Small Molecule Transcriptional Activators: Lead Agents for Gene Therapy
Summary One difficulty that has been encountered with the use of transcriptional activation systems in gene therapy is that overexpression of transcriptional activators in cells typically inhibits gene expression-a process known as squelching. Researchers at Harvard University have overcome this problem by developing novel transcriptional activators that, when bound to a specific DNA binding domain, stimulate the transcription of specific in vivo target genes without attenuating the expression of other important genes, such as those necessary for cell survival. The small molecule transcriptional activators also form the foundation of a three-hybrid system for detecting protein-protein interactions between transcriptional activators.
Invention: PCR is used to link random oligonucleotides, either 18-24 nucleotides long, to DNA encoding a DNA binding domain, so that hybrid genes are produced that encoded a fusion protein consisting of a DNA binding domain and either a 6mer or 8-mer peptide. The hybrid genes are expressed in yeast and the library is screened for peptides that can stimulate transcription of a reporter gene harboring their respective DNA binding sites (see chart below). One small molecule transcriptional activator (P201) demonstrated higher reporter activity than the positive control. P201 also did not squelch transcriptional activation from a co-transfected LexA transcriptional activation system, suggesting that P201 functions through a distinct target.
Applications Small molecule transcriptional activators are to be utilized in gene therapy to increase the expression of specific genes associated with disease. In another example, the transcriptional activator forms the core of a three hybrid system for detecting protein-protein interactions between transcriptional activators. This is important because in standard yeast two hybrid systems, false positives are common. Moreover, these systems cannot be used to identify the interaction targets of transcriptional activators because the activator is fused to the DNA binding moiety which in turn activates transcription. Any artificial sequence that interacts with the holoenzyme can be tested for its ability to be used as the transcriptional activation domain in this improved protein-protein interaction systems.
Publications and Patents
Mapp AK, Ansari AZ, Ptashne M, Dervan PB. Activation of gene expression by small molecule transcription factors. Proc Natl Acad Sci U S A. 2000 Apr 11; 97(8):3930-5.
Patent Status: Pending
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