While it is relatively easy to deliver plasmids to a target cell, the transgenes are still susceptible to epigenetic silencing inside the nucleus. Indeed, it has been previously shown that plasmids are frequently associated with specific histone tail modifications that induce silencing (H3K9me2, H3K9me3, H4K20me2, and H4K20me3). The goal of this work is to prevent epigenetic silencing by adding transcription factor binding sites (TFBS) at different locations in a transgene expression plasmid. Specifically, binding sites for 14 different epigenetically active proteins were inserted upstream and downstream of two common promoters used in mammalian cell transfections: the elongation factor 1α (EF1α) and cytomegalovirus (CMV) promoters. Adding the sites upstream of the CMV promoter did not have any significant effects on transgene (luciferase) expression in prostate cancer cells (PC3), but a few of the sites (e.g. NF-Y, HNF4) did significantly enhance transgene expression from the EF1α promoter. The strongest enhancement (10-fold) was observed with the binding site for nuclear factor Y (NF-Y). NF-Y is a complex of three subunits (A, B, and C) which bind a specific CCAAT sequence (NF-YA) and mimic the H2A/B histone subunits (NF-YB and NF-YC) to upregulate gene expression. Interestingly, the NF-Y sequence had no effect on transgene expression when inserted downstream of the EF1α promoter, but several other sites did enhance transgene expression, including the CCCTC-binding factor (CTCF, 14-fold enhancement). CTCF is commonly referenced as an insulator, since it forms chromatin loops that prevent the spread of heterochromatin.
Since multiple NF-Y sites are often found near promoters in genomic DNA, pairs of NF-Y sites within the same or adjacent nucleosomes were also tested, but we observed that a single NF-Y binding site provided the strongest enhancement in plasmid DNA. In addition, point mutations of the canonical CCAAT binding sequence eliminated the observed enhancement. Similar experiments with the CTCF binding site revealed that the core CTCF binding site (CCGCGNGGNGGCAG) is sufficient to enhance transgene expression. To confirm that the enhancement observed after adding these sequences is due to an alteration in epigenetic regulation, we are currently conducting chromatin histone immunoprecipitation (ChIP) experiments to confirm NF-Y and CTCF binding to plasmid DNA, along with other histone modifications. Overall, however, our results clearly show that transgene expression can be easily and significantly enhanced by adding short sequences upstream and downstream of the EF1α promoter.
