Abstract
Human gene therapy approaches involving transcription factors often rely on artificial activation domains for transcriptional activation. These domains are often large (e.g., 80 amino acids for VP16), recruit multiple co-activation complexes at once, and offer no fine control over the level of transcription. In an attempt to understand the sequence and structural requirements of a minimal mammalian activator, we employed a molecular diversity approach with a peptide phage display library composed of random eight–amino acid peptides. Using the KIX domain of the mammalian co-activators p300 and CBP as target, we discovered a family of synthetic binding peptides. These peptides share significant homology with natural KIX domain ligands, and are shown to bind an overlapping, yet distinct, surface of p300/CREB-binding protein (CBP). When fused to a heterologous DNA binding domain, these synthetic peptides function as titratable, modular, and potent transcriptional activators in living cells through specific recruitment of p300/CBP, with the level of transcriptional activation proportional to the affinity of the synthetic peptide for the KIX domain. Taken together, our data demonstrate that a molecular diversity approach can be used to discover minimal, co-activator domain-specific synthetic activators, and that transcriptional activation can be modulated as desired at the level of co-activator recruitment.
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Acknowledgements
We thank Daniel S. Kemp (MIT) for assistance with molecular modeling and peptide helicity issues, Brian K. Kay (University of Wisconsin) and Keyong Du (Joslin Diabetes Center) for many helpful discussions, David Parker for GST/KIX domain mutants and assistance with fluorescence polarization, Paul K. Brindle (St. Jude's) for c-Myb reagents, Susan Taylor (UC San Diego) for protein kinase A, Ron Evans (Salk Institute) for the G5B-luciferase plasmid, Katiri W. Wagner, Xu Mei Li, and Floretta V. Lake for technical assistance, and Ligita Stukuls and Chi Lam for administrative assistance. J.V.F. is a postdoctoral fellow of the Howard Hughes Medical Institute, and is also generously supported by the Paul D. and Lovie S. Kemp Career Development Fund for Prostate Cancer, the Hershey Family Foundation, and the Rita Leabman Memorial Fund. M.R.M. and L.C.C. (GM 56203) are supported by grants from the NIH.
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Frangioni, J., LaRiccia, L., Cantley, L. et al. Minimal activators that bind to the KIX domain of p300/CBP identified by phage display screening. Nat Biotechnol 18, 1080–1085 (2000). https://doi.org/10.1038/80280
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DOI: https://doi.org/10.1038/80280
