Fig 2: AML1-ETO and miR-29b-1 form a regulatory circuit that modulates leukemic phenotypeOur results establish a regulatory circuit between the AML1-ETO oncogene and the miR-29b-1 tumor suppressor; AML1-ETO transcriptionally regulates miR-29b-1, and miR-29b-1 translationally inhibits AML1-ETO. MicroRNA-29b-1 has been implicated in hematological malignancies and is a direct transcriptional target of the hematopoietic C/EBPa transcription factor [29]. Our results suggest that AML1-ETO regulates the expression of miR-29b-1 directly by binding to the promoter regulatory region of the miR-29a/b-1 locus, as well as indirectly through down-regulation of C/EBPa, which activates the miR-29a/b-1 locus [29]. It has been shown that miR-29b-1 directly inhibits the expression of Myc, a key regulator of cell cycle and cell growth, Akt2, an upstream sensor in the DNA repair/damage pathways and a regulator of cell apoptosis, and Cyclin D2 (CCND2), which mediates cell cycle control [30]. The current study provides evidence of a complex interplay between AML1-ETO and miR-29b-1 that leads to partial apoptosis, decreased BrdU incorporation, and release from AML1-ETO-mediated myeloid differentiation block. Our study and others together implicate miR-29b-1 as a key modulator of leukemic phenotype in acute myeloid leukemia. Green arrows indicate an activating event, while red symbols show an inhibitory event.

Fig 3: AML1-ETO occupies the miR-29a/b-1 locus and transcriptionally down regulates the miR-29a/b-1 primary transcriptA. Top schematic shows the miR-29a/b-1 locus on human chromosome 7 (Accession Number EU154353) [45]. Runx binding sites are indicated. RNAs originating from the locus are shown; orange blocks indicate exons, while the orange line shows intronic regions. Genomic tracks from ChIP-seq dataset from Trombly et al [35] were re-analyzed for occupancy of the miR-29a/b-1 locus by AML1-ETO, NCoR, or the activating H3K4me3 and suppressive H3K27me3 histone modifications. B. Actively proliferating Kasumi-1 cells were subjected to chromatin immunoprecipitation with two separate antibodies against ETO (black bars), a rabbit polyclonal antibody against carboxy-terminus of RUNX1, which is not present in AML1-ETO (white bar), and an IgG isotype control (gray bar). Immuno-enriched chromatin was analyzed by quantitative PCR using primers encompassing Runx binding sites in the regulatory region of the miR-29a/b-1 locus. Bar graph shows immuno-enriched chromatin as a percentage of input. C. Western blot analysis of SKNO-1 cells infected with a non-silencing short hairpin RNA (shNS), or an shRNA selectively targeting AML1-ETO (shA/E). Significant knock down of the AML1-ETO protein was reproducibly obtained 5 days post-infection. GAPDH was used as protein loading control. D. Total cellular RNA from SKNO-1 cells infected with shNS and shA/E was analyzed for expression of the pri-miR-29a/b-1, as well as of CDKN1A, a known AML1-ETO target gene. Bar graph represents an average of three independent experiments, and error bars indicate standard deviation (SD). Student's t-test shows highly significant changes in the expression of pri-miR-29b-1 and CDKN1A (p > 0.01) upon AML1-ETO knockdown.

Fig 4: miR-29b-1 targets the 3’UTR of the AML1-ETO transcript and down-regulates AML1-ETO protein in leukemia cellsA. The ETO/RUNX1T1 transcripts have seed sequences in 3’UTRs for conserved miR families that can potentially down-regulate the proteins. The seed sequence for the miR-29 family is shown. B. 293T cells were co-transfected with a ETO 3’UTR-luciferase reporter construct and NS, miR-15 or miR-29 mimics. Cells were harvested 48 hours post-transfection and the luciferase activity was measured. Bar graphs represent two independent experiments, and show that miR-29 targets the ETO 3’UTR as indicated by a significant decrease in luciferase activity (p > 0.01) C. SKNO-1 cells expressing the indicated miRs were harvested for western blot analysis 5 days post infection. Protein levels for ETO were determined by antibodies from two independent sources (see Materials and Methods for details). Levels of CDK6, a known target of miR-29a, were also examined. Tubulin was used as a loading control. Western blot represents results from two biological replicates.