Figure 2.

Unequal impact of Runx1 deficiency on Runx1 binding at the Cebpa and Cebpe enhancers and on Cebpa and Cebpe activation in response to G-CSF. (a) Time-course expression of Hoxb4 (key stem cell transcription factor), Cebpa (key lineage-commitment transcription factor) and Cebpe (key late-differentiation transcription factor) in WT and Runx1+/− lineage-negative (Lin−) cells cultured with G-CSF for 15 days. SYBR Green bound to double-stranded DNA was detected in real-time with the 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative expression values were calculated by raising 2 to the power of the negative value of delta-delta CT for each sample. Primer sequences: HoxB4: forward (F): 5′-CCAGAATCGGCGCATGA-3, reverse (R): 5′-CCCGAGCGGATCTTGGT-3′ Cebpa: F: 5′-CAAAGCCAAGAAGTCGGTGGACAA-3′ R: 5′-TCATTGTCACTGGTCAACTCCAGC-3′ Cebpe: F: 5′-GCTACAATCCCCTGCAGTACC-3′ R: 5′-TGCCTTCTTGCCCTTGTG-3′ GAPDH: F: 5′-ACCACACTCCATGCCATCAC-3′, R:5′-TCCACCACCCTGTTGCTGTA-3′. Mean±standard deviation. **P<0.01, Student's t-test. Experiments performed in triplicate. (b) Cebpa and Cebpe protein expression in WT and Runx1+/− Lin− cells cultured for 15 days with G-CSF. Western blot analyses with the following primary antibodies: anti-Cebpa, anti-Cebpe (Santa Cruz Biotechnology, Dallas, TX, USA; #SC-61, #SC-25770), anti-β-actin (Sigma-Aldrich, St Louis, MO, USA, A3854); secondary antibodies: anti-Rabbit (GE Healthcare, Waukesha, WI, USA; NA934) and anti-Mouse (GE Healthcare, NXA931) at 1:5000 and 1:10 000 dilutions, respectively. (c) Gene expression of CEBPA, CEBPE and HOXB4 in primary AML cells with normal cytogenetics and RUNX1 mutation (TCGA database, n=14). Heatmap by Arraystar software. (d) Chromatin immunoprecipitation (ChIP) analysis of Runx1+/− and WT cells to evaluate Runx1 binding at the Cepba and Cebpe enhancers. Enhancers of Cebpa and Cebpe were identified by others using ChIP coupled with deep sequencing.²⁸ ChIP performed using ChIP Assay Kit (cat#17-295, EMD Millipore, Billerica, MA, USA). 5 × 10⁶ cells bone marrow (BM) cells from WT and Runx1+/1 fresh BM cells were resuspended in 10 ml of media and formaldehyde was added to a final concentration of 1% followed by 10 min incubation at 37 °C. The media containing formaldehyde was removed and the cells were washed twice with ice-cold PBS containing protease inhibitor (cat#P8340, Sigma-Aldrich) and phosphatase inhibitor cocktails (cat#P0044, Sigma-Aldrich). Crosslinked cells were resuspended in SDS lysis buffer (Millipore, #20–163) and incubated for 10 min on ice. These cell pellets were sonicated at 15 s pulses with 45 s hold on ice for a total of 10 min of pulsing at full power (Fisher-Scientific Model #550 Sonic Dismembrator equipped with a microtip probe). Genomic DNA fragments of 200-bp and 1-kb in size were obtained (confirmed by agarose gel electrophoresis). After centrifugation at 4 °C, the supernatant was pre-cleared with salmon sperm DNA/Protein A agarose-50% slurry (Millipore, #16–157) for 30 min at 4 °C with agitation. A 10% aliquot (vol) of the precleared protein/DNA mixture was removed and used for subsequent reverse transcription–PCR input quantification. 10 μg of anti-Runx1 ChIP grade antibody (cat#ab23980, Abcam, Cambridge, MA, USA) was added to the remaining protein/DNA mixture and incubated at 4 °C overnight with rotation. Salmon sperm DNA/Protein A agarose-50% slurry was added and incubated for 1 h at 4 °C followed by washes with the ChIP assay kit provided buffers. The immunoprecipitated (IP) complexes were reverse crosslinked at 65 °C for 4 h and DNA was recovered by phenol/chloroform extraction protocol (Sigma). The IP products were amplified and quantified using real-time PCR. ChIP primer sequences are:²¹ cepba enhancer left primer, 5′- TTCCCGTTTCTGAAATCTGC-3′, cebpa enhancer right primer, 5′-GGTTGTGGCAAGAAGGTCAC-3′, cebpe enhancer left primer, 5′-GTGTCATGGTCACCCTAGCC-3′ and cebpe enhancer right primer, 5′-CTGGAGCTAGCAGGGGTTTT-3′. Mean±standard deviation from three independent ChIP experiments. **P<0.001, NS=not significant, Tukey–Kramer HSD test.