Scientific Report 2005

Molecular Biology

Chemical Regulation of
Gene Expression

J.M. Gottesfeld, D. Alvarez-Carbonell, R. Burnett, J. Chou, D. Herman, K. Jennsen, S. Ku, P.B. Dervan*, K. Luger**

* California Institute of Technology, Pasadena, California
** Colorado State University, Fort Collins, Colorado

Transcription Regulation with Small Molecules

Pyrrole-imidazole polyamides are the only available class of synthetic small molecules that can be designed to bind predetermined DNA sequences with affinities comparable to those of cellular gene regulatory proteins. In collaboration with P.B. Dervan and colleagues at the California Institute of Technology, we showed that polyamides inhibit the DNA-binding activities of various transcriptional regulatory proteins and can be used to inhibit transcription in cell culture experiments. Previous studies established that transcription can be inhibited with polyamides by targeting the binding sites for essential transcription regulatory proteins in gene promoters in the cell nucleus. We also found that site-specific DNA alkylation by polyamide-chlorambucil conjugates within a coding region of a gene strongly blocks transcription elongation by mammalian RNA polymerase II, both in vitro and in reporter gene transfection experiments in cell culture.

We screened a series of polyamide-chlorambucil conjugates with different DNA sequence specificities for effects on morphology and growth characteristics of human colon carcinoma cell lines. We identified a compound that causes cells to arrest in the G₂/M stage of the cell cycle, without any apparent cytotoxic effects. This change in growth properties required both the DNA-binding specificity of the polyamide and the alkylator moiety, suggesting that growth arrest is due to the silencing of a set of specific genes by site-specific alkylation.

Surprisingly, DNA microarray analysis indicated that only a few genes of about 18,000 genes probed were significantly downregulated by this polyamide, and reverse transcriptase–polymerase chain reaction and Western blotting experiments confirmed that among these genes, a member of the human gene family that encodes histone H4, an essential component of chromatin, is significantly downregulated. This particular gene, the gene for histone H4c, is actively transcribed in various cancer cell lines but is only moderately transcribed in normal cells and tissues. Downregulation of H4c mRNA by small interfering RNA yielded the same cellular response, providing target validation. The gene for histone H4c contains binding sites for the active polyamide, and DNA alkylation within the coding region of the gene was confirmed in cell culture by using ligation-mediated polymerase chain reaction.

Cells treated with this polyamide-chlorambucil conjugate did not grow in soft agar and did not form tumors in nude mice, indicating that polyamide-treated cells are no longer tumorigenic. The compound is active in vivo, blocking tumor growth in mice, without any obvious toxic effects. We extended these studies to various cell lines representing various types of human cancers, including solid tumors of the breast, cervix, lung, pancreas, prostate, and bone and blood cancers, such as leukemias. Our results suggest that polyamide-DNA alkylators may lead to a new class of cancer chemotherapeutic agents.

Polyamides As Activators Of Gene Expression

In several human diseases, activation of a repressed gene might be useful as a therapeutic approach. One example is the neurodegenerative disease Friedreich’s ataxia, in which gene silencing caused by an unusual DNA structure is the primary cause of the disease. The DNA abnormality found in 98% of patients with Friedreich’s ataxia is the unstable hyperexpansion of a GAA triplet repeat in the first intron of the frataxin gene, which adopts a triplex DNA structure, resulting in decreased transcription and reduced levels of frataxin protein. Frataxin is a mitochondrial protein that functions in iron homeostasis, and decreased levels of frataxin lead to neurodegeneration and cardiomyopathies.

We designed pyrrole-imidazole polyamides to target GAA repeats in DNA with high affinity, and we found that these molecules relieved transcription inhibition of the frataxin gene in cell lines and in primary lymphocytes derived from patients with Friedreich’s ataxia. These molecules localize in the cell nucleus, as determined by fluorescence deconvolution microscopy with polyamide-dye conjugates, and most likely reverse repression of the frataxin gene by stabilizing canonical Watson-Crick B-type DNA. Changing the sequence specificities of the molecules abolished their ability to induce frataxin expression. These molecules are a first step toward therapeutic agents for treatment of Friedreich’s ataxia.

DNA Recognition Within Chromatin

Biochemical and x-ray crystallography studies indicate that nucleosomal DNA is largely available for molecular recognition by pyrrole-imidazole polyamides. Polyamide binding sites that are located 80 bp apart on linear DNA lie across the 2 gyres of the DNA superhelix in the nucleosome, forming a supergroove that is unique to the nucleosome. On the basis of this observation, we developed bivalent pyrrole-imidazole polyamide clamps that bind with high specificity across the nucleosomal supergroove. X-ray crystallography studies performed in the laboratory of our collaborator, K. Luger, Colorado State University, indicated that the clamps bind as designed and effectively cross-link the 2 gyres of the DNA superhelix in the nucleosome and stabilize nucleosomal DNA from dissociation. These molecules are useful probes of chromatin structure and dynamics and are tools for regulation of nucleosome mobility during transcription.

Publications

Beltran, A.C., Dawson, P.E., Gottesfeld, J.M. Role of DNA sequence in the binding specificity of synthetic basic-helix-loop-helix domains. Chembiochem 6:104, 2005.

Dickinson, L.A., Burnett, R., Melander, C., Edelson, B.S., Arora, P.S., Dervan, P.B., Gottesfeld, J.M. Arresting cancer proliferation by small-molecule gene regulation. Chem. Biol. 11:1583, 2004.

Edayathumangalam, R.S., Weyermann, P., Dervan, P.B., Gottesfeld, J.M., Luger, K. Nucleosomes in solution exist as a mixture of twist-defect states. J. Mol. Biol. 345:103, 2005.

Gearhart, M.D., Dickinson, L., Ehley, J., Melander, C., Dervan, P.B., Wright, P.E., Gottesfeld, J.M. Inhibition of DNA binding by human estrogen-related receptor 2 and estrogen receptor with minor groove binding polyamides. Biochemistry 44:4196, 2005.