Rational Design, Selection and Specificity of Artificial Transcription Factors (ATFs): The Influence of Chromatin in Target Gene Regulation

Abstract

Artificial Transcription Factors (ATFs) are engineered DNA-binding proteins designed to bind specific sequences of DNA. ATFs made of Zinc Finger (ZF) domains have been developed to regulate specific genes and phenotypes both in cells and whole organisms. Recently, an emerging application of engineered DNA-binding domains include the specific editing of the genome, the ability to specifically cut, recombine, modify DNA and image protein-nucleic acid interactions in living cells. In this review we will summarize the techniques used for the rational design, screening and functional selection of ZF proteins in mammalian cell systems and their applications in areas of biotechnology, functional genomics and molecular therapeutics. The in vivo specificity of the engineered ATFs will be discussed, with particular emphasis on epigenetic modifications influencing ATF-DNA interactions.

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

“…Six zinc finger (6ZF) arrays read an 18-base pair (18-bp) sequence that is potentially unique in the human genome and provide a high degree of genomic specificity and selectivity (12). Engineering binding specificity is achieved by grafting the ␣-helical domain of each ZF known to interact with the target DNA triplet (13). We have constructed multimodular 6ZF proteins referred as artificial transcription factors (ATFs), recognizing sequences in targeted promoters with dissociation constants in the picomolar range (10,(13)(14)(15)(16)(17)(18).…”

“…Engineering binding specificity is achieved by grafting the ␣-helical domain of each ZF known to interact with the target DNA triplet (13). We have constructed multimodular 6ZF proteins referred as artificial transcription factors (ATFs), recognizing sequences in targeted promoters with dissociation constants in the picomolar range (10,(13)(14)(15)(16)(17)(18). In an ATF, the 6ZF scaffold can be linked to a variety of protein modules to promote transcriptional activation (19 -23), repression (24,25), and more recently, epigenetic editing (26).…”

Breaking through an epigenetic wall

“…Moreover, like their natural counterparts, artificial ZFP require specific activators and repressor domains to promote differential expression of targeted genes. 35,36 More recently, we have described nearly 95% repression of Sox2 via ZFP attached to a Kruppel associated Box (KRAB or SKD domain 37 ) and ~60% repression with the same ZFPs linked to a Dnmt3a catalytic domain, 38 in breast cancer cells. KRABcontaining ZFPs are traditionally regarded as potent transcriptional repressors via KAP-1-mediated recruitment of Histone deacetylase NuRD complex, Histone deacetylases (HDACs), histone methyltransferase SETDB1 and heterochromatin protein 1 (HP1).…”

Engineering Transcription Factors in Breast Cancer Stem Cells

“…This has facilitated the construction of arrays of ZFs to recognize large DNA sequences using simple and relatively quick molecular biology approaches. Nevertheless, proteins of improved DNA-binding specificity can be further optimized by randomization and subsequent selection of the ZF backbone, using both in vitro and in vivo approaches (for an overview of these, see references [7,86,87]). Our laboratory has focused on six-ZF arrays recognizing 18-bp sequences because of the high affinity of these proteins (with K d s in the picomolar range).…”

“…ATFs are typically composed of arrays of specific six Zinc Finger (ZF) domains, and are designed to bind, unique eighteen base pairs (bps) in targeted promoters. These ZFs are linked to several effector domain functions, which mediate activation, repression or epigenetic regulation of the gene of interest [7]. We will review ATFs generated in our laboratory and others, able to reprogram the epigenetic status of endogenous promoters, oncogenes and tumor suppressors, to revert some of the phenotypic hallmarks of aggressive cancer cells.…”