Scientific Report 2006

Molecular Biology

Chemical Regulation of Gene Expression

D. Alvarez, R. Burnett, C.J. Chou, D. Herman, K. Jenssen, S. Ku, E. Soragni, J. Puckett,* S. Tsai,* M. Farkas,* P.B. Dervan,* J.M. Gottesfeld

* California Institute of Technology, Pasadena, California

The ability to control gene expression at will has been a longstanding goal in molecular biology and human medicine. We focus on pyrrole-imidazole polyamides, a class of small molecules that can be programmed by chemical synthesis to recognize a wide range of DNA sequences. The following is a summary of our recent efforts to develop polyamides as therapeutic agents for human disease and to identify another class of small molecules that offer promise in the treatment of neurodegenerative diseases.

Blocking Cancer Cell Proliferation with a Polyamide-Chlorambucil Conjugate

The nitrogen mustard chlorambucil is a common DNA alkylator used to treat a variety of lymphatic cancers. Because chlorambucil alkylates DNA at all potentially available guanine residues in the genome, coupling of chlorambucil to a polyamide will increase the DNA-sequence specificity and perhaps decrease unwanted side effects while retaining the ability of the compound to kill cancer cells. We recently found that a specific polyamide-chlorambucil conjugate called 1R-Chl alters the morphology and growth characteristics of colon carcinoma cells in culture and causes the cells to arrest in the G₂/M stage of the cell cycle, without any apparent cytotoxic effects.

Cells treated with 1R-Chl do not grow in soft agar and do not form tumors in nude mice, indicating that polyamide-treated cells are no longer tumorigenic. The compound blocks proliferation of metastatic colon carcinoma cells in immunocompromised mice, and no apparent toxic effects occur at doses required for a therapeutic effect. Importantly, this gene-targeted small molecule requires no delivery vehicle because the molecule is cell permeable and localizes in the nucleus of various cancer cell lines. Using microarray analysis, we found that the gene target of 1R-Chl is the gene for histone H4c, a member of the gene family that encodes a critical component of cellular chromatin and a gene that is highly expressed in a wide range of cancer cells. Reduction in histone H4 protein by polyamide treatment was confirmed in cells treated with 1R-Chl, which caused chromatin decondensation.

To confirm that downregulation of histone H4c transcription is the primary event leading to cell-cycle arrest by 1R-Chl, we turned to short interfering RNAs directed toward H4c mRNA. Unlike 1R-Chl, which arrests cells at the G₂/M phase of the cell cycle, the H4c short interfering RNA arrests cells at the G₁/S phase. However, G₂/M arrest by 1R-Chl and downregulation of the H4c gene can be confirmed in other tumorigenic cell lines. We found that 1R-Chl causes extensive DNA damage in colon cancer cells, leading to phosphorylation of histone H2A.X at serine 139 and recruitment of the DNA repair protein Nbs1 to discrete sites in the genome. These events are hallmarks of the cellular DNA damage response pathway. Control polyamide-Chl conjugates that lack binding sites in the H4c gene and have no antiproliferative effects by themselves can cause G₂/M cell-cycle arrest when used in combination with short interfering RNAs to histone mRNAs.

On the basis of these findings, we propose that 1R-Chl exerts its antiproliferative effect through a novel 2-hit mechanism. The highly transcribed H4c gene in several cancer cell lines is a primary target for DNA alkylation by 1R-Chl, resulting in downregulation of H4c transcription and histone H4 protein. Loss of his tone protein leads to a transition from condensed to open chromatin, exposing otherwise hidden binding sites for 1R-Chl. These sites are then alkylated by 1R-Chl, causing widespread DNA damage and a cascade of events leading to G₂/M arrest and loss of tumorigenicity.

Our findings indicate how a single molecule can target cancer cells because of a specific gene expression profile and block cancer cell proliferation. Ongoing studies are aimed at the development of 1R-Chl as a potential human cancer therapeutic agent.

Polyamides as Activators of Gene Expression

The neurodegenerative disease Friedreich’s ataxia is caused by gene silencing through expansion of GAA-TTC triplet repeats in the first intron of a nuclear gene that encodes the essential mitochondrial protein frataxin. Normal frataxin alleles have 6–34 repeats whereas alleles from patients with Friedreich’s ataxia have 66–1700 repeats. Longer repeats cause a more profound frataxin deficiency and are associated with earlier onset and increased severity of the disease. Two models have been proposed to account for gene silencing by expanded GAA-TTC repeats: unusual DNA structures and repressive heterochromatin.

Molecules that reverse formation of unusual DNA structures and/or heterochromatin in the gene for frataxin most likely increase transcription through expanded GAA-TTC repeats, thereby relieving the deficiency in frataxin mRNA and protein in cells from patients with Friedreich’s ataxia. We found that polyamides targeting GAA-TTC repeats partially alleviated transcription repression of frataxin in a cell line derived from white blood cells from a patient with Friedreich’s ataxia. These molecules also increased frataxin protein levels in these cells, and microarray studies showed that a limited number of genes in the human genome were affected by polyamides targeting GAA-TTC repeat DNA.

We hypothesize that polyamides might act as a thermodynamic “sink” and lock GAA-TTC repeats into double-stranded B DNA. Such an event would disfavor duplex unpairing, which is necessary for formation of the unusual DNA structures associated with expanded triplet repeats. Alternatively, polyamides may relieve heterochromatin-mediated repression by opening the chromatin domain containing frataxin. To explore this last hypothesis, we turned to another class of small molecules.

Histone Deacetylase Inhibitors that Reverse Frataxin Silencing

We used antibodies to the various modification states of the core histones and chromatin immunoprecipitation methods to examine the chromatin structure of the gene for frataxin in normal cells and in cell lines derived from patients with Friedreich’s ataxia. We found that gene silencing at expanded frataxin alleles was accompanied by hypoacetylation of histones H3 and H4 and methylation of histone H3 at lysine 9, consistent with a heterochromatin-mediated repression mechanism.

These findings suggest that histone deacetylase inhibitors, compounds that reverse heterochromatin, might activate frataxin. We identified a commercial histone deacetylase inhibitor, BML-210, that partially reverses silencing in the Friedreich’s ataxia cell line. On the basis of the structure of this compound, we synthesized and assayed a series of derivatives of BML-210 and identified histone deacetylase inhibitors that reverse frataxin silencing in primary lymphocytes from patients with Friedreich’s ataxia. These molecules act directly on the histones associated with frataxin, increasing acetylation at particular lysine residues on histones H3 and H4. Unlike many triplet-repeat diseases (e.g., the polyglutamine expansion diseases such as Huntington’s disease and the spinocerebellar ataxias), expanded GAA-TTC triplets do not alter the coding potential of frataxin. Thus, gene activation would be of therapeutic benefit. Studies in animals are under way to explore the bioavailability and efficacy of these histone deacetylase inhibitors.

Publications

Alvarez, D., Chou, C.J., Latella, L., Zeitlin, S.G., Ku, S., Puri, P.L., Dervan, P.B., Gottesfeld, J.M. A two-hit mechanism for pre-mitotic arrest of cancer cell proliferation by a polyamide-alkylator conjugate. Cell Cycle 5:1537, 2006.

Burnett, R., Melander, C., Puckett, J.W., Son, L.S., Wells, R.D., Dervan, P.B., Gottesfeld, J.M. DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA-TTC repeats in Friedreich’s ataxia. Proc. Natl. Acad. Sci. U. S. A. 103:11497, 2006.

Herman, D., Jenssen, K., Burnett, R., Soragni, E., Perlman, S.L., Gottesfeld, J.M. Histone deacetylase inhibitors reverse gene silencing in Friedreich’s ataxia. Nat. Chem. Biol. 2:551, 2006.

Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, D.A., Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of 5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006.

Trzupek, J.D., Gottesfeld J.M., Boger D.L. Alkylation of duplex DNA in nucleosome core particles by duocarmycin SA and yatakemycin. Nat. Chem. Biol. 2:79, 2006.