Designer TALEs team up for highly efficient gene induction

Abstract

Transcription activator–like effectors (TALEs) can be programmed to specifically bind any DNA, which makes these proteins broadly usable for biotechnology applications in many organisms. One can generate highly specific activators, repressors and nucleases by fusing the TALE DNA-binding domain to different functional protein domains1. In this issue, two groups report that TALE activators can achieve even higher levels of gene induction than envisaged before if several are used simultaneously2,3. These results increase the power of TALEs as tools for targeted gene control. TALE proteins have a unique DNA-binding mode. Tandem 34-amino-acid repeats each recognize one DNA base pair in a contiguous sequence, and repeat-variable diresidues (RVDs) at positions 12 and 13 specify which base is bound (Fig. 1a). The last repeat is conserved only for its first part and termed a halfrepeat. This one-to-one relation of RVD and DNA base makes it possible to use a simple pickand-match approach to build artificial TALEs with just about any desired DNA-binding specificity4. Natural TALEs from plant-pathogenic bacteria function as transcriptional activators in plants, but they can be exploited as designer transcription factors in other organisms when fused to broadly active transcription activation domains such as herpes simplex virus VP16 or its tetrameric repeat VP64 (ref. 5). However, the induction rates have often been relatively low so far. The two papers in this issue explore novel ways to achieve efficient synthetic transcriptional activators2,3. Maeder et al.2 generated a total of 70 designer TALEs to induce three human genes: VEGFA, NTF3 and the microRNA cluster MIR302–367. They achieved induction rates of up to 114-fold over basal transcription. One reason might be that they chose DNase I hypersensitive regions within promoters that have an open chromatin conformation and are often targets of endo genous transcription factors. This approach makes sense considering that TALEs could not induce expression of silent Oct4 in murine neural stem cells unless epigenetic modifiers were inhibited by chemicals6. Maeder et al. successfully designed TALEs that bind either DNA strand upor downstream of the natural transcription start site2. This emphasizes that TALEs can support transcription from very different positions. The authors further analyzed whether the total number of repeats per TALE influences its activity. Among TALEs with 14.5–24.5 repeats, those with 16.5– 18.5 repeats had a higher activity in some but not all instances2. In contrast, 10.5 repeats has already been shown to be sufficient for full activity in plants7. In addition, it is not only the repeat number but also the RVD composition8,9 that controls activity: that is, NN (Asn-Asn) and HD (His-Asp) repeats contribute most to overall TALE function. Researchers are well advised to use TALEs with about 17.5 repeats, an obvious choice anyway if specificity needs to be balanced with TALE size. It is known that transcriptional activators can act synergistically to induce much higher expression than what the individual contributions would suggest10,11. So far, these studies have relied mostly on heterologous GAL4– (ref. 10) or zinc finger–activation domain fusions11. To test whether this effect also applies to TALEs, Maeder et al. simply transfected combinations of five or six TALEs for each of the three genes studied (Fig. 1). Indeed, the TALE combinations elevated transcription of the microRNA cluster substantially (from approximately 3to 46-fold individually up to 283-fold together)2, but the effect on the other genes was minor. In the other article, Perez-Pinera et al.3 targeted four human genes (IL1RN, KLK3, CEACAM5 and ERBB2) with 6–8 TALEs each. Annekatrin Richter and Jens Boch are in the Department of Genetics, Martin Luther University Halle-Wittenberg, Halle, Germany. e-mail: jens.boch@genetik.uni-halle.de Figure 1 | Synthetic TALE activators act synergistically to express human genes. (a) Cartoon of a TALE. The indicated amino acids in each repeat recognize the base below. NLS, nuclear localization sequence; VP64/p65, activation domains (ADs). (b) Single TALEs induce target human genes with variable efficiencies. (c) Combinations of TALEs targeting either DNA strand allow for much higher gene induction rates. a