News and Publications

Protein-Nucleic Acid Interactions in Transcriptional Regulation

J.M. Gottesfeld, A. Beltran, L.M. Burke, L.A. Dickinson, J. Ehley, P. Dawson,* J.M. Belitsky,** N. Wurtz,** C. Melander,** P.B. Dervan,** K. Luger***

* Department of Cell Biology, TSRI
** California Institute of Technology, Pasadena, CA
*** Colorado State University, Fort Collins, CO

SEQUENCE-SPECIFIC INHIBITION OF GENE TRANSCRIPTION WITH DESIGNED LIGANDS

The 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 pyrrole-imidazole polyamides can bind their target sequences in the context of cellular chromatin and are effective inhibitors of gene transcription.

Polyamides were designed and synthesized to target unique sequences within the HIV type 1 (HIV-1) promoter and enhancer elements. Using a series of DNA constructs in which a polyamide binding site was placed at various distances upstream and downstream from the HIV-1 TATA box, we determined the optimal distance and orientation for inhibition of interactions between the TATA-box binding protein and DNA and for inhibition of transcription by RNA polymerase II.

Additionally, the DNA-binding activities of various transcription factors, including lymphoid enhancer factor-1, Ets-1, estrogen receptor, estrogen-related receptor, and basic helix-loop-helix (bHLH) proteins, and viral activators and repressors have been inhibited with polyamides. Importantly, a combination of polyamides targeting the HIV-1 enhancer and promoter can selectively inhibit HIV-1 replication in primary human lymphocytes.

Although polyamides inhibit transcription when they interfere with the DNA-binding activity of essential components of the gene regulatory machinery, polyamides bound within the RNA-coding regions of genes are not an obstacle to an advancing RNA polymerase. When the DNA alkylating agent chlorambucil (bis(dichloroethylamino)benzene) is attached to a polyamide, site-specific DNA alkylation results in a strong block to transcription elongation by both bacteriophage T7 RNA polymerase and mammalian RNA polymerase II. These polyamide conjugates were also effective in blocking transcription by RNA polymerase II in reporter gene transfection experiments in cell culture. Thus, polyamides can be designed to inhibit the transcriptional activity of selected genes in living cells. Our results also suggest that these molecules may be useful as therapeutic agents for human disease.

DNA RECOGNITION WITHIN CHROMATIN

Because approximately 95% of genomic DNA is packaged in nucleosomes (the complex of an octamer of histone proteins and 147 bp of DNA), a major issue in gene regulation is how regulatory DNA-binding proteins can recognize their target sequences in nucleosomal DNA. We used a series of pyrrole-imidazole polyamides as sequence-specific probes for nucleosome structure and DNA accessibility in the nucleosome. We found that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible to these molecular probes. Polyamides only failed to bind where sites were completely blocked by interactions with the histone octamer. These results suggest that much of the DNA in the nucleosome is freely accessible for molecular recognition, at least by these small ligands.

To explore the functional consequences of polyamide binding to nucleosomal DNA, we determined the effect of polyamides on RNA transcription with a chromatin template. Two polyamides that bind to a nucleosome positioning sequence inhibit both heat-induced nucleosome sliding and transcription by bacteriophage T7 RNA polymerase from the nucleosomal template but not from histone-free DNA. These polyamides also prevent repositioning of the histone octamer by RNA polymerase and thereby inhibit passage of the elongating polymerase through nucleosomal DNA. These results establish the requirement for octamer mobility for transcription of nucleosomal templates by T7 RNA polymerase and suggest a mechanism for inhibition of transcription of target genes at the level of chromatin structure.

MOLECULAR MECHANISMS OF DNA BINDING AND PROTEIN DIMERIZATION BY bHLH TRANSCRIPTION FACTORS

Our focus in other studies is identification of amino acid residues of bHLH regulatory proteins that mediate both DNA binding and protein dimerization. Members of the bHLH family of transcriptional regulators are involved in many aspects of development and tissue-specific gene expression, and mutations and rearrangements in the genes encoding bHLH proteins are involved in numerous developmental diseases and cancer. Our studies have been made possible through the use of solid-phase peptide synthesis methods and powerful new approaches for chemical mutagenesis of protein domains.

The bHLH domains of the ubiquitously expressed factors E12 and E47, the tissue-specific factor Tal 1, and the repressor Id 1 have been prepared and characterized. Electrophoretic mobility shift assays and fluorescence anisotropy were used to determine the dissociation constants of the E12 homodimer and E12-Tal 1 heterodimer bound to DNA. Electrophoretic mobility shift assays also indicated inhibition of the E12 homodimer with Id 1 and inhibition of the E12-Tal 1 heterodimer with Id 1. Rigorous thermodynamic analysis of the dimer-DNA interaction was done for both the E12 homodimer and the E12-Tal 1 heterodimer.

We are also interested in determining the molecular basis for homodimerization versus heterodimerization in bHLH proteins. We hypothesize that a specific amino acid code for dimerization exists within the helical segments, and our experiments are aimed at elucidating this code for homodimerization versus heterodimerization. Combinatorial libraries of mutant bHLH domains from Tal 1, E12, and E47 and the dominant repressor protein Id will be prepared to determine the amino acids that specify homodimerization for E12/E47 and heterodimerization of E12/E47 with Tal 1 and of E12/E47 with Id.

PUBLICATIONS

Dickinson, L.A., Edgar, A.J., Ehley, J., Gottesfeld, J.M. Cyclin L: an RS domain protein involved in pre-mRNA splicing. J. Biol. Chem., in press.

Ehley, J.A., Melander, C., Herman, D., Baird, E.E., Ferguson, H.A., Goodrich, J.A., Dervan, P.B., Gottesfeld, J.M. Promoter scanning for transcription inhibition with DNA-binding polyamides. Mol. Cell. Biol. 22:1723, 2002.

Gottesfeld, J.M., Luger, K. Energetics and affinity of the histone octamer for defined DNA sequences. Biochemistry 40:10927, 2001.

Turner, J.M., Gottesfeld, J.M. Regulation of gene expression with synthetic DNA-binding ligands. Chemtracts 14:563, 2001.