Repression of DNA-binding-dependent glucocorticoid receptor-mediated gene expression

摘要

Gene expression is controlled by transcription factors that regulate the rates at which genes are expressed either by recruiting or inhibiting protein complexes that bind to the promoters or enhancers of target genes. Molecules that can specifically modulate these protein-DNA interfaces show promise as tools for understanding gene regulation pathways and may have application in human medicine. Hairpin pyrrole-imidazole polyamides are programmable oligomers that bind the DNA minor groove in a sequence-specific manner with affinities comparable to those of natural DNA-binding proteins. These cell-permeable small molecules have been shown to enter the nuclei of live cells, disrupt protein-DNA interactions, and downregulate endogenous gene expression. This thesis describes the use of polyamides to modulate gene expression in order to probe gene regulation mechanisms of several different biologically relevant systems. A polyamide is designed to target the glucocorticoid receptor transcription factor DNA binding site located in the promoter of the glucocorticoid-induced leucine zipper gene. This polyamide is shown to bind with high affinity to the promoter sequence, modulate the expression of this gene, and disrupt the binding of the protein to the gene’s promoter. Examination of the global effects of this polyamide on mRNA transcription is used to elucidate a list of genes that are regulated by a glucocorticoid receptor protein-DNA dependent mechanism. Also in this thesis, the specificities of a Cy3-labeled polyamide known to downregulate expression of the Vascular Endothelial Growth Factor is examined using DNA microarrays composed of hairpins harboring all 524,800 unique 10 base pair DNA sequences. We experimentally verify the correlation of Cy3 fluorescence intensity with quantitative DNase I footprint-derived binding affinities. Additionally, progress is made towards the polyamide-mediated inhibition of Myc/Max transcription factor gene regulation.