The Basics of Gene Regulation: DNA, the Genome,
Genes and Gene Regulation
Deoxyribonucleic acid, or DNA, is present in all cells and is responsible for determining the inherited characteristics of all living organisms. A cell’s DNA is arranged as chromosomes and comprises individual units called genes. The complete compliment of genes is known as the genome. The human genome has been sequenced, as have the genomes of several other species. genes encode proteins, which are assembled through the processes of transcription, whereby DNA is transcribed into ribonucleic acid, (RNA), and subsequently translation, whereby RNA is translated into protein.

All cells in an individual's body contain the same set of genes. However, only a fraction of these genes are turned on, or expressed, in an individual cell at any given time. It is the pattern of gene expression that determines the structure, biological function, and health of all cells, tissues, and organisms. The aberrant expression of certain genes can lead to disease.

Transcription factors are proteins that bind to DNA and regulate gene expression. In higher organisms, transcription factors typically consist of two principal components: a DNA-binding domain, recognizes and binds to a specific DNA sequence within or near a particular gene and a functional domain that causes the target gene to be activated (turned on) or repressed (turned off).


What are ZFP TFs?
ZFP TFs are novel transcription factors designed and engineered by Sangamo scientists to regulate the expression of target endogenous genes.


Figure 1: ZFP transcription factors have two domains: A recognition domain that recognizes and binds to a specific DNA sequence, and a functional domain that provides a specific activity for the protein.

Our technology is based upon the engineering of a naturally occurring class of DNA transcription factors called zinc finger DNA-binding proteins, or ZFPs. The DNA recognition and binding function of ZFPs can be used to target a variety of functional domains to a gene-specific location. The two-component structure of our engineered ZFP TFs is modeled on the structure of naturally occurring transcription factors.

Consistent with the two-domain structure of ZFP TFs, we take a modular approach to their design. The recognition domain is composed of two or more zinc fingers; each finger recognizes and binds to a three base pair sequence of DNA and multiple fingers can be linked together to more precisely recognize longer stretches of DNA. By modifying those portions or the critical amino acid contacts of a ZFP that interact with DNA, we can engineer novel ZFPs capable of recognizing defined DNA sequences in any gene.

We can combine engineered ZFPs with a variety of different functional domains to generate proteins that can activate or repress gene expression. In addition, we can engineer a ZFP TF with a functional domain that has a “switch” component, enabling us to regulate its activity and thus the expression of its target gene using a small molecule drug (Regulatable Gene Therapy 76k PDF). The capability for regulated expression is important particularly for the use of ZFP TFs in gene therapy applications (as ZFP Therapeutics) as it allows control of both the duration of the exposure to the therapeutic agent and gives the flexibility of more precise dosing.

What are ZFNs?
Another application of our technology combines the gene targeting function of an engineered ZFP and the functional domain of a restriction endonuclease, an enzyme that cuts DNA. The resulting protein, a ZFP nuclease or ZFN may be used to modify a specific DNA sequence within a gene enabling corrrection or disruption of a gene or the addition of a new DNA sequence.


Why ZFPs?
Of the many different DNA binding motifs that have evolved over time, C2 H2 zinc finger DNA binding proteins have proven to be the most versatile and Nature has fully exploited their useful properties.

(Fig. 2: Genome-wide comparison of transcriptional activator families in eukaryotes. This histogram shows that C2H2 zinc finger class of DNA binding proteins is the most abundant class of transcription factor in all of the species that they authors surveyed. Tupler R, Perini G, Green MR (2001). Nature 409: 832-833.)

C2H2 zinc fingers are found in 2% of all human genes, and they are by far the most abundant class of DNA-binding domains found in human transcription factors. Their structure makes them an ideal framework for engineering to bind to selected target sequences. This is because of their compact modular structure and the fact that they can be stitched together to bind to longer DNA sequences in a predictable way. Each “module” or finger recognizes and binds to 3 base pairs of DNA. Three such fingers can be joined together to bind a 9-base pair sequence and correspondingly 6 fingers an 18 base pair sequence. From the structural analysis of such proteins the actual amino acids that make contact with the DNA have been identified and are located at the same position in each finger. Varying these amino acids enables the ZFP to bind to different DNA sequences.


What are the advantages of ZFP TFs?
We believe that our ZFP TF technology platform has several technical advantages compared with other technologies. Among the advantages of our ZFP TF-based approach to gene regulation are:

What are the advantages of ZFNs?
We believe that our ZFN technology platform has several technical advantages including:

• ZFNs can be used to specifically correct or disrupt genes directly in cells.
• ZFNs can also be used to specifically insert a DNA sequence into a defined site in the genome.
• Permanent gene correction, disruption or insertion requires only transient cellular expression of ZFNs.