Fluorescence imaging 5.0x10^4 of HEK-293T cells were plated in a 35 mm fluorescence cell culture imaging dish the day before transfection and incubated overnight at 37°C in 5% CO2. The next day, 1 mg of BII-tdT-GFP vector containing a bidirectional promoter was mixed with 1 mL of jetOPTIMUS and diluted in 100 mL of jetOPTIMUS buffer. The mixture was incubated at room temperature for 10 min, added to the cells, and incubated at 37°C in 5% CO2 for 48 h before imaging. Fluorescence images were directly observed under a Revolve fluorescence microscope. Fluorescence images were acquired using the GFP, RFP, and transparent filters. Time-lapse imaging of red and green fluorescence was performed on a CytoSMART Lux3 live cell imager using a 20 min capture interval. Cell transfection and luciferase reporter assay HeLa, HEK293T and HepG2 cells were plated at 2.0x10^5 cells per well in a 24-well plate the day before transfection and incubated overnight at 37°C in 5% CO2. For each well, 400 ng of pGL3 reporter construct plus 100 ng of Renilla luciferase pRL-SV40 control DNA was diluted in 100 mL of buffer. 1 mL of jetOPTIMUS was added and incubated at room temperature for 10 min. The DNA mixture containing was then added to each well and incubated at 37°C in 5% CO2 for 48 h before analysis. For suspension cell line transfection, 53105 Jurkat or YTS cells were seeded per well in a 24-well plate the day before transfection. For Jurkat cells, 4 mg of reporter construct DNA, 400 ng of Renilla DNA, 5 mL of jetOPTIMUS were diluted in 200 mL buffer and incubated at room temperature for 10 min before addition to each well. For YTS cells, 4.5 mg of reporter construct DNA, 400 ng of Renilla DNA, 6 mL of jetOPTIMUS were diluted in 150 mL buffer and incubated at room temperature for 10 min before addition to each well. For primary fibroblast transfection, early passage cells were seeded the day before transfection in a 24-well plate at a density of 50,000 cells/well. The cells were transfected when cells reached a 50–60% confluency level. For each well, 2 mg of reporter construct DNA, 150 ng of Renilla DNA, 2 mL of jetOPTIMUS were diluted in 50 mL buffer and incubated at room temperature for 10 min before addition to the well. The DNA mixture containing plasmid DNA was then added to each well and incubated at 37°C in 5% CO2 for 12 h before Luciferase activity analysis. Luciferase activity was assayed using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions. Briefly, the culture medium was removed 48 h post-transfection and cells were washed with 0.5 mL of phosphate buffered saline (PBS, pH 7.4). Cells were then lysed with passive lysis buffer. The suspensions were centrifuged at 14000 rpm for 1 min. A total of 20 mL of cell lysate supernatant was mixed with 100 mL of luciferase substrate, and the light units were measured on a luminometer. Measurement of the firefly luciferase activity of the promoter constructs was normalized relative to the activity of the Renilla luciferase produced by the pRLSV40 control vector and each construct was tested in duplicate in at least three independent experiments.

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34⁺ hematopoietic progenitors in vitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.