Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the hTERT Locus

Abstract

The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.

Figure 1

Approximate location and sequence of the human telomerase reverse transcriptase 5 (hTERT5) and hTERT6 sites in the hTERT locus. (a) The hTERT5 and hTERT6 zinc finger nuclease (ZFN) full target sites were identified in the hTERT promoter and exon-1. Genomic sequences are provided in the traditional 5′-3′ orientation of the top strand and 3′-5′ of the bottom strand with position is given relative to the translational start codon. (b) Schematic of hTERT promoter locus showing relative position of hTERT6L, hTERT6R, and hTERT5R zinc finger transcription factor-binding sites at scale. The arrow represents the site of the initiation ATG.

Figure 2

Screening human telomerase reverse transcriptase (hTERT) zinc finger transcription factors (ZFTFs) for transcriptional activation activity. (a) Schematic of the pMC6 transcriptional reporter, which contains 3 kb of the hTERT promoter and exon-1 sequence upstream of a green fluorescent protein (GFP) reporter gene. When cotransfected with a ZFTF-expressing plasmid, the ZFTF will bind to the pMC6 reporter plasmid and recruit the cellular machinery necessary to express GFP. (b) Flow cytometry plots demonstrating the increase in GFP⁺ cells after cotransfection of 100 ng of the MC6 reporter plasmid with 700 ng of plasmid expressing the KW602 ZFTF in HEK293 cells. Here, the x-axis plots cellular autofluorescence as “orange” (abscissa) and the y-axis plots the fluorescent intensity of GFP expressed by pMC6 as “green”. (c) All ZFTFs listed in Table 2 were screened for transcriptional activation activity using pMC6. Our results identify which ZFPs efficiently recognize and bind the hTERT target half-sites as ZFTFs to stimulate increases in %GFP⁺ over background (mean ± SD). ZFP, zinc finger protein.

Figure 3

Human telomerase reverse transcriptase (hTERT) zinc finger transcription factors (ZFTFs) exhibit an additive response. The best performing ZFTF for each target half-site were transfected in increasing amounts of ZFTF-expressing plasmid (200, 400, and 600 ng) with 100 ng of pMC6 into HEK293 cells. The KW602, KW620, and KW641 ZFTFs were also cotransfected in pairs or all three combined (200 ng each, mean ± SD).

Figure 4

Diagram of the green fluorescent protein (GFP)-based single-strand annealing (SSA) assay. For the purpose of screening nuclease activities of various zinc finger nuclease (ZFN) pairs, we inserted the GFP1/2 ZFN full site and a human telomerase reverse trancriptase (hTERT) ZFN full site between two repeated sequences (hatch boxes) within a GFP reporter gene. Cotransfection of this reporter plasmid (20 ng) with ZFN-expressing plasmids (100 ng each) into HEK293 cells results in the delivery of a double-stranded break between the repeated GFP sequences which is then repaired by the endogenous SSA pathway (after the repeat sequences have annealed to each other) to produce a functional GFP reporter gene.

Figure 5

Schematic of the green fluorescent protein (GFP)-based gene-targeting reporter cell line. Based on a previously described strategy, we rendered a GFP reporter gene non-functional through the insertion of a stop codon, an I-SceI recognition site, a frame shift nucleotide, and the human telomerase reverse transcriptase 6 (hTERT6) zinc finger nuclease (ZFN) full site. Using this construct, we generated a stably integrated clonal HEK293 cell line in which we cotransfected pairs of ZFN-expressing plasmids and a repair donor plasmid. Nuclease activity of the ZFN pair is measured by the ZFN-mediated HR repair event between the cut GFP reporter and the donor plasmid producing a functional GFP reporter gene. The CAG promoter used in these experiments is a hybrid of the CMV intermediate-early enhancer and the chicken β-actin promoter. CMV, cytomegalovirus; DSB, double-stranded break; HR, homologous recombination.

Figure 6

Zinc finger nuclease (ZFN)-induced modification of the genomic human telomerase reverse transcriptase (hTERT) locus in K562 cells. ZFN-treated cell populations were treated with either the hTERT5 (KW744/KW664) or hTERT6 ZFNs (KW635/KW613) and screened in bulk for mutations in the genomic that arose after repair of the ZFN-induced double-stranded break by NHEJ pathways. Mismatches in the mutated sequences are detectable by digestion with a form of the Cel1 nuclease (Surveyor). We included the use of human codon-optimized Fn domains in the ZFNs and expected digest products are given to the right of the figure. NHEJ pathway, non-homologous end joining pathway.