Polydactyl Zinc Finger Gene Switches as a New Software for the Genome

In all organisms, from the simplest to the most complex, proteins that bind nucleic acids control the expression of genes. The nucleic acids DNA and RNA are the molecules that store the recipes of all life forms. The fertilized egg of a human contains the genetic recipe for the development and differentiation of a single cell into two cells, four cells, and so on, finally yielding a complete individual. The coordinated expression or reading of the recipes for life allows cells containing the same genetic information to perform different functions and to have distinctly different physical characteristics. Proteins that bind nucleic acids enable this coordinated control of the genetic code. Lack of coordination due to genetic defects or to viral seizure of control of the cell results in disease.

One major effort in this laboratory involves the development of methods to produce proteins that bind to specific nucleic acid sequences. The production of these new proteins will enable us to address fundamental questions concerning this binding, i.e. to study protein recognition of nucleic acids but also to create a powerful tool and therapeutic strategy. These proteins will be used as specific genetic switches to turn on or turn off genes on demand creating an operating system for genomes. Towards this end we have made significant progress in selecting and designing specific zinc finger domains that will constitute an alphabet of 64 domains that will allow any DNA sequence to be bound selectively. The prospects for this "second genetic code" are fascinating and promise a major impact on basic and applied biology.

In order to create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. We have shown that our domains are functionally modular and may be recombined with one to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity like the one pictured here.

We demonstrated for the first time that transcription factors based on modified zinc finger DNA-binding domains can be used to regulate endogenous genes or transgenes. These studies have been extended by several groups demonstrating transcriptional activation or repression of endogenous genes in plant and animal cells.

Recently we reported the development of the first transgenic organisms incorporating this technology. We have also engineered novel transcription factors that can be directly regulated with small molecules using a variety of strategies. This advance now allows for endogenous gene to be placed under chemical control. The ability to study and manipulate genes in their native chromatin environment was previously an unapproachable task. The availability of rapid and robust methods for controlling gene function are of prime importance in the post-genomic era, not only for assigning functions to newly discovered genes, but also for therapeutic intervention. Novel approaches to gene discovery based on retroviral delivery of libraries of transcription factors are also being developed. This approach allows for each gene in the genome of the target organism to be silenced or activated and the resulting cell population selected or screened for novel phenotypes. We are continuing to advance this approach to control genes associated with Cancer, to inhibit HIV transcription, and to provide a new approaches to genetic diseases like sickle cell anemia...but much more remains to be done and to be discovered...

Laboratory Achievements: 
-Developed and Patented the First Artificial Zinc Finger Transcription Factors in 1993 
-Developed the First Polydactyl Zinc Finger Transcription Factors for Genome-Specific Targeting (six-finger proteins)
-Developed the First Artificial Transcription Factors Capable of Endogenous Gene Regulation 
-Developed Validated Modular Zinc Finger Domains that Enable Targeting of most DNA Sequences 
-Developed the First Transgenic Organisms with Artificial Zinc Finger Transcription Factors 
-Developed a Wide-Range of Chemically Regulated Factors including Novel Single-Chain Steroid Hormone Receptor Ligand Binding Domain Fusion Proteins 
-Developed the First Genome Wide Approaches using Transcription Factor Libraries