Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody

Abstract

Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.

FACT is a sensor of DNA torsional stress in eukaryotic cells

“…Indirect ways to detect Z-DNA utilized reporter plasmids with Z-DNA-prone inserts or boosting rate of transcription at certain genomic regions. In both cases, however, Z-DNA emerges locally and its detection is either indirect (mutations in reporter (85) or poorly detectable (1,86). Therefore, finding that CBL0137 induces massive stable and long-lasting transition of genomic DNA into the Z-form at a scale that enables easy detection by specific antibodies as whole nucleus staining in CBL0137-treated cells was surprising.…”