Exploring Strategies for the Design of Artificial Transcription Factors

Abstract

Artificial transcription factors can be engineered to interact with specific DNA sequences to modulate endogenous gene expression within cells. A significant hurdle to implementation of this approach is the selection of the appropriate DNA sequence for targeting. We reasoned that a good target site should be located in chromatin, where it is accessible to DNA-binding proteins, and it should be in the close vicinity of known transcriptional regulators of the gene. Here we have explored the efficacy of these criteria to guide our selection of potential regulators of ␥-globin expression. Several zinc finger-based transcriptional activators were designed to target the sites proximal to the ؊117-position of the ␥-globin promoter. This region is proximal to the binding sites of known and potential natural transcription factors. Design and study of three transcription factors identified the potent transcriptional activator, gg1-VP64-HA. This transcription factor was able to interact directly with the ␥-globin promoter and upregulate expression of reporter gene constructs as well as the endogenous gene in a selective manner. Transfection of a gg1-VP64-HA expression vector or retroviral delivery of this transcription factor into the erythroleukemia cell line K562 resulted in an increase of fetal hemoglobin. The ␥-globin content of cells expressing gg1-vp64-HA showed up to 16-fold higher levels of fetal hemoglobin than the native K562 cell line. These transcriptional activators constitute a novel class of regulators of the globin locus that may be suitable for treatment of diseases arising from mutations in this locus such as sickle cell disease and thalassemic diseases.

A zinc-finger transcriptional activator designed to interact with the γ-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts

“…38 We have used a relatively low concentration of SCF of 10 ng/mL in our cultures to achieve a low baseline level of HbF production, whereas a concentration of 50 ng/mL is typically used when HbF synthesis is maximized. 38 The zinc-finger transcriptional factor that we have shown induces HbF in maturing erythroblasts derived from adult CD34 ϩ cells was designed to function as a transcriptional activator ( Figure 6A); indeed, it has been shown to enhance expression from a minimal ␥-promoter in a reporter assay 15 (supplemental Figure 2B). However, the region in the ␥-globin gene promoter to which it binds includes a sequence called the direct repeat element because it is tandemly repeated in the ⑀ promoter ( Figure 6B).…”

“…The ␤-spectrin promoter was excised as a 568-bp EcoRI-EcoRI fragment from pCR2.1-Topo clone described in "GFP vectors" and cloned between MfeI and EcoRI sites of an intermediate plasmid 5Ј to the internal ribosome entry site GFP sequences to create pSp-iG. This vector was linearized with EcoRI (blunt) to allow for insertion of the 803-bp GG1-VP64 coding sequences recovered as a BamHI-EcoRI fragment from a retroviral expression vector analogous to pMx-gg1-VP64-HA 15 and rendered blunt with Klenow DNA polymerase generating pSp-GG1-VP64-iG. The Sp-iG or Sp-GG1-VP64-iG intermediates were excised as StuI-SnaBI fragments and cloned into pCL20cMp-GFP digested with MluI (blunt)-EcoRI (blunt) replacing the Mp-GFP cassette to create pCL20cSp-iG or pCL20cSp-GG1-VP64-iG, respectively.…”

“…16 Zinc finger domains linked to a transcriptional activator domain were constructed to bind to 18-bp segments of the proximal ␥-globin gene promoters. 15 Of the 3 independent artificial transcriptional factors that were assembled, one binding to the 18-bp segment of the ␥-promoter, which includes position Ϫ117, proved to be the most active at augmenting ␥-globin production in K562 human erythroleukemia cells. The Ϫ117 position of the A ␥-promoter is the site of a naturally occurring mutation resulting in hereditary persistence of HbF 17,18 ; thus, this region of the ␥-globin promoter is known to be relevant to modulation of ␥-globin gene expression.…”

“…15 Advances in the engineering of polydactyl zinc-finger transcription factors has made it possible to produce zinc-finger proteins capable of recognizing any 18-bp stretch of DNA. 16 Zinc finger domains linked to a transcriptional activator domain were constructed to bind to 18-bp segments of the proximal ␥-globin gene promoters.…”

Development of Zinc Finger Domains for Recognition of the 5′-CNN-3′ Family DNA Sequences and Their Use in the Construction of Artificial Transcription Factors

“…Likewise, we have recently reported the construction of the ␥-globin-targeting protein gg1 incorporating the 5Ј-CTG-3Ј domain together with 5Ј-ANN-3Ј and 5Ј-GNN-3Ј domains. This protein binds the sequence 5Ј-GTC AAG GCA AGG CTG GCC-3Ј with a 0.7 nM dissociation constant (42). This protein was shown by DNase I footprinting and chromatin immunoprecipitation to bind the targeted sequence in vitro and in vivo, and transcription factors based on this protein were robust regulators of the endogenous human ␥-globin gene.…”