Gottesfeld Lab Research Interests

Small Molecule Regulation of Gene Expression
The ability to control gene expression at will has been a longstanding goal in molecular biology and human medicine. Studies in our laboratory are focused pyrrole--imidazole polyamides, a class of small molecules that can be programmed by chemical synthesis to recognize a wide range of DNA sequences. Below we summarize recent efforts toward the development of polyamides as therapeutics for human disease, and the identification of another class of small molecules that offer promise in the treatment of neurodegenerative diseases.

Identification of Histone Deacetylase Inhibitors that Reverse Frataxin Gene Silencing

We examined the chromatin structure of the frataxin gene in normal and FRDA cell lines using antibodies to the various modification states of the core histones and chromatin immunoprecipitation methods. We find that gene silencing at expanded frataxin alleles is 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 (HDAC) inhibitors, compounds that reverse heterochromatin, might activate the frataxin gene. We identified one commercial HDAC inhibitor, BML--210, that partially reverses silencing in the FRDA cell line. Based on the structure of this compound, we synthesized and assayed a series of derivatives of BML-210 and identified HDAC inhibitors that reverse frataxin silencing in primary lymphocytes from Friedreich’s patients. These molecules act directly on the histones associated with the frataxin gene, increasing acetylation at particular lysine residues on histones H3 and H4 (H3-K14, H4-K5 and H4-K12). Unlike many triplet-repeat diseases (for example, the polyglutamine expansion diseases such as Huntington’s disease and the spinocerebellar ataxias), expanded GAA·TTC triplets do not alter the coding potential of the frataxin gene; thus, gene activation would be of therapeutic benefit. Animal studies are currently underway to explore the bioavailability and efficacy of these molecules.

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Blocking Cancer Cell Proliferation with a Polyamide-Chlorambucil Conjugate
One common DNA alkylator in clinical use for a variety of lymphatic cancers is the nitrogen mustard chlorambucil. Since 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 reported that a specific polyamide-chlorambucil conjugate called 1R--Chl alters the morphology and growth characteristics of colon carcinoma cells in culture, and causes these cells to arrest in the G2/M stage of the cell cycle, without any apparent cytotoxicity. 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 no longer tumorigenic. The compound blocks proliferation of metastatic colon carcinoma cells in immunocompromised mice, and no apparent toxicity is observed at doses required for a therapeutic effect. Importantly, this gene-targeted small molecule requires no delivery vehicle as the molecule is cell permeable and localizes in the nucleus of various cancer cell lines. The gene target of 1R--Chl was identified as histone H4c by microarray analysis, 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 this agent, causing chromatin decondensation.

To confirm that down regulation of histone H4c transcription is the primary event leading to cell cycle arrest by 1R--Chl, we turned to siRNAs directed toward H4c mRNA. Unlike 1R--Chl, which arrests cells at the G2/M phase of the cell cycle, the same cells treated with the H4c siRNA are arrested at the G1/S phase. However, G2/M arrest by 1R--Chl and down regulation of the H4c gene can be confirmed in other tumorigenic cell lines. We find that 1R--Chl causes extensive DNA damage in colon cancer cells, leading to phosphorylation of histone H2A.X at Ser 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 anti--proliferative effects by themselves, are able to cause G2/M cell-cycle arrest when cells are treated with these polyamide conjugates and siRNAs to histone mRNAs.

Based on these findings, we propose that 1R--Chl exerts its anti-proliferative effect through a novel two--hit mechanism. The highly transcribed H4c gene in several cancer cell lines is a primary target for DNA alkylation by 1R--Chl, resulting in down-regulation of H4c transcription and histone H4 protein. Loss of histone protein leads to a transition from condensed to open chromatin, exposing otherwise hidden 1R--Chl binding sites. 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 tumorigenecity. Our findings demonstrate how a single molecule can target cancer cells due to 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.

Polyamides as Activators of Gene Expression
The neurodegenerative disease Friedreich’s ataxia (FRDA) 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 while FRDA patient alleles 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 unusual DNA structures and/or heterochromatin formation in the frataxin gene would likely increase transcription through expanded GAA•TTC repeats, thereby relieving the deficiency in frataxin mRNA and protein in FRDA cells. We find that polyamides targeting GAA•TTC repeats partially alleviate transcription repression of the frataxin gene in a cell line derived from white blood cells from a FRDA patient. These molecules also increase frataxin protein levels in these cells, and microarray studies show that a limited number of genes in the human genome are 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 the frataxin gene. To explore this latter hypothesis, we turned to another class of small molecules.