Figure 1.

Results for engineered ZF-TFs targeting endogenous OCT4 in HEK293 cells. (A) Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) results for OCT4 mRNA levels. Expression plasmids encoding ZF-TFs with the NF-ĸB p65 activation domain were transfected into HEK293 cells. Control cells were transfected with vectors encoding GFP (Green Fluorescent Protein). After 48 h, the cells were harvested and total RNA was isolated. The levels of OCT4 mRNA and PPIA (cyclophilin A) endogenous control mRNA were measured by qRT-PCR. The OCT4/PPIA ratio was used for normalizing OCT4 expression. Values plotted are the fold OCT4 expression compared to the GFP control. ZF-TFs that bind on the forward strand (closed circles) and reverse strand (open circles) were graphed according to their target location between −2500 bp and +500 bp of the major OCT4 TSS. Those used for FLAG-ChIP-qPCR are indicated with their position in green for active ZF-TFs and red for inactive ZF-TFs. Positions of the distal enhancer (DE), proximal enhancer (PE) and proximal promoter (PP), also known as conserved regions 4, 2 and 1, respectively (25,40), are indicated below the x-axis. Positions of ChIP-qPCR amplicons are also indicated below the x-axis and are named according to the midpoint of the amplicon. (B) FLAG-ChIP-qPCR results. Chromatin immunoprecipitation was performed with cross-linked chromatin from HEK293 cells transfected with each of the 11 ZF-TF constructs indicated in (A) and anti-FLAG M2 antibody, as described in the Materials and Methods section. qPCR was performed with a series of primers across the ZF-TF target region on OCT4 [indicated below the x-axis in (A)], as well as primers that amplify a closed chromatin region on MYT where the ZF-TFs should not bind. Percent of input was calculated and plotted for each. Percent input was ≤0.003% for all negative control, mouse IgG ChIPs (not shown) and for vector alone with anti-FLAG.