The design and development of artificial transcription factors

The exquisite specificity with which gene networks are expressed to determine cellular fate and function is mediated by regulatory proteins called transcription factors (TFs). The central role of TFs in the regulation of specific genes and gene networks is underscored by the fact that their malfunction is linked to metabolic and developmental disorders, cancer, and diabetes. Thus, a major goal at the interface of chemistry, biology, and molecular medicine is the generation of synthetic molecules that function to modulate the activity of TFs.;To address this need, we developed a new class of artificial transcription factors known as protein-DNA dimerizers (PDDs). PDDs take advantage of the modularity and cooperativity found in natural TFs to enhance the DNA binding affinity and specificity of their intended TF target. To achieve this, the PDDs are composed of a DNA binding domain (DBD) for specific DNA targeting and a protein interaction domain (hook) which mediates the interaction with specific TFs and provides spatial control over PDD activity.;In this thesis, I describe the creation of PDDs that have been designed to mimic the natural Hox family of developmental regulators in their ability to recruit Exd to DNA. Examination of the linker between the DBD and hook domains demonstrated that PDDs are flexible in the positioning of the hook peptide, and long linkers conferred temperature sensitivity on PDD function, imparting temporal control over their function. The PDDs were used to examine the role of the Ubx Ia linker in Exd-Ubx complex formation. PDDs also permitted reaching DNA binding sites that are more favorable for specific binding but are not directly adjacent to the target TF. Cognate site identifier (CSI) analysis provided rapid assessment of the DNA affinity and specificity of new DBDs for PDDs and identified a new binding mode for Exd-PDD complex formation. CSI-FID (fluorescent intercalator displacement) is a new technology for the label-free analysis of the DNA binding properties of untagged biomolecules and small molecule DBDs. These studies describe the design and optimization of PDDs, towards modulating transcriptional activity for the analysis of gene networks or the regulation of aberrant gene activity.