in primary AML blasts but not in normal hematopoietic precursors, thus suggesting a role for the epigenetic repression of the RA signaling pathway in myeloid leukemogenesis.
Highlights
Introduction target gene mediatingretinoic acid (RA) activity/resistance in cells
Northern blot analysis revealed that the siAGF1 oligonucleotide (siA/E)-RNA transcripts were expressed in SKNO-1 cells (Figure 1A), while quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blot analyses assessed a diminution of about 50% in the AML1/ETO mRNA and fusion protein levels in si-A/E cells as compared with mock-infected cells (Figure 1B-C)
The AML1/ETO knockdown restored the differentiation response of SKNO-1 cells to RA as shown by the specific induction of the expression levels of CD11b in RA-treated si-A/E cells, while the levels of CD14 remained unchanged (Figure 1E). These data suggested that threshold levels of AML1/ETO are required for the silencing of the RA signaling pathway and for inducing the differentiation block, which occurs toward the granulocytic lineage
Summary
At these sites, AML1/ETO recruits histone deacetylase, DNA methyltransferase, and DNA-methyl-CpG binding activities that promote a repressed chromatin conformation. The link among AML1/ETO, heterochromatic RAR2 repression, RA resistance, and myeloid differentiation block is indicated by the ability of either siRNA-AML1/ETO or the DNA methylation inhibitor 5-azacytidine to revert these epigenetic alterations and to restore RA differentiation response in AML1/ETO blasts. RAR2 is commonly silenced by hypermethylation in primary AML blasts but not in normal hematopoietic precursors, suggesting a role for the epigenetic repression of the RA signaling pathway in myeloid leukemogenesis. These events are essential during development and for the maintenance of tissue- and cell-type–specific functions.[1,2,3] The contribution of epigenetic mechanisms for a correct cell function is highlighted by the effects of their deregulation that in cooperation with genetic alterations lead to the establishment and progression of tumors.[4,5,6,7,8,9]
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