Murine Hepatic Cells Research Articles

P13-11 Hepatic metabolism of copaiba oil and resin oil-based formulation: analysis of the impact on cytotoxicity and cell cycle of human and murine hepatic cells with and without metabolic activation

P13-11 Hepatic metabolism of copaiba oil and resin oil-based formulation: analysis of the impact on cytotoxicity and cell cycle of human and murine hepatic cells with and without metabolic activation

Limited disassembly of cytoplasmic hepatitis B virus nucleocapsids restricts viral infection in murine hepatic cells.

Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells. Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pregenomic RNA (pgRNA), covalently closed circular DNA (cccDNA), and different relaxed circular DNA (rcDNA) intermediates were produced in vitro . The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine, hepatic cells. HBV replication-competent plasmid, cccDNA, and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo . In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein-linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine, but not in human, hepatic cells. Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.

Functional Comparison of Human and Murine Equilibrative Nucleobase Transporter 1

BackgroundThiopurines are a key component for the immunosuppressive therapy of various leukemias, inflammatory bowels disease, and other autoimmune disorders. Thiopurine therapy discontinuation/interruption is associated with a higher risk of relapse, but is common due to the prevalence of severe and potentially fatal adverse events, such as hepatotoxicity.Drug transporter expression is a known factor for patient variability in drug response and toxicity. We have recently established that the SLC43A3‐encoded transporter, equilibrative nucleobase transporter 1 (ENBT1), is the primary mechanism by which the thiopurine, 6‐mercaptopurine (6‐MP) enters cells. ENBT1 is known to be highly expressed in human hepatocytes, however, the relationship between ENBT1 and thiopurine‐induced hepatotoxicity has not been explored in the literature.To investigate this paradigm, our lab has proposed developing a novel SLC43A3 knockout mouse model. However, the functional differences between human and murine ENBT1, in mediating 6‐MP transport, has equally not been explored. Evidence that suggests ENBT1 is functionally similar between species, would be an essential foundation to assist bridging the gap between our proposed animal model and clinical studies within the literature.HypothesisWe hypothesize that hSLC43A3‐encoded hENBT1 and mslc43a3‐encoded mENBT1 are functionally similar and will have non‐significantly different 6‐MP transport kinetics and resulting cytotoxicity.MethodsThe ENBT1‐deficient human embryonic kidney 293 (HEK293) cell line was stably transfected with either hSLC43A3 or mslc43a3. [3H]Adenine and [3H]6‐MP were used in an oil‐stop centrifugation assay to assess ENBT1‐mediated transport activity in wildtype and transfected HEK293 cell lines. To determine cytotoxicity, wildtype and transfected HEK293 cell lines were incubated for 48 hours with a range of 6‐MP concentrations (78 nM – 1.28 mM) before being assessed via MTT cell viability assay.ResultsOil stop centrifugation assay of ENBT1‐mediated [3H]adenine and [3H]6‐MP transport revealed that hENBT1 and mENBT1 have similar transport kinetics (Km and Vmax), where resulting Michaelis‐Menten curves were not significantly different (unpaired T‐test: Adenine ‐ t16=0.32, p=0.76, n=5 & 6‐MP ‐ t16=0.64, p=0.53, n=4). Adenine inhibition of ENBT1‐mediated [3H]6‐MP transport and 6‐MP inhibition of ENBT1‐mediated [3H]adenine transport also showed that hENBT1 and mENBT1 have concurring inhibition kinetics (Ki), inhibition curves were not significantly different (unpaired T‐test: Adenine: t17=0.29, p=0.77, n=5 & 6‐MP: t14=0.18, p=0.86, n=4). Subsequent MTT cell viability assay showed that wildtype HEK293 are relatively 6‐MP resistant, while the transfected‐HEK293s are 6‐MP sensitive and have non‐significantly different cell viability curves (unpaired T‐test: t28=0.13, p=0.89, n=5).ConclusionOur results show that hENBT1 and mENBT1 are functionally similar in regards to ENBT1‐mediated adenine and 6‐MP transport in a stably transfected cell line. Additional work is necessary to investigate whether this is the case in human and murine hepatic cell lines/primary hepatocytes that have endogenous SLC43A3/ENBT1 expression and a larger range of xenobiotic metabolizing enzymes that may alter the equilibrium of 6‐MP within cells.

Antimicrobial activity and mechanisms of multiple antimicrobial peptides isolated from rockfish Sebastiscus marmoratus

Pathogenic disease is a major factor affecting the aquaculture of the rockfish Sebastiscus marmoratus, an important commercial species inhabiting the nearshore waters of the Western Pacific Ocean. Antimicrobial peptides (AMPs), as critical components of innate immunity, have been considered as promising antibiotic substitutes. The aims of this study were 1) to identify major AMPs in the rockfish, 2) to assess their antimicrobial activity and 3) to evaluate their potential therapeutic application. Six AMPs were identified, Hepcidin 1, liver-expressed antimicrobial peptide 2 (LEAP-2), Piscidin, Moronecidin, NK-lysin and β-defensin through analysis of the liver transcriptome of S. marmoratus. The transcriptional expression profiles of these AMPs were investigated by real-time quantitative PCR (RT-qPCR). These AMPs showed tissue-specific distribution patterns, and S. marmoratus displays a time-, dose- and tissue-dependent expression of AMPs in response to lipopolysaccharide (LPS) challenge. While the synthetic peptides of LEAP-2 and Moronecidin exerted broad-spectrum antimicrobial activity against important aquatic pathogens in vitro by directly disrupting microbial membrane, and no cytotoxicity against murine hepatic cells was observed at the effective concentrations from 5 μM to 40 μM. The existence of multiple AMPs and their distinct tissue distribution patterns and inducible expression patterns suggests a sophisticated, highly redundant, and multilevel network of antimicrobial defensive mechanisms of S. marmoratus. Therefore, S. marmoratus-derived AMPs appear to be potential therapeutic applications against pathogen infections in aquaculture.

Assessing the role of surface glycans of extracellular vesicles on cellular uptake

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. However, there is ambiguity around the fundamental mechanisms by which these effects are transduced, particularly in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Lectin microarray technology was employed to define the surface glycosylation patterns of EVs. Glycosidases PNGase F and neuraminidase which cleave N-glycans and terminal sialic acids, respectively, were used to analyze the relevance of these modifications to EV surface glycans on the uptake of fluorescently labelled EVs by a panel of cells representing a variety of tissues. Flow cytometry revealed an increase in affinity for EVs modified by both glycosidase treatments. High-content screening exhibited a broader range of responses with different cell types preferring different vesicle glycosylation states. We also found differences in vesicle charge after treatment with glycosidases. We conclude that glycans are key players in the tuning of EV uptake, through charge-based effects, direct glycan recognition or both, supporting glycoengineering as a toolkit for therapy development.

A Novel Fluorescence-Based Assay for the Measurement of Biliverdin Reductase Activity.

Biliverdin reductase (BVR) is the enzyme responsible for the last step in the production of bilirubin from the breakdown of heme. Bilirubin is one of the most potent antioxidant molecules in the body. Monitoring BVR activity is essential in studying the antioxidant capacity of cells and tissues. Traditional methods of determining BVR activity have relied on the measurement of bilirubin converted from biliverdin using absorbance spectroscopy. The approach has limited sensitivity and requires large quantities of cells or tissues. We have developed a novel fluorescence-based method utilizing the eel protein, UnaG, for the detection of bilirubin produced by BVR. The UnaG protein only fluoresces by the induction of bilirubin. We have also used this approach to measure intracellular bilirubin content of cultured cells. We validated this assay using cell lysates from mouse liver and immortalized murine hepatic cell line (Hepa1c1c7) and kidney cell line (MCT) in which BVR isoform A (BVRA) was either knocked out via CRISPR or stably overexpressed by lentivirus. Also, we tested the method using previously reported putative BVRA inhibitors, Closantel and Ebselen. These studies show a new method for measuring bilirubin intracellularly and in lysates.

Mimicking liver sinusoidal structures and functions using a 3D-configured microfluidic chip.

Physiologically, four major types of hepatic cells - the liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and hepatocytes - reside inside liver sinusoids and interact with flowing peripheral cells under blood flow. It is hard to mimic an in vivo liver sinusoid due to its complex multiple cell-cell interactions, spatiotemporal construction, and mechanical microenvironment. Here we developed an in vitro liver sinusoid chip by integrating the four types of primary murine hepatic cells into two adjacent fluid channels separated by a porous permeable membrane, replicating liver's key structures and configurations. Each type of cells was identified with its respective markers, and the assembled chip presented the liver-specific unique morphology of fenestration. The flow field in the liver chip was quantitatively analyzed by computational fluid dynamics simulations and particle tracking visualization tests. Intriguingly, co-culture and shear flow enhance albumin secretion independently or cooperatively, while shear flow alone enhances HGF production and CYP450 metabolism. Under lipopolysaccharide (LPS) stimulations, the hepatic cell co-culture facilitated neutrophil recruitment in the liver chip. Thus, this 3D-configured in vitro liver chip integrates the two key factors of shear flow and the four types of primary hepatic cells to replicate key structures, hepatic functions, and primary immune responses and provides a new in vitro model to investigate the short-duration hepatic cellular interactions under a microenvironment mimicking the physiology of a liver.

Abstract 461: Hepatic Nuclear Factor 4 Alpha as a Regulator of Alanine: Glyoxylate Aminotransferase 2 Expression and Systemic Levels of Endogenous Methylarginines

Introduction: Endogenous methylarginines have been proposed as markers and potentially mediators of cardiovascular diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of regulation of plasma levels of all three endogenous methylarginines. It has also been demonstrated that AGXT2 and its alternative substrate beta-aminoisobutyric acid (BAIB) can play an important modulatory role in lipid metabolism. Using bioinformatic analysis we identified a highly conserved putative binding site for the diabetes-associated transcription factor hepatic nuclear factor 4 alpha (HNF4A) in the mammalian AGXT2 promoter region. The aim of this study was to test the hypothesis that HNF4a is the major regulator of AGXT2 expression and activity. Methods and results: We introduced several point mutations in the putative HNF4A binding site and investigated their influence on activity of the murine Agxt2 promoter using luciferase reporter assay. The mutated constructs decreased the activity of the reporter gene by 75% as compared to the native promoter sequence. We showed direct binding of HNF4a to Agxt2 promoter using chromatin immunoprecipitation. We were able to demonstrate that siRNA-mediated knockdown of HNF4a leads to 50% reduction of Agxt2 expression in the murine hepatic cell line Hepa 1-6. In the in-vivo part of the project we showed that liver-specific Hnf4a knockout mice have a 90% reduction in liver Agxt2 mRNA levels, a 85% decrease in liver AGXT2 activity and significantly increased plasma levels of endogenous methylarginines and BAIB. Conclusions: In the current study we showed direct binding of HNF4a to the mammalian AGXT2 promoter region. We also demonstrated using in-vitro and in-vivo approaches that HNF4A is the major regulator of Agxt2 expression and has direct influence on systemic levels of endogenous methylarginines and BAIB. These findings suggest a novel link between NO-mediated impairment of vascular and renal function and lipid metabolism.

Cyanidin-3-O-β-glucoside and protocatechuic acid activate AMPK/mTOR/S6K pathway and improve glucose homeostasis in mice

AMP-activated protein kinase (AMPK) activation is an established treatment for diabetes. Here the effects of anthocyanin extract from blood orange juice, i.e. cyanidin-3-O-β-glucoside (C3G) and its secondary metabolite protocatechuic acid (PCA), on AMPK signalling in murine hepatic cell line and in the liver of C57BL/6 mice were investigated. Results showed for the first time that C3G and PCA activate AMPK and suppress its downstream kinase mTOR/S6K both in vitro and in vivo systems. Then, C3G and particularly PCA increased expression of glut 1 and glut 4 in the liver and improved glucose tolerance in normal and obese mice. Finally, the consumption of blood orange juice was observed to increase insulin sensitivity in mice. These findings indicate that C3G and PCA are natural AMPK activators and dietary consumption of food sources of C3G and PCA improves glucose homeostasis. Mainly, blood orange juice has beneficial effect for type 2 diabetes.

Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction.

Poly(I:C) treatment leads to interferon-dependent clearance of hepatitis B virus in a hydrodynamic injection mouse model.

We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro. Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo. The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-α/βR-, IFN-γ-, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-α/βR-, IFN-γ-, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients. It has become well accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus in an HBV mouse model. For the first time, we showed that HBV clearance in this model is dependent not only on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T-cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection.

Murine hepatic miRNAs expression and regulation of gene expression in diet-induced obese mice.

MicroRNAs are short, non-coding RNA molecules that regulate gene expression primarily by translational repression or by messenger RNA degradation. MicroRNAs play crucial roles in various biological processes. However, little is known regarding their role in obesity. We investigated differences of microRNA (miRNA) expression in liver tissue from diet-induced obese mice and potential effects of them on gene and protein expression. We used a miRNA microarray and quantitative RT-PCR to determine miRNA expression in murine liver tissue. Gene and protein expression were determined by qRT-PCR and Western blot analysis. Effects of miRNA by knock-down using RNAi or overexpression on putative target genes and/or proteins in a murine hepatic cell line were also investigated. MicroRNA array and qRT-PCR analsysis revealed that > 50 miRNAs were down- or upregulated more than 2-fold in the liver of diet-induced obese mice. While changes in expression of many genes were observed at the mRNA level, some were only altered at the protein level. Overexpression or knock-down of miR-107 in murine hepatic cells revealed that the expression of its putative target, fatty acid synthase, was dramatically decreased or increased, respectively. In conclusion, more than 50 hepatic miRNAs were dysregulated in diet-induced obese mice. Some of them regulate protein expression at translation level and others regulate mRNA expression at transcriptional level. MiR-107 is downregulated while FASN, a putative target of miR-107, was increased in diet-induced obese mice. These findings provide the evidence of the correlation of miRNAs and their targets in diet-induced obese mice.

Effects of S‐adenosylhomocysteine and homocysteine on DNA damage and cell cytotoxicity in murine hepatic and microglia cell lines

Limited research has been performed on S-adenosylhomocysteine (SAH) or homocysteine (Hcy)-evoked cell damage in hepatic and neuronal cells. In this study, we assessed effects of SAH or Hcy on cell cytotoxicity and DNA damage in hepatic and neuronal cells and attempted to find the underlying mechanism. Cell cytotoxicity and DNA damage were evaluated in murine hepatic cells (BNL CL.2 cell line) and microglia cells (BV-2 cell line) with SAH or Hcy treatment for 48 h. The influences of SAH or Hcy on lipid peroxidation and DNA methylation were also measured in both cell lines. SAH (5-20 microM) or Hcy (1-5 mM) dose dependently inhibited cell cytotoxicity and enhanced DNA damage in both types of cells. Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, whereas Hcy caused minimal effects on these two parameters at much higher concentrations. Hcy significantly induced lipid peroxidation, but not SAH. The present results show that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA hypomethylation, resulting in irreversible DNA damage and increased cell cytotoxicity. In addition, higher Hcy could induce cellular DNA damage through increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is a better marker of cell damage than Hcy in hepatic and microglia cells.

Urate Oxidase Knockdown Decreases Oxidative Stress in a Murine Hepatic Cell Line

Humans, birds, and some primates do not express the uric acid degrading enzyme urate oxidase (UOX) and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a health advantage to animals without UOX, knockout mice lacking UOX develop pathological complications including gout and kidney failure. As an alternative to the knockout model, RNA interference was used to decrease UOX expression using stable transfection in a mouse hepatic cell line (ATCC, FL83B). Urate oxidase mRNA was reduced 66% (p < 0.05) compared to wild type, as measured by real time RT-PCR. To determine if UOX knockdown resulted in enhanced protection against oxidative stress, cells were challenged with hexavalent chromium (Cr(VI)) or 3-morpholinosydnonimine hydrochloride (SIN-1). Compared to wild type, cells with UOX knockdown exhibited a 37.2 +/- 3.5% reduction (p < 0.05) in the electron spin resonance (ESR) signal after being exposed to Cr(VI) and displayed less DNA fragmentation (p < 0.05) following SIN-1 treatment. Cell viability decreased in wild type cells (p < 0.05), but not cells with UOX knockdown, after treatment with SIN-1. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress.

α-Aminoadipate δ-Semialdehyde Synthase mRNA Knockdown Reduces the Lysine Requirement of a Mouse Hepatic Cell Line

α-Aminoadipate δ-semialdehyde synthase (AASS) is the bifunctional enzyme containing the lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first 2 steps in the irreversible catabolism of lysine. A rare disease in humans, familial hyperlysinemia, can be caused by very low LKR activity and, as expected, reduces the lysine “requirement” of the individual. This concept was applied to a murine hepatic cell line (ATCC, FL83B) utilizing RNA interference (RNAi) to achieve AASS mRNA knockdown. Cells were antibiotic selected for stable transfection of 2 plasmids that express different short hairpin RNA sequences for AASS knockdown. Compared with the wild-type cell line, AASS mRNA abundance was reduced 79.0 ± 6.4% (P < 0.05), resulting in a 29.8 ± 5.2% (P < 0.05) reduction in AASS protein abundance, 41.3 ± 10.0% (P < 0.05) less LKR activity, and a reduction in lysine oxidation by 50.7 ± 11.8%. To determine the effect of AASS knockdown on the lysine requirement, cells were grown in media containing 12.5, 25.0, 50.0, 100, or 200 μmol/L lysine. Using a segmented model approach for growth rate analysis, the lysine requirement of the cell line with AASS silencing was 43.4 ± 1.7 μmol/L, ∼26% lower (P < 0.05), than the lysine requirement of the wild-type cell line. These results indicate AASS knockdown decreases the lysine requirement of the cell via a reduction of lysine catabolism through the saccharopine pathway, providing the initial proof in principle that RNAi can be used to reduce the nutrient requirement of a system.

α‐Aminoadipate δ‐semialdehyde synthase mRNA knockdown reduces the lysine requirement of a murine hepatic cell line

α-Aminoadipate δ-semialdehyde synthase (AASS) is the bifunctional enzyme containing the lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first tw...

Searching for DNA-protein interactions by lambda phage display.

We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.