Faculty, Graduate Program
Chemical Regulation of Gene Expression
Research in our laboratory concerns protein-DNA interactions involved in regulation of gene expression in mammalian cells and the development of small molecules to regulate gene expression at will. Rules have been developed for sequence-specific recognition of DNA in the minor groove with synthetic peptide-like compounds containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids. These molecules have affinities and specificities for their target DNA sequences that are comparable to those of natural DNA-binding transcriptional regulatory proteins. We have shown that designed Py-Im polyamides inhibit both transcription factor-DNA interactions and gene transcription both in vitro and in cell culture experiments.
Our current research focuses on the development of Py-Im polyamides as new therapeutics for human diseases, including cancer, neurodegenerative diseases and infectious diseases. By screening a small library of polyamide-DNA alkylator conjugates, we identified a lead compound that alters the morphology and growth characteristics of various cancer cell lines in culture. This compound causes cells to arrest in the G2/M stage of the cell cycle, without apparent cytotoxicity. Microarray analysis indicates that only one gene in the human genome is significantly down regulated by this polyamide, and RT-PCR and western blotting experiments confirm that histone H4c mRNA and total H4 protein levels are reduced in treated cells. Cells that are unable to form their full complement of nucleosomes, due to a lack of histone protein, will be unable to proceed through the cell cycle and hence we expect that cells treated with this compound will be non-tumorigenic. As expected, cells treated with this compound fail to grow in soft agar, and do not form tumors in nude mice, indicating that polyamide-treated cells are indeed no longer tumorigenic. The compound is active in vivo, blocking tumor growth in mice, without obvious toxicity to the animals. Our results suggest that polyamide-DNA alkylators may lead to a new class of cancer chemotherapeutics.
A large class of neurodegenerative diseases is caused by the expansion of DNA triplets within the coding region of critical genes. One such disease is Friedreich's ataxia (FRDA), which is caused by the expansion of the triplet GAA within a mitochondrial gene involved in iron homeostasis known as the frataxin gene. Expanded GAA repeats result in decreased transcription and reduced levels of frataxin protein in affected individuals, either by adopting unusual DNA structures or repressive chromatin. We designed pyrrole-imidizole polyamides to target GAA repeats with high affinity. Flourescent polyamide analogues localize in the nucleus of lymphoid cell lines derived from individuals affected with FRDA, and the GAA-specific molecules relieve transcription inhibition of the frataxin gene in cell culture. These studies demonstrate that polyamides can also be used to induce gene expression and represent a promising first step toward development of gene-specific therapeutics for FRDA and other trinucleotide expansion diseases.
Ph.D., California Institute of Technology, 1975
Editorial Boards, Journal of Biological Chemistry, Gene Expression.
For a complete list of publications: http://www.scripps.edu/gottesfeld/publications.html
Edayathumangalam, R.S., Weyermann, P., Gottesfeld, J.M., Dervan, P.B., Luger, K. Molecular recognition of the nucleosomal "supergroove". Proc. Natl. Acad. Sci. USA 101: 6864-9, 2004.
Dudouet, B., R. Burnett, L.A. Dickinson, M.R. Wood, C. Melander, J.M. Belitsky, B. Edelson, P.B. Dervan, and J.M. Gottesfeld. Accessibility of nuclear chromatin by DNA-binding polyamides. Chem. & Biol. 10: 859-67, 2003.
Gottesfeld, J.M., C. Melander, R.K. Suto, H. Raviol, K. Luger, and P.B. Dervan. Sequence-specific Recognition of DNA in the Nucleosome by Pyrrole-Imidazole Polyamides, J. Mol. Biol., 309: 615-629, 2001.
Dickinson, L.A., J.W. Trauger, E.E. Baird, R.J. Gulizia, D.E. Mosier, J.M. Gottesfeld and P.B. Dervan. Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands. Proc. Natl. Acad. Sci. USA, 95: 12890-12895, 1998.