ZFP226 is a novel artificial transcription factor for selective activation of tumor suppressor KIBRA

KIBRA has been suggested as a key regulator of the hippo pathway, regulating organ size, cell contact inhibition as well as tissue regeneration and tumorigenesis. Recently, alterations of KIBRA expression caused by promotor methylation have been reported for several types of cancer. Our current study aimed to design an artificial transcription factor capable of re-activating expression of the tumor suppressor KIBRA and the hippo pathway. We engineered a new gene named ‘ZFP226′ encoding for a ~23 kDa fusion protein. ZFP226 belongs to the Cys2-His2 zinc finger type and recognizes a nine base-pair DNA sequence 5′-GGC-GGC-GGC-3′ in the KIBRA core promoter P1a. ZFP226 showed nuclear localization in human immortalized kidney epithelial cells and activated the KIBRA core promoter (p < 0.001) resulting in significantly increased KIBRA mRNA and protein levels (p < 0.001). Furthermore, ZFP226 led to activation of hippo signaling marked by elevated YAP and LATS phosphorylation. In Annexin V flow cytometry assays ZFP226 overexpression showed strong pro-apoptotic capacity on MCF-7 breast cancer cells (p < 0.01 early-, p < 0.001 late-apoptotic cells). We conclude that the artificial transcription factor ZFP226 can be used for target KIBRA and hippo pathway activation. This novel molecule may represent a molecular tool for the development of future applications in cancer treatment.

acute lymphocytic leukemia 24,25 . Furthermore, alterations of KIBRA expression in clear cell renal cell carcinoma (ccRCC) have been analyzed in whole-genome expression profiling using Illumina BeadChip technology. The gene expression profiles of 101 ccRCC and adjacent tissue sample pairs of the K2 series suggested KIBRA downregulation in this series using locus-specific probes 26 . In this line, we observed that inactivated KIBRA expression depends on promoter methylation in ccRCC 27 .
Since aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during tumorigenesis, regaining expression and effective normalization of function offers a unique opportunity for targeted therapies 28 . Novel and more precise medical strategies may involve artificial TFs for locus-specific modulation of gene expression and the reactivation of tumor suppressor function. This approach has been successfully used for the experimental treatment of breast, ovarian and cervical cancer cell lines with an artificial TF re-activating EPB41L3 expression even when expression was silenced by promoter hypermethylation 29 . Here, we describe the activation of the tumor suppressor KIBRA using a novel artificial TF named ZFP226. ZFP226 induced KIBRA mRNA and protein, resulting in increased YAP phosphorylation and thus activation of hippo signaling. Furthermore, ZFP226 was capable of reducing the viability of MCF-7 breast cancer cells.

Results
Design of the artificial zinc finger ZFP226. KIBRA, a hippo pathway regulator, has been identified as a central TSG, which is frequently affected by epigenetic silencing in different types of cancer [1][2][3] . We recently reported that human KIBRA expression depends on a complex alternative CpG-rich promoter system 30 . Moreover, we observed that inactivated KIBRA expression depends on promoter methylation in ccRCC 27 . Based on this work, we generated an artificial zinc finger TF for KIBRA expression reactivation. The artificial three zinc finger ZFP226 belongs to the Cys2-His2 type and was constructed to target the DNA sequence 5′-GGC-GGC-GGC-3′ located within the human KIBRA core promoter P1a (Fig. 1A). This DNA target sequence was chosen for its location within a transcriptionally active region with high sensitivity for promoter methylation 30 . The ZFP226 sequence consists of 279 bp encoding for 93 amino acids. A nuclear localization signal ('PKKKRKV'), a VP64 activator domain of four VP16 motifs ('DALDDFDLDML') and a HA-tag ('YPYDVPDYA') were linked to the ZFP226, resulting in a ~23 kDa (522 bp) fusion protein (Fig. 1B, Supplementary Fig. S1). The Cys2-His2 class of zinc finger proteins was chosen with respect to very specific characteristics. In brief, the structural stability of the Cys2-His2 zinc finger ββα-fold is based on hydrophobic interactions and chelation of a zinc ion by the Cys2-His2 residues 31 . Amino acid side chain contacts created by the α-helix of the domain are responsible for nucleic acid recognition 31 . The covalent linkage of multiple zinc finger domains subsequently allows for the recognition of extended asymmetric DNA sequences 31 such as the KIBRA target 5′-GGC-GGC-GGC-3′ motif.
The artificial zinc finger ZFP226 was readily expressed in IHKE cells transfected with the generated expression plasmid (pZFP226) as detected by western blot targeting the HA-tag (Fig. 1C). Nuclear localization was confirmed by immunofluorescence using anti-HA antibody (Fig. 1D). WGA was used to stain cellular membranes (nuclear envelope, endoplasmic reticulum [ER], plasma membrane, vesicles). The merged image suggests ZFP226 localization also to membranous compartments such as the ER, endosomes and lysosomes within the cytosol (Fig. 1D, enlarged detail of merged image).
Expression of ZFP226 leads to activation of the KIBRA core promoter. Luciferase-based cotransfection experiments using a KIBRA core promoter construct as reporter and the ZFP226 expression vector resulted in a ~1.7-fold increase of transcriptional activity for promoter construct −730/+186 compared to shuttle vector control (p < 0.001, Fig. 2) in IHKE cells. Consistently, ZFP226 binding-site mutation prevented the activating effect of ZFP226 (p = 0.0731, Fig. 2). This result suggests that ZFP226 is able to drive KIBRA gene expression by target promoter activation.

ZFP226 induces the upregulation of endogenous KIBRA.
To study the cellular consequences induced by the generated artificial zinc finger ZFP226, we analyzed KIBRA mRNA and protein expression levels in transfected IHKE cells. Real-time PCR analysis revealed significantly increased KIBRA mRNA expression levels 48 hrs after ZFP226 transfection compared to shuttle vector control (all p < 0.001, Fig. 3A). Of note, our previous analyses revealed that ubiquitous TF SP1 is a strong activator of KIBRA expression on mRNA and protein level 27 . Therefore, SP1 overexpression served as positive control in real-time PCR and western blot experiments. KIBRA protein was significantly increased by SP1 and ZFP226 compared to shuttle vector control (p < 0.001, Fig. 3B). These results suggest that the artificial TF ZFP226 exhibits comparable biological activity to drive KIBRA expression as TF SP1.
ZFP226 activates YAP and LATS1 phosphorylation. Since active hippo signaling leads to LATS1 phosphorylation und subsequent YAP inactivation with cytosolic sequestration and eventual degradation, relative LATS1 and YAP phosphorylation (pLATS1, pYAP) are important indicators of hippo pathway activity. We were able to show that relative levels of pLATS1 and most importantly pYAP were significantly increased 48 hrs after ZFP226 transfection in IHKE cells (both p < 0.001, Fig. 4A,B), suggesting an activation of hippo signaling by elevated KIBRA expression. Of Note, YAP expression was unaffected by pZFP226 transfection.

ZFP226 induces apoptosis of breast cancer cells. In cancer, defects in different apoptotic pathways
can disturb the balance between cell proliferation and apoptosis, and thus allow the survival of cells with genetic abnormalities 32 . The hippo pathway has been shown to regulate cell number by modulating proliferation, differentiation, and apoptosis [1][2][3][4] . Furthermore, impaired hippo signaling has been reported in a variety of different cancers, including breast cancer 13,14 , linking dysregulated hippo signaling to tumor initiation and progression [15][16][17][18][19] . Therefore, we hypothesized that ZFP226 may induce apoptosis in human MCF-7 breast adenocarcinoma cells by activating hippo signaling.  Cells were transfected with pZFP226, incubated for 48 hrs and analyzed by flow cytometry (Fig. 5A,B, Supplementary Fig. S4). The number of apoptotic cells within quadrant Q2 (early-apoptotic cells) and quadrant Q3 (late-apoptotic/necrotic cells) was significantly increased after ZFP226 transfection in MCF-7 cells compared to shuttle vector control (p Q2 < 0.05, p Q3 < 0.01; Fig. 5B). Additionally, real-time PCR analysis of apoptotic markers revealed significantly increased pro-apoptotic BAX as well as decreased anti-apoptotic BCL-2 expression after ZFP226 transfection compared to shuttle vector control (p BAX < 0.01, p BCL-2 < 0.001; Fig. 5C).

Discussion
In the current study we report the design, construction and functional characterization of the novel artificial TF ZFP226. ZFP226 was capable to (1) activate the KIBRA core promoter, a tumor suppressor and upstream regulator of the hippo pathway, resulting in (2) significantly increased KIBRA mRNA as well as protein levels, (3)  activation of hippo signaling marked by elevated LATS1 and YAP phosphorylation and (4) reduced viability of breast cancer cells.
In humans, impaired hippo signaling has been reported for several types of cancer 16,33 such as renal cell carcinoma 11 , hepatocellular carcinoma 12 and breast cancer 13,14 . In line with this observation, components of the hippo pathway are target of aberrant gene methylation and epigenetic silencing also in humans 17 as already reported for LATS1/2 20,21 , MST1/2 22,23 and KIBRA 24,25 . We recently reported that human KIBRA expression depends on a complex alternative CpG-rich promoter system 30 with inactivated KIBRA expression induced by promoter methylation in ccRCC 27 . Based on this work, we generated the artificial zinc finger ZFP226 to activate the expression of tumor suppressor KIBRA and (re-)activation of the hippo pathway. Artificial zinc finger TFs have been suggested as a powerful molecular tools to modulate target gene expression in cells and organisms. These TFs are designed to specifically recognize target sites within the promoter region of interest and effectively up-or downregulate expression of their target genes not only in vitro, but also in vivo [34][35][36][37][38] . This approach has been successfully used for the experimental treatment of breast, ovarian and cervical cancer cell lines with an artificial TF re-activating EPB41L3 expression even when expression was silenced by promoter hypermethylation 28 . Furthermore, Cori et al. 35 engineered an artificial zinc finger-based TF ("Jazz") for upregulation of the human and mouse utrophin expression 35,36 . Mattei et al. 37 generated transgenic mice that specifically overexpressed "Jazz" in skeletal muscle. Subsequently, crossing the "Jazz" transgenic mice with the Duchenne muscular dystrophy mouse model resulted in a strong amelioration of the dystrophic phenotype 37,38 . These examples provide evidence for the promising therapeutic approach based on artificial TFs.
ZFP226 belongs to the Cys2-His2 zinc finger type and recognizes a nine base pair DNA sequence 5′-GGC-GGC-GGC-3′ in the KIBRA core promoter P1a, which is well-characterized 30 . Artificial TFs are designed to target a single promoter but may have multiple DNA targets by chance. Assuming random base distribution, a nine base pair DNA sequence such as 5′-GGC-GGC-GGC-3′, is present in the human genome about 1.3 × 10 4 times 35 . However, only a small portion of these sequences is accessible to DNA-binding proteins with an active role in gene expression regulation. Even if we were able to demonstrate the ability of ZFP226 to activate the KIBRA core promoter resulting in significantly increased KIBRA mRNA and protein levels, further improvement of ZFP226 specificity by target sequence extension is mandatory.
The hippo pathway negatively regulates the activity of two main downstream mediators, YAP and its family member TAZ [7][8][9] . YAP and TAZ are inactivated by phosphorylation through LATS kinases with subsequent inhibition of proliferation 10 . Therefore, YAP protein level and YAP phosphorylation are important indicators of hippo pathway activity. Of note, in datasets of breast cancer patients, elevated expression of gene signatures for YAP/ TAZ activity correlated with high histological grade, enrichment of stem cell signatures, metastasis development and progression, as well as poor outcome [39][40][41] . To this respect, enriched TAZ nuclear staining has been detected in high-grade breast cancer 40,42 and is associated poor clinical outcome 42,43 . Furthermore, in primary breast cancer specimens reduced KIBRA expression has been correlated with the claudin-low subtype, an aggressive sub-group with epithelial-to-mesenchymal transition features and a poor prognosis 44 . In our experiments, we detected a significant activation of hippo signaling in ZFP226-transfected cells marked by elevated LATS1 and YAP phosphorylation. Of note, LATS1 protein levels tended to be decreased in individual ZFP226 experiments suggesting a potential feedback mechanism to restore hippo pathway homeostasis 5 . However, this trend was not statistically significant over all analyzed experiments. Most importantly, we found that ZFP226 was able to induce apoptosis by activating hippo signaling in human breast adenocarcinoma cells as we observed a significantly increased number of early-and late-apoptotic cells after transfection with ZPF226. These findings were supported by significantly elevated expression of pro-apoptotic BAX and decreased expression of anti-apoptotic BCL-2 within ZFP226-transfected cells.

Conclusion
ZFP226 is a novel artificial TF, which was capable to activate the KIBRA core promoter, to significantly increase KIBRA mRNA as well as protein levels, thereby activating hippo signaling marked by elevated LATS1 and YAP phosphorylation. ZFP226 reduced the viability of breast cancer cells in vitro. This novel molecule may represent a molecular tool for the development of future applications in cancer treatment and needs further investigation.

Methods
Construction of the ZFP226 transcription factor. The artificial zinc finger protein ZFP226 was constructed using ZF tools 3.0 as described previously 45 . ZFP226 was engineered to drive transcription from the 9 base pair DNA sequence 5′-GGC-GGC-GGC-3′ located to KIBRA promoter P1a 30 . The resulting ZFP226 sequence consists of 279 bp encoding for 93 amino acids. A nuclear localization signal ('PKKKRKV'), a VP64 activator domain of four VP16 motifs ('DALDDFDLDML') and an HA-tag ('YPYDVPDYA') were fused C-terminal to the ZFP226, resulting in a ~23 kDa (522 bp) protein. Off-target binding was controlled using data from ELISA specificity graphs (implemented in ZF tools 3.0). The final sequence was synthesized and ligated into the pEX-A2 vector by Eurofins Genomics. ZFP226 cDNA was then hydrolyzed using XhoI and KpnI and subcloned into the pCMV-driven eukaryotic expression vector pcDNA3.1+ (Thermo Scientific; Supplementary Fig. S6). Sequence accuracy and identity was controlled by direct sequencing of both DNA strands.
Real-time PCR. Total RNA was extracted using the NucleoSpin RNA Kit (Macherey-Nagel). First strand cDNA synthesis was performed using MuLV Reverse Transcriptase (Thermo Scientific) and 1 µg of total RNA. cDNA was amplified in a 384-well format (standard real-time PCR conditions) in duplicates using Power SYBR Green (Thermo Scientific) on an Applied Biosystems 7500 Fast real-time PCR system. Relative quantification was calculated using the 2 −ΔΔCt method and S18 as endogenous control. The absence of non-specific amplification products was confirmed by agarose gel electrophoresis and generation of melting curves using the Applied Biosystems software. Oligonucleotides had an amplification efficiency of ≥90%. BAX and BCL-2 were used as standard markers for apoptosis as described 52 (oligonucleotide sequences are given in Supplementary  Table S1).
Annexin V apoptosis assay. For Annexin V apoptosis assays, MCF-7 cells were transfected with pZFP226 or pcDNA3.1 as shuttle vector control using the Neon Transfection System (Thermo Scientific) according to the manufacturer's protocol. In brief, a total of 5 × 10 6 cells/ml was transfected with 1 μg of plasmid DNA using the following configuration: 1100 V, 2 pulse, 30 ms. Then, 5 × 10 4 cells/ml were seeded on a 6-well plate and incubated for 48 hrs. Cells were harvested using trypsin, spun down and washed with PBS twice. Cells were labelled for Annexin V as described 53 using APC (allophycocyanin)-conjugated Annexin V (1:20; BD biosciences). Cells were DAPI -stained directly before measurement. Cells were sorted using BD FACSCanto II (BD biosciences). Diva software v6.1.3 was used and data analysis was performed with FlowJo software v9.5.1.
Statistical analysis. Data are given as mean ± SD p-values were calculated by unpaired, two-tailed Student's t-test or one-way ANOVA where appropriate. p-values < 0.05 were considered significant. Data availability. The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.