Artificial Transcription Factors Research Articles

Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni–NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.

Su1991 Reactivation of Reprimo by an Artificial Transcription Factor in Gastric Cancer Cell Lines

Introduction: Gastric cancer (GC) is the second cause of cancer-related death worldwide and the leading one in Latin America. It has been proposed that the silencing of Reprimo (RPRM), by epigenetic mechanisms, is a putative biomarker of GC. RPRM is a p53 dependent G2 arrest mediator and is considered to be tumor suppressor gene involved in the pathogenesis of numerous cancers such as gastric, colon, pituitary, among others. We propose that the reactivation of RPRM expression, by means of an activator Artificial Transcription Factor (ATF), modifies the proliferation rate of GC cell lines in vitro. Methods: An activator ATF for RPRM was designed using the method proposed by zincfingertools.org. Thus, an ATF with a unique domain of six zinc-finger capable of binding specifically to 18 consecutive base pairs, located in the promoter region of RPRM, was engineered. This ATF was then inserted into a modified pcDNA3.1+ vector coupled with a potent transcription activator domain (VP64). A luciferase reporter assay was performed using a pGL3 vector (Promega), linked to a 634 base pairs region of the promoter region of RPRM, in order to evaluate the efficiency and specificity of the synthetized ATF. Finally, the ATF was transiently transfected with lipofectamine2000 (Invitrogen). Subsequently, RPRM mRNA expression was evaluated by a RT-qPCR and proliferation rate was evaluated by a MTS assay. The experiments were conducted on AGS and MKN28 GC cell lines. Results: The ATF designed was able to significantly increase the luciferase activity vs controls, both in MKN28 and AGS cell lines. Accordingly, RPRM mRNA was upregulated in the presence of the ATF. These findings were associated with a lower proliferation rate in both cell lines. Since the AGS cell line is highly methylated in the promoter region of RPRM, it might explain a significantly lower response to the presence of ATF, in comparison with that of MKN28. Discussion: Our work suggests that a RPRM specific ATF could be used as a novel tool to reactivate this epigenetically silenced tumor suppressor gene. Moreover, the upregulation of RPRM modifies one of the hallmarks of cancer pathways by reducing the proliferation rate of cancerous cell lines in vitro. Grant Support: Fondecyt #1111014 and Fondef #D09I1137 from the Government of Chile to AHC.

Creation of a Six-fingered Artificial Transcription Factor That Represses the Hepatitis B Virus HBx Gene Integrated into a Human Hepatocellular Carcinoma Cell Line

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection–related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection–related HCC.

Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors

C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression.Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, re-activation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets.

Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer

Background:The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear.Methods:Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain.Results:Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival.Conclusion:The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer.

UtroUp is a novel six zinc finger artificial transcription factor that recognises 18 base pairs of the utrophin promoter and efficiently drives utrophin upregulation

BackgroundDuchenne muscular dystrophy (DMD) is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin. Currently there is no effective treatment for DMD. Among the different strategies for achieving a functional recovery of the dystrophic muscle, the upregulation of the dystrophin-related gene utrophin is becoming more and more feasible.ResultsWe have previously shown that the zinc finger-based artificial transcriptional factor “Jazz” corrects the dystrophic pathology in mdx mice by upregulating utrophin gene expression. Here we describe a novel artificial transcription factor, named “UtroUp”, engineered to further improve the DNA-binding specificity. UtroUp has been designed to recognise an extended DNA target sequence on both the human and mouse utrophin gene promoters. The UtroUp DNA-binding domain contains six zinc finger motifs in tandem, which is able to recognise an 18-base-pair DNA target sequence that statistically is present only once in the human genome. To achieve a higher transcriptional activation, we coupled the UtroUp DNA-binding domain with the innovative transcriptional activation domain, which was derived from the multivalent adaptor protein Che-1/AATF. We show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, possesses a low dissociation constant that is consistent with a strong affinity/specificity toward its DNA-binding site. When expressed in mammalian cell lines, UtroUp promotes utrophin transcription and efficiently accesses active chromatin promoting accumulation of the acetylated form of histone H3 in the utrophin promoter locus.ConclusionsThis novel artificial molecule may represent an improved platform for the development of future applications in DMD treatment.

Network Inference Analysis Identifies an APRR2-Like Gene Linked to Pigment Accumulation in Tomato and Pepper Fruits

Carotenoids represent some of the most important secondary metabolites in the human diet, and tomato (Solanum lycopersicum) is a rich source of these health-promoting compounds. In this work, a novel and fruit-related regulator of pigment accumulation in tomato has been identified by artificial neural network inference analysis and its function validated in transgenic plants. A tomato fruit gene regulatory network was generated using artificial neural network inference analysis and transcription factor gene expression profiles derived from fruits sampled at various points during development and ripening. One of the transcription factor gene expression profiles with a sequence related to an Arabidopsis (Arabidopsis thaliana) ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-LIKE gene (APRR2-Like) was up-regulated at the breaker stage in wild-type tomato fruits and, when overexpressed in transgenic lines, increased plastid number, area, and pigment content, enhancing the levels of chlorophyll in immature unripe fruits and carotenoids in red ripe fruits. Analysis of the transcriptome of transgenic lines overexpressing the tomato APPR2-Like gene revealed up-regulation of several ripening-related genes in the overexpression lines, providing a link between the expression of this tomato gene and the ripening process. A putative ortholog of the tomato APPR2-Like gene in sweet pepper (Capsicum annuum) was associated with pigment accumulation in fruit tissues. We conclude that the function of this gene is conserved across taxa and that it encodes a protein that has an important role in ripening.

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

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.

A Potential New Therapeutic Approach for Friedreich Ataxia: Induction of Frataxin Expression With TALE Proteins

TALEs targeting a promoter sequence and fused with a transcription activation domain (TAD) may be used to specifically induce the expression of a gene as a potential treatment for haploinsufficiency. This potential therapeutic approach was applied to increase the expression of frataxin in fibroblasts of Friedreich ataxia (FRDA) patients. FRDA fibroblast cells were nucleofected with a pCR3.1 expression vector coding for TALEFrat#8 fused with VP64. A twofold increase of the frataxin mRNA (detected by quantitative reverse transcription-PCR (qRT-PCR)) associated with a similar increase of the mature form of the frataxin protein was observed. The frataxin mRNA and protein were also increased by this TALE in the fibroblasts of the YG8R mouse model. The addition of 5-aza-2′-deoxycytidine (5-Aza-dC) or of valproic acid (VPA) to the TALE treatment did not produce significant improvement. Other TADs (i.e., p65, TFAP2α, SRF, SP1, and MyoD) fused with the TALEFrat#8 gene did not produce a significant increase in the frataxin protein. Thus the TALEFrat#8-VP64 recombinant protein targeting the frataxin promoter could eventually be used to increase the frataxin expression and alleviate the FRDA symptoms.

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

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

Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast

A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys2His2 zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.

Design of Artificial DNA Binding Proteins toward Control and Elucidation of Cellular Functions

An artificial transcription factor that can regulate the expression of specific genes at a desired time is very useful for research in chemical biology, cell biology, and future gene therapy. A C2H2 zinc finger motif, one of zinc-containing proteins, is known as the most ubiquitous DNA binding motif. The motif is attractive for designing artificial transcription factors with desired DNA binding specificities because of its characteristic DNA binding properties: (1) recognition of 3 bp per motif, (2) tandemly connected modular structure, and (3) binding to non-palindrome sequences as a monomer. Taking advantage of these properties, artificial DNA binding proteins with new DNA binding characteristics have been designed. By changing the linker region between two 3-zinc finger domains, artificial 6-zinc finger proteins were developed and shown to skip DNA sequences. Zinc-responsive transcription factors were created by altering one of the zinc ligands. An artificial zinc finger transcription factor targeting a core clock gene induced phase shifts of the cellular "circadian rhythm". Herein, I will summarize creation and function of the above-mentioned artificial zinc finger-type DNA binding proteins and transcription factors.

The p16-Specific Reactivation and Inhibition of Cell Migration Through Demethylation of CpG Islands by Engineered Transcription Factors

Methylation of CpG islands inactivates transcription of tumor suppressor genes including p16 (CDKN2A). Inhibitors of DNA methylation and histone deacylation are recognized as useful cancer therapeutic chemicals through reactivation of the expression of methylated genes. However, these inhibitors are not target gene-specific, so that they lead to serious side effects as regular cytotoxic chemotherapy agents. To explore the feasibility of methylated gene-specific reactivation by artificial transcription factors, we engineered a set of Sp1-like seven-finger zinc-finger proteins (7ZFPs) targeted to a 21-bp sequence of the p16 promoter and found that these 7ZFPs could bind specifically to the target p16 promoter probe. Then the p16-specific artificial transcription factors (p16ATFs) were made from these 7ZFPs and the transcription activator VP64. Results showed that transient transfection of some p16ATFs selectively up-regulated the endogenous p16 expression in the p16-active 293T cells. Moreover, the transient transfection of the representative p16ATF-6I specifically reactivated p16 expression in the p16-methylated H1299 and AGS cells pretreated with a nontoxic amount of 5'-aza-deoxycytidine (20 and 80 nM, respectively). In addition, stable transfection of the p16ATF induced demethylation of p16 CpG island and trimethylation of histone H3K4, and inhibited recruitment of DNA methyltransferase 1 and trimethylation of H3K9 and H3K27 in the p16 promoter in H1299 cells without 5'-aza-deoxycytidine pretreatment. Notably, inhibition of cell migration and invasion was observed in these p16-reactivated cells induced by transient and stable p16ATF transfection. These results demonstrate that p16ATF not only specifically reactivates p16 expression through demethylation of CpG islands, but also restores methylated p16 function.

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.

A Designed Zinc-finger Transcriptional Repressor of Phospholamban Improves Function of the Failing Heart

Selective inhibition of disease-related proteins underpins the majority of successful drug–target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.

Significant Rewiring of the Transcriptome and Proteome of an Escherichia coli Strain Harboring a Tailored Exogenous Global Regulator IrrE

Cell reprogramming for microorganisms via engineered or artificial transcription factors and RNA polymerase mutants has presented a powerful tool for eliciting complex traits that are practically useful particularly for industrial strains, and for understanding at the global level the regulatory network of gene transcription. We previously further showed that an exogenous global regulator IrrE (derived from the extreme radiation-resistant bacterium Deinococcus radiodurans) can be tailored to confer Escherichia coli (E. coli) with significantly enhanced tolerances to different stresses. In this work, based on comparative transcriptomic and proteomic analyses of the representative strains E1 and E0, harboring the ethanol-tolerant IrrE mutant E1 and the ethanol-intolerant wild type IrrE, respectively, we found that the transcriptome and proteome of E. coli were extensively rewired by the tailored IrrE protein. Overall, 1196 genes (or approximately 27% of E. coli genes) were significantly altered at the transcriptomic level, including notably genes in the nitrate-nitrite-nitric oxide (NO) pathway, and genes for non-coding RNAs. The proteomic profile revealed significant up- or downregulation of several proteins associated with syntheses of the cell membrane and cell wall. Analyses of the intracellular NO level and cell growth under reduced temperature supported a close correlation between NO and ethanol tolerance, and also suggests a role for membrane fluidity. The significantly different omic profiles of strain E1 indicate that IrrE functions as a global regulator in E. coli, and that IrrE may be evolved for other cellular tolerances. In this sense, it will provide synthetic biology with a practical and evolvable regulatory “part” that operates at a higher level of complexity than local regulators. This work also suggests a possibility of introducing and engineering other exogenous global regulators to rewire the genomes of microorganism cells.

Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer

The transcription factor (TF) SOX2 is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition to its normal stem cell function, SOX2 over-expression is associated with cancer development. The ability to selectively target this and other oncogenic TFs in cells, however, remains a significant challenge due to the ‘undruggable’ characteristics of these molecules. Here, we employ a zinc finger (ZF)-based artificial TF (ATF) approach to selectively suppress SOX2 gene expression in cancer cells. We engineered four different proteins each composed of 6ZF arrays designed to bind 18 bp sites in the SOX2 promoter and enhancer region, which controls SOX2 methylation. The 6ZF domains were linked to the Kruppel Associated Box (SKD) repressor domain. Three engineered proteins were able to bind their endogenous target sites and effectively suppress SOX2 expression (up to 95% repression efficiencies) in breast cancer cells. Targeted down-regulation of SOX2 expression resulted in decreased tumor cell proliferation and colony formation in these cells. Furthermore, induced expression of an ATF in a mouse model inhibited breast cancer cell growth. Collectively, these findings demonstrate the effectiveness and therapeutic potential of engineered ATFs to mediate potent and long-lasting down-regulation of oncogenic TF expression in cancer cells.

Abstract 3146: Awake the sleeping: Gene specific re-expression of epigenetically silenced candidate tumor suppressor genes in cervical cancer by artificial transcription factors

Abstract Recently, we reported diagnostic marker gene promoters that are hypermethylated in cervical cancer. As hypermethylation is often seen for tumor suppressor genes (TSGs), such marker genes might exhibit tumor suppressive activities upon re-expression. In this study, we aim to specifically upregulate candidate TSGs (C13ORF18, EPB41L3 and CCNA1) using self-designed Artificial Transcription Factors (ATFs). An advantage of this approach is that genes are re-expressed from their natural promoter allowing all splice variants to be expressed in natural ratios In cervical cancer cell lines, DNA methylation status (methylation-specific PCR, bisulfite sequencing) and gene (re)expression levels (qRT-PCR) were determined, also after treatment with a DNA demethylating agent (5-Aza-2′-Deoxycytidine (DAC)) and a histone deacetylase inhibitor (Trichostatin A (TSA)). Sequence specific DNA-binding domains were constructed by engineering zinc finger proteins and fused to a strong transcriptional activator. Functional effects of re-expression of the genes were studied using cell survival/apoptosis/cell growth assays. Histone marks were identified using chromatin IP, before and after treatment with ATFs. C13ORF18, EPB41L3 and CCNA1 silencing in cervical cancer was associated with DNA hypermethylation and these genes could be re-expressed in a dose dependent manner using DAC. Gene-specific re-activation of the genes could be achieved by the ATFs: Up to 300-fold in negative (methylated) cells and up to 80-fold in positive (unmethylated) cells. Co-treatment of ATFs with DAC or TSA further enhanced the effect of the ATFs. The hypermethylation status of the genes was associated with low levels of histone acetylation and repressive histone marks (H3K9me3, H3K27me3). Interestingly, preliminary data show that re-activation of the silenced genes increased the histone acetylation, while repressive histone marks decreased, indicating that ATF-induced re-expression was associated with changes in the epigenetic code. Furthermore, it was found that specific re-activation of the genes inhibited cell growth and induced apoptosis. These data confirm our hypothesis that hypermethylated cancer-specific genes can be re-activated, resulting in a strong anti-tumor effect. Furthermore, this is the first time that these genes are reported as TSGs in cervical cancer. The great advantage of gene-specific DNA targeting is that only the TSGs of interest are upregulated, while current genome-wide epigenetic drugs also target metastasis inducing genes, and can therefore enforce cancer growth. Currently, the DNA binding domains are fused to epigenetic effector domains to explore the ability of gene specific epigenetic editing. Financed by NWO-VIDI to MR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3146. doi:1538-7445.AM2012-3146

Combinatorial targeting of ribbon–helix–helix artificial transcription factors to chimeric recognition sites

Artificial transcription factors (ATFs) are potent synthetic biology tools for modulating endogenous gene expression and precision genome editing. The ribbon–helix–helix (RHH) superfamily of transcription factors are widespread in bacteria and archaea. The principal DNA binding determinant in this family comprises a two-stranded antiparallel β-sheet (ribbons) in which a pair of eight-residue motifs insert into the major groove. Here, we demonstrate that ribbons of divergent RHH proteins are compact and portable elements that can be grafted into a common α-helical scaffold producing active ATFs. Hybrid proteins cooperatively recognize DNA sites possessing core tetramer boxes whose functional spacing is dictated by interactions between the α-helical backbones. These interactions also promote combinatorial binding of chimeras with different transplanted ribbons, but identical backbones, to synthetic sites bearing cognate boxes for each protein either in vitro or in vivo. The composite assembly of interacting hybrid proteins offers potential advantages associated with combinatorial approaches to DNA recognition compared with ATFs that involve binding of a single protein. Moreover, the new class of RHH ATFs may be utilized to re-engineer transcriptional circuits, or may be enhanced with affinity tags, fluorescent moieties or other elements for targeted genome marking and manipulation in bacteria and archaea.

A Designer Artificial Transcription Factor Stably Reprograms Cancer Cells by Targeted DNA Methylation

The ability to stably alter gene expression states via epigenetic reprogramming hold significant implications for controlling breast cancer and other cancers. Herein, we describe a novel approach using artificial transcription factors (ATFs) to epigenetically target and stably down‐regulate genes that are aberrantly expressed in breast cancer. Our objective was to develop ATFs that contain zinc finger proteins coupled with DNMT3a to target and epigenetically repress Maspin (tumor suppressor) and SOX2 (oncogene). Upon ATF transduction in SUM159 cells that express Maspin at a moderate level, we significantly down‐regulate Maspin expression levels and repression was directly associated with ATF‐mediated DNA methylation adjacent to the ATF binding site. ATF‐transduced cells formed colonies in soft agar and selected clonal populations expanded from these colonies displayed persistent, heritable silencing of Maspin over many cell generations due to promoter methylation. Upon ATF transduction in MCF7 cells that overexpress SOX2, we observed a significant down‐regulation of SOX2 followed by cell arrest and death. These observations combine to strongly suggest that this technology will enable directed DNA methylation that can be utilized to modify the epigenetic status of any target gene in any cancer cell type. A.G.R. supported by ACS Postdoctoral Fellowship 116066‐PF‐08‐166‐01‐GMC.