Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system

THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2007; 9: 308–318.Published online 2 March 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1012

Generation and characterization of tTS-H4: a noveltranscriptional repressor that is compatible withthe reverse tetracycline-controlled TET-ON system

Ernesto Bockamp1*Cerstin Christel1

Dorothe Hameyer1

Andriy Khobta2

Marko Maringer1

Marco Reis1

Rosario Heck1

Nina Cabezas-Wallscheid1

Bernd Epe2

Barbara Oesch-Bartlomowicz1

Bernd Kaina1

Steffen Schmitt3

Leonid Eshkind1

1Institute of Toxicology/MouseGenetics, Johannes GutenbergUniversity, D-55131 Mainz, Germany2Institute of Pharmacy, JohannesGutenberg University, D-55099Mainz, Germany3FACS and Array Facility, JohannesGutenberg University, D-55131Mainz, Germany

*Correspondence to:Ernesto Bockamp, Institute ofToxicology/Mouse Genetics,Johannes Gutenberg University,Obere Zahlbacher Str. 67, D-55131Mainz, Germany. E-mail:[email protected]

Received: 18 December 2006Accepted: 11 January 2007

Abstract

Background Conditional gene regulatory systems ensuring tight andadjustable expression of therapeutic genes are central for developing futuregene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option.Moreover, the tightness of tetracycline-regulated gene switches can besubstantially improved by complementing transcriptional activators withantagonizing repressors.

Methods To develop novel tetracycline-responsive transcriptional repres-sors, we fused various transcriptional silencing domains to the TetR (B/E)DNA-binding and dimerization domain of the Tn10-encoded tetracyclineresistance operon (TetR (B/E)). The resulting fusion proteins were individ-ually tested for their ability to repress transcription of the constitutivelyactive hypoxanthine phosphoribosyltransferase (HPRT) promoter. In addi-tion, compatibility with the commonly used reverse tetracycline-controlledtransactivator system (rtTA-system) and responsiveness to the pharmaco-logical effector doxycycline (DOX) were evaluated. Finally, inducibility,effector-dependent promoter activity and the modification of histone H3and H4 of the active versus the repressed target promoter were determined.

Results Fusion of the human deacetylase 4 (HDAC4) carboxy-terminalsilencing domain to TetR (B/E) resulted in a functional transcriptionalrepressor. This novel repressor, termed tTS-H4, efficiently reduced theactivity of the murine HPRT promoter and a constitutively active humancytomegalovirus (hCMV) minimal promoter. Furthermore, combining tTS-H4with the rtTA transcriptional activator allowed for grading, turning off andresuming target gene expression over several orders of magnitude withoutbackground.

Conclusions The tTS-H4 repressor is compatible with the commonly usedrtTA transcriptional activation system and is a versatile new tool for tightlyand adjustably regulating conditional gene expression. Copyright 2007John Wiley & Sons, Ltd.

Keywords conditional gene repression; tetracycline system; gene therapy;doxycycline; transcription; histone modification

Introduction

One of the key issues in successfully translating experimental genetherapy strategies into the clinical setting is to tightly control

Copyright 2007 John Wiley & Sons, Ltd.

Characterization of the tTS-H4 repressor 309

the expression of transgenes. In order to reach adequatetherapeutic levels and to avoid undesired toxicity manygene therapeutic strategies will also require precisefine-tuning of expression levels. In this regard drug-inducible transcriptional regulators have been effectivelyused to control gene expression in tissue cultureexperiments and transgenic animals and might thereforealso be instrumental as genetic switches for futuregene therapeutic applications [1–5]. Among severalligand-responsive gene regulatory systems, tetracycline-controlled (tet-controlled) gene expression has emergedas a safe and reliable option with in vivo preclinicaltrials using tet-controlled transgene expression alreadyin progress [3]. Furthermore, it is to be envisaged thattransgenic and gene therapy strategies might benefitfrom conditional expression of small interfering RNAs(siRNAs) as a potential new class of drugs [6–8]. To thisend several reports using tet-controlled transcriptionalrepressor systems showed tight regulation of both RNApolymerase II and III (RNA pol II and III) driven smallhairpin (shRNAs) and microRNAs (miRNAs) [9–11].

Despite major improvements to gene regulatorysystems, several key issues can often not be properlyaddressed. For example, application of the tet-system canresult in good transgene expression and gene switching.However, initially, the tet-system system was troubled byleaky gene regulation in the non-induced state mainlydue to residual binding of the transactivator in theabsence of a pharmacological inducer [12]. Furthermore,dose-response experiments with the tet-system usingintravenous injection of a helper-dependent adenovirusdemonstrated that tight control over transgene expressionmight be lost at higher vector concentrations thus directlylimiting the amount of vector that can be used for delivery[13]. However, the unwanted leakiness of tet-regulatorysystems can be shielded by combining transcriptionalactivators with antagonizing repressors [14,15]. Inaddition, the immunogenicity of transcriptional regulatorsmight trigger the unwanted destruction of expressingcells thus preventing efficient long-term somatic genetransfer and treatment [16–18]. For all these reasonsthe development of novel and alternative transcriptionalactivators and repressors is important as it augmentsthe repertoire of basic experimental tools which mightbe useful for future gene therapeutic applications.Consequently, novel regulators should then be evaluatedalongside already established systems in terms of theirpotential for future gene therapeutic strategies and inparticular be tested for undesired cell functions andunwanted side effects [19].

Here we present tTS-H4, a novel tet-controlled tran-scriptional repressor, which is compatible with the reversetet-controlled transactivator (rtTA) system. This newrepressor was efficient in down-regulating the activity ofthe constitutively active mammalian hypoxanthine phos-phoribosyltransferase (HPRT) promoter and completelyrepressed an actively transcribing and commonly usedhuman cytomegalovirus (hCMV) minimal promoter. Fur-thermore, combination of tTS-H4 with the rtTA-system

facilitated tight transcriptional regulation over two ordersof magnitude. These findings indicate that the tTS-H4repressor is suitable for controlling target gene expressionin mammalian cells.

Materials and methods

Plasmid construction

DNA encoding the human HDAC4 was a gift from TonyKouzarides [20]. The pCMB tTS-H4-hygro expressionplasmid is based on the pCAG expression vector [21]and was generated by in-frame inserting the HDAC4coding region (amino acids 530–1084) into pCMB-TetR-hygro. The parental pCMB-TetR-hygro expression vectorwas constructed by inserting the TetR domain togetherwith the class B DNA-binding domain (amino acids 1–50)and the E dimerization domain (amino acids 51–211)of the Tn10-encoded tetracycline resistance operon fromEscherichia coli [22] into the pCMB1-hygro expressionvector. In-frame fusions of TetR to the repression domainsof MeCP2 [23], HDAC1 [24], HDAC3 [25], Sin3A [26],EED polycomb group protein [27] and the enzymaticde novo methyltransferase domain of Dnmt3 [28] werealso cloned into the pCMB TetR-hygro expression vector.The pCMV-tTS plasmid was a gift from Hermann Bujard[14]. To obtain the pCMV-tTS-H4 expression plasmidthe tTS-H4 coding region was inserted into pcDNA3.1 (Invitrogen). Details of all constructs are availableupon request. All fusion constructs were confirmed bynucleotide sequencing.

To generate the pHPRT-dEGFP/luc responder plasmid,the murine HPRT promoter sequence was amplified with5′-CGGGATCCCTCCTACCTCTGTAGTGCTGGG-3′ and 5′-CGGGATCCCTCCTACCTCTGTAGTGCTGGG-3′ as poly-merase chain reaction (PCR) primers using a murinecosmid library (Library 121, German Resource Center andPrimary Database, Berlin). The resulting PCR fragmentcontaining the murine HPRT promoter was 5′ insertedinto the modified pIRESneo (Clontech) containing thedestabilized d2EGFP coding region (from pd2EGFP-1,Clontech) followed by an intronic sequence, an intrari-bosomal entry site (IRES) and the firefly luciferase frompGL-2 basic (Promega).

Cell culture

NIH 3T3 mouse fibroblasts (ATCC CRL-1658), HRL9 HeLa[14] and tTS-H4 expressing clonal HRL9 cell lines werecultured using standard conditions in Dulbecco’s modifiedEagle’s medium supplemented with 5% fetal calf serum(Invitrogen). Transient transfections were performedusing Polyfect transfection reagent (Qiagen) accordingto the manufacturer’s instructions with 3.5 µg expressionplasmid, 0.5 µg pHPRT-dEGFP/luc responder plasmid and25 ng pEF-BOSS-renilla expression vector (provided byRalph Ross, Dermatology, Mainz University) in 60–70%

Copyright 2007 John Wiley & Sons, Ltd. J Gene Med 2007; 9: 308–318.DOI: 10.1002/jgm

310 E. Bockamp et al.

confluent 60 mm culture dishes. In all transient assayscells were harvested 24 h after transfection and assayedfor luciferase activity.

To generate triple transgenic HRL9-H4 HeLa cell lines(termed HRL9-H4), HRL9 cells were transformed with10 µg linearized pCMB tTS-H4 expression plasmid and1 µg linearized pPur vector (Clontech) using the calciumphosphate based ProFectin transfection system (Promega)according to the manufacturer’s instructions.

Luciferase reporter gene assays

To determine the luciferase activity of transformedcells a combined firefly/renilla luciferase reporter assaysystem was used (dual luciferase reporter assay system,Promega). The transfected cells growing in 60 mm disheswere harvested, washed once with phosphate-bufferedsaline (PBS) and subsequently lysed in 300 ml of lysisbuffer. A linear relationship between light units andvolume was confirmed in all experiments. In transientassays the relative luciferase light units (RLU) werenormalized for transfection efficiency against renilla lightunits in each sample. When stable cell lines were analyzed,RLU values were normalized against the amount ofa 10 µl protein aliquot in each sample. Mean valuesand standard deviations were calculated using the Excelsoftware program from Microsoft.

Chromatin immunoprecipitation (ChIP)and quantitative real-time (qrt)-PCRanalyses

Chromatin preparation and ChIP were essentially per-formed as previously described [29]. In brief, cells werefixed with 1% formaldehyde for 15 min at room tem-perature and DNA-chromatin complexes fractionated bysonication to an average DNA fragment size of 300–400bp. Samples were pre-cleared with non-immune rabbitserum (Rockland). Then equal amounts of pre-clearedchromatin were incubated with the anti-acetyl-histoneH3 (K 9,14), anti-acetyl-histone H4 (K5,8,12,16) antibod-ies (both Upstate), non-immune rabbit serum, or saved asnon-ChIPed input. In each reaction an excess of IgG (10 µgper ChIP sample) was used. Immunoprecipitated DNA (IP-DNA) was purified after reversal of the crosslinks (65 ◦C,overnight) and proteinase K treatment (0.6 mg/ml, 50 ◦C,3 h) using the Min-Elute reaction cleanup kit (Qiagen).

Analyzed genomic regions were the hCMV promoter ofthe luciferase transgene and the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene promoter. For ampli-fication of the hCMV promoter region oligonu-cleotides 5′-ACCCGGGTCGAGTAGGCGTGTA-3′ and 5′-GGCGTCTTCCATTTTACCAACAGT-3′ were used whichgive rise to a 234 bp amplification product. As GAPDH-specific primers 5′- TAGCTCAGGCCTCAAGACCTT-3′and 5′-AAGAAGATGCGGCTGACTGTC-3′ were employedwhich resulted in a 161 bp GAPDH-specific fragment. The

ChIP recoveries of the indicated DNA fragments were mea-sured by qrt-PCR with LightCycler 1.5 and FastStart DNAMaster SYBR Green I kit (both Roche Diagnostics). Reac-tions contained 3 mM MgCl2 and 350 nM of each primer.Fluorescence acquisition temperature during cycling was88 ◦C. For each measurement, at least four known dilu-tions of the relevant input DNA were run to generate astandard curve (linear regression coefficients were in allcases ≥ 0.97). DNA recovery in ChIP samples was mea-sured for every mode of cell treatment as percentageof correspondent input DNA. Recovery of the referenceGAPDH fragment was used as internal sample processingcontrol for hyperacetylated histones H3 and H4. Speci-ficity of the obtained PCR products was controlled bymelting curve analyses and agarose gel electrophoresis.

Results

Generation and functional analysis ofTetR transcriptional repressors

To develop novel tet-responsive transcriptional repres-sors, we generated fusions of known silencing domains tothe TetR (B/E) DNA-binding and dimerization domain ofthe Tn10-encoded tet-resistance operon from Escherichiacoli [22]. The resulting TetR (B/E) fusion constructswere individually tested for their potential to repressthe constitutively active HPRT promoter. To this endexpression vectors for each novel fusion were transientlyco-transfected with a reporter construct containing theHPRT promoter driving the expression of luciferase underthe control of a tetO DNA-binding site (see Figure 1 for aschematic representation of the experimental approach).We chose the HPRT gene promoter as a target for testingthe different candidate repressors since this promoter iswell characterized and constitutively active in mammaliancells [30,31]. As indicated on the right of Figure 2, onlyco-transfection with the TetR-HDAC4 construct encodingthe tTS-H4 repressor resulted in a reduction in luciferasereporter gene activity of about 50%. By contrast, co-transfection with all other TetR fusions, with a controlvector expressing the TetR domain or with the bluescriptplasmid, did not result in significant down-regulation ofluciferase activity, indicating that these constructs failedto induce repression of the HPRT promoter (Figure 2).In addition to luciferase, the HPRT promoter constructwas designed to also express a destabilized enhancedgreen fluorescent protein (EGFP) by virtue of a bicistronicmRNA. However, visual inspection of NIH 3T3 fibrob-lasts transiently transfected with the pHPRT-dEGFP/lucresponder plasmid did not reveal any EGFP fluorescence(data not shown). The cause for this lack of EGFP expres-sion was not further addressed and only luciferase activitywas used as readout for transcriptional HPRT promoteractivity.

Next, we wanted to test if the tTS-H4 repressor wouldallow for conditional doxycycline-dependent (DOX-dependent) transcriptional control of the HPRT promoter,



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Figure 1. Schematic representation of the strategy used for the experimental screening of novel TetR (B/E) repressors. CAGpromoter, cytomegalovirus immediate early enhancer combined with the chicken beta-actin promoter element; TetR, Tet (B/E)domain; pA, polyA signal; DOX, doxycycline; tetO, DNA-binding consensus for TetR (B/E) repressor homodimers; pHPRT,hypoxanthine phosphoribosyltransferase (HPRT) promoter

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Figure 2. Screening of different TetR fusions identifies the tTS-H4 repressor. Graphical representation showing the different TetRfusions. In each case TetR was fused in frame to the MeCP2, HDAC1, HDAC3, HDAC4, Sin3a, EED polycomb group protein repressiondomains and the enzymatic de novo methyltransferase domain of Dnmt3 and cloned into the pCMB expression vector. Numbersassociated to each repression domain indicate the amino acid positions of the parental protein fused to TetR. Numbers on the rightshow the mean values and the standard deviation for co-transfections using individual pCMB-TetR expression constructs togetherwith the tetracycline-dependent pHPRT-dEGFP/luc responder plasmid. Luciferase activities are expressed as RLU (relative lightunits) and were calculated in relation to the activity obtained by co-transfection of pHPRT-dEGFP/luc with the bluescript vectorwhich was defined to be 100%. Transfections were normalized to renilla light units. All experiments were performed in the absenceof DOX. Mean values and standard deviation were calculated in each case from ten independent experiments (n = 10)

as illustrated in Figure 3A. Comparison of luciferaseactivity between co-transfected 3T3 murine fibroblastcells maintained in the presence of 1000 ng/ml DOX(+DOX) with cells grown without DOX (−DOX) revealeda repression of about 50% only in cells cultured withoutDOX (left part of Figure 3B). To also compare thenovel tTS-H4 with the commonly used tTS tet-dependent

repressor [14], 3T3 fibroblasts were co-transfected withthe pCMV-tTS expression vector in parallel experiments.The results of these experiments are depicted on theright of Figure 3B and demonstrated that the classicaltTS silencer did repress the HPRT target promoter toabout 20% residual activity. Taken together the transienttransfection experiments established that the tTS-H4



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Figure 3. DOX-dependent repression of the HPRT promoter withthe tTS-H4 and tTS repressor. (A) Schematic representationshowing DOX-responsive recruitment of the tTS-H4 repressor(consisting of the TetR DNA-binding (TetR) domain and theHDAC4 repression domain (HDAC4)), to the tetO operatorsequence upstream of the HPRT promoter. In the presenceof DOX, the tTS-H4 repressor will not bind to the tetO DNAsequence and the HPRT promoter is actively transcribingthe luciferase reporter gene (above). In the absence of DOXthe tTS-H4 fusion is recruited to the tetO element resultingin transcriptional repression of the HPRT promoter (below).(B) Measured HPRT promoter repression with and withoutDOX. The 3T3 murine fibroblast line was co-transfected withthe pCMV-tTS-H4 or the pCMV-tTS expression vector, thepHPRT-dEGFP/luc luciferase reporter plasmid and the pEF-BOSSrenilla expression vector and grown for an additional 24 h inDOX-free medium (−DOX) or in the presence of 1000 ng/ml DOX(+DOX). A marked reduction in luciferase activity of about 50%was observed for the tTS-H4 repressor only when cells weregrown without DOX (left). Co-transfection with the classicalpCMV-tTS in the absence of DOX resulted in about 20% ofresidual luciferase activity (right). Relative light units (RLU)from ten different transfections (n = 10) were normalized withrenilla light emission and are represented on a percentage scale

fusion did repress the transcriptional activity of theHPRT target promoter to about 50% in a DOX-dependentfashion. Furthermore, the experiments conducted inparallel with the tTS repressor suggested that in transienttransfection assays and under the conditions used here,the classical tTS will be somewhat more effective.

TTS-H4 represses transcription of astably integrated hCMV minimalpromoter, is compatible with the rtTAtranscriptional activation system andallows for dose-dependent grading ofgene expression

Although the experiments reported above demonstratedthat the tTS-H4 repressor silenced transcription in tran-sient transfection experiments, we wanted to assesswhether tTS-H4 can efficiently repress a target promoterwhich is integrated into chromatin. This is an importantquestion as many applications, in particular those involv-ing transgenic animals and gene therapy approaches,will require the stable integration of transgenic con-structs into chromosomal DNA. Furthermore, we wantedto determine if tTS-H4 is compatible with the commonlyused tet-dependent rtTA transactivator thus facilitatingco-ordinated antagonistic transcriptional control over tar-get promoters. To clarify these issues, we made use ofthe HRL9 cell line. In previous experiments the HRL9 cellline had been specifically selected because it contained aconstitutively active hCMV minimal promoter expressingluciferase next to a tetO DNA-binding site [14]. In additionto the active hCMV promoter, the HRL9 cell line stablyexpresses the tet-dependent rtTA transcriptional activa-tor. Previous experiments with HRL9 cells established thatthe rtTA transactivator will significantly increase the tran-scriptional activity of the per se moderately active hCMVpromoter in the presence of high concentrations of DOX[14]. Furthermore, Freundlieb and colleagues showedthat at concentrations of 10 ng/ml DOX or below rtTAwill not or only to a very low extent bind to the tetO con-sensus and thus not substantially activate transcription ofthe hCMV promoter [14].

To test the effectiveness of tTS-H4 for repressing thehCMV promoter, the HRL9 cell line was stably transfectedwith the pCMB tTS-H4 expression vector and severalpuromycin-resistant clones were established and assayedfor down-regulation of the luciferase reporter gene. Theinitial analysis of different clonal cell lines revealeda broad distribution including clones with no, low,moderate and complete repression of luciferase activityprobably due to different levels of expressed repressorprotein. Of all tested stable clonal cell lines (n = 120) theHRL9-H4/102 and the HRL9-H4/42 lines exhibited thehighest degree of reporter gene repression and were usedin the following experiments.

On the left of Figure 4 the three theoretically possibleDOX-dependent promoter occupancies are shown. Toevaluate if hCMV promoter activity can be graded by



administering different DOX concentrations, the HRL9-H4/102 cell line was grown for 48 h without DOX or in thepresence of 10 ng/ml or 1000 ng/ml DOX. As expected,virtually no luciferase activity was measured in DOX-freemedium demonstrating the potential of tTS-H4 to activelyrepress the constitutive hCMV promoter in the context ofchromatin (Figure 4D). By contrast, cultivating this cellline in the presence of 10 ng/ml DOX resulted in modestluciferase activity (Figure 4E). To also determine whetherhigh concentrations of DOX would further increase hCMVpromoter activity, cells were maintained for 48 h in thepresence of 1000 ng/ml DOX. As shown in Figure 4F,under high DOX concentrations transcriptional activity ofthe target promoter was substantially increased by a factorof 102 as compared to the repressed state. The same tightregulation without background and equally high DOX-dependent induction was independently confirmed with

the second HRL9-H4/42 cell line (data not shown). Theresults of these experiments clearly demonstrated thatdose-dependent hCMV target promoter activation withvirtually no background can be achieved by combiningthe rtTA activator with the tTS-H4 repressor.

Kinetics of the tTS-H4/rtTA-regulatorysystem

To assess whether the known rapid induction kinetics ofthe rtTA transactivator were affected by the presence ofthe tTS-H4 silencer, previously repressed HRL9-H4/102cells were shifted to medium containing 1000 ng/mlDOX and luciferase activity was monitored during a20 h period. As seen in Figure 5A, after 5 h about 40%luciferase activity and after 10 h maximal luciferase

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Figure 4. TTS-H4 represses the hCMV promoter in HRL9-H4 cells and is compatible with the rtTA transcriptional regulatory system.Boxes on the left show schematic representations of the three possible promoter states (A–C). Diagrams on the right representthe measured luciferase activity at different concentrations of DOX (D–F). (A) Repressed promoter with tTS-H4 bound to the tetOoperator sequence. (B) Basal constitutive promoter activity. In this case neither the tTS-H4 repressor nor the rtTA activator isbound to the tetO DNA sequence. (C) RtTA-activated hCMV promoter element expressing high levels of luciferase. (D) No DOX:Repressed luciferase expression. (E) Low DOX (10 ng/ml): Moderate luciferase expression. (F) High DOX (1000 ng/ml): Highluciferase activity. For each DOX concentration the mean of ten independent experiments (n = 10) is shown. Luciferase activity wasnormalized against protein concentration and is represented on a logarithmic scale



Figure 5. Kinetics of luciferase induction and repression in HRL9-H4 cells. (A) Switch-on kinetics: Repressed HRL9-H4/102 cellswere plated in 60 mm dishes (time = 0 h) and cultivated in medium containing 1000 ng/ml DOX. Each point represents the meanand the standard deviation of normalized luciferase activity from at least five independently grown dishes (n = 5) which wereharvested at the indicated time. (B) Switch-off kinetics: Induced HRL9-H4/102 cells were plated in 60 mm dishes (time = 0 h) andcultivated in medium without DOX. Each point represents the mean and the standard deviation of normalized luciferase activityfrom at least five independently grown dishes (n = 5) which were harvested at the indicated time. Luciferase activity (RLU activity)was normalized against protein concentration and is represented on a percentage scale

expression levels were reached. Longer incubation timesof 15 and 20 h did not further increase luciferase values.These results demonstrated that the known inductionkinetics of the rtTA transactivator [14,32] were notaffected by the tTS-H4 repressor. To next address theturn-off kinetics of the system, luciferase activity wasdetermined during a period of 30 h after DOX withdrawal.The results of this experiment are shown in Figure 5B. Inthis case no decrease in luciferase activity was observedduring the first 10 h. However, after 15 h the measuredluciferase activity dropped to about 30% and reachedbackground levels after 30 h. This observed time coursedemonstrated that for abrogating luciferase reporter geneactivity a minimum of 30 h is required. Nevertheless,because shut-down kinetics will also depend on thespecific half-life time of the target, the exact time neededto completely turn off the gene function should bedetermined for each individual experimental setting.

TTS-H4-mediated transcriptionalrepression is reversible

For any conditional gene regulatory system it is essentialto know whether the executed molecular switch isreversible or will result in permanent epigenetic changes.To examine the effects of several consecutive cyclesof repression and induction, HRL9-H4/102 cells were

repeatedly shifted from medium containing 1000 ng/mlDOX to DOX-free medium. Figure 6A shows the resultof these experiments demonstrating that repetitive cyclesof maximal target gene activation followed by completereduction to background levels can be induced by virtueof DOX administration and withdrawal. These findingsalso established that repetitive switching of gene activitywith the tTS-H4/rtTA system does not affect the maximalrange of target gene expression.

To next determine if promoter activity can be restoredeven after prolonged tTS-H4 repression, we cultivatedthe tTS-H4-expressing HRL9-H4/102 cell line for 25 daysin DOX-free medium. Following this extended periodof promoter repression, different DOX concentrationswere applied. As seen in Figure 6B, incubation with1000 ng/ml DOX resulted in the re-establishment ofstrong transcriptional activity (100-fold over background,black bar on the right). These observed high luciferaseinduction levels were very similar to those obtainedprior to long-term repression (compare the black to thewhite bar in Figure 6B). To determine whether low DOXconcentrations were also sufficient for inducing hCMVpromoter activity, long-term repressed cells were exposedto 10 ng/ml DOX and luciferase activity was measured.At 10 ng/ml DOX the transcriptional activity of the hCMVpromoter was restored to moderate levels which wereagain comparable to those induction levels seen beforelong-term repression (Figure 6B, centre). This result


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Figure 6. TTS-H4-induced transcriptional repression of thehCMV promoter is completely reversible. (A) The tTS-H4-expressing HRL9-H4/102 clonal cell line was subjected toconsecutive cycles of hCMV minimal promoter repression (33 hin DOX free medium) and induction (15 h in medium containing1000 ng/ml DOX) and luciferase activity determined at theend of each cycle. Bars represent the mean and the standarddeviation of normalized luciferase activity from at least fiveindependently grown dishes (n = 5) which were harvested at theindicated time points. Luciferase activity was normalized againstprotein concentration and is represented on a percentage scale.(B) To long-term repress luciferase activity, HRL9-H4/102 cellswere maintained for 25 days in DOX-free medium. Subsequently,cells were cultivated for 24 h in DOX-free medium (no DOX)or either shifted to 10 ng/ml DOX or to 1000 ng/ml DOX.White columns represent luciferase induction measured beforeand black columns after long-term repression. For each DOXconcentration the mean and the standard deviation of fiveindependent experiments is shown (n = 5). Luciferase activitywas normalized against protein concentration and is representedon a logarithmic scale. Note that DOX-dependent induction levelswere similar before and after long-term repression

demonstrated that moderate transcriptional cues weresufficient to reconstitute low-level transgene expression.To also confirm that the repressed state was preservedduring the experiment, luciferase activity was measuredfor cells permanently maintained in DOX-free medium.As expected no activity was obtained from these cells(Figure 6B, left).

Taken together our results demonstrated that tTS-H4-mediated repression of the hCMV target promoter wascompletely relieved by DOX even after prolonged periodsof silencing. This finding is important as it shows that thertTA/tTS-H4 combination will allow for long periods of

repression followed by efficient re-activation to differentexpression levels.

TTS-H4 repression did induceacetylation changes of histones H3 andH4

For some chimeric transcriptional repressors gene silenc-ing has been reported to be linked to epigenetic changes ofthe target promoter [33,34]. To determine if and to whatextent modifications in histone acetylation are involved intTS-H4 repression, chromatin immunoprecipitation assays(ChIP assays) were performed. Using the clonal HRL9-H4/102 cell line, acetylation levels of histones H3 andH4 in the hCMV target promoter were determined. Inthe hyper-activated state (1000 ng/ml DOX) the levelsof acetylated histones H3 and H4 observed in the hCMVpromoter region were similarly high as those seen for theconstitutively active GAPDH promoter, which was usedas a reference fragment (compare hCMV- and GAPDH-specific ChIP recoveries in Figure 7, black bars). Thisfinding suggested a fairly accessible chromatin structurefor the hCMV promoter. Reduction of DOX concentrationto 10 ng/ml (moderately active hCMV promoter) resultedin a decrease of histone H3 and histone H4 acetyla-tion at the hCMV promoter, but did not modulate thesteady-state acetylation of the GAPDH control (Figure 7,hatched bars). Finally, after complete withdrawal of DOXfrom the medium (repressed hCMV promoter), we founda more than 3-fold decreased hCMV fragment recoverywith the antibody to acetyl-histone H3 and an about 2-fold decrease for the acetyl-histone H4 as compared tothe fully induced promoter (white bars in Figure 7). Asexpected, with the GAPDH promoter no substantial overallacetylation alteration was found at different DOX concen-trations (Figure 7, compare black, hatched and whitebars for GAPDH). Our ChIP experiments demonstratedthat tTS-H4 repression was accompanied by a quantita-tive decrease in acetylation of histones H3 and H4 at thehCMV target promoter. Since the tTS-H4 fusion proteincontains the canonical deacetylase domain of the humanHDAC4 protein, it is reasonable to assume that tTS-H4transcriptional silencing is, at least in part, mediated byspecific deacetylation of histones.

Discussion

Tetracycline (tet)-responsive gene regulatory systems arewidely used to regulate gene expression in tissue cultureexperiments and transgenic animals and might also berequired for tightly controlling and adequately adjustinggene function in future gene therapeutic settings [1–5].Here we describe a novel tet-dependent transcriptionalrepressor, termed tTS-H4, which is compatible with thertTA transactivation system [35]. We demonstrate thatantagonizing the tTS-H4 repressor with the rtTA activator



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Figure 7. TTS-H4-mediated gene repression of the hCMV promoter is associated with deacetylation of core histones.Differences in the acetylation of histones H3 (left) and H4 (right) between the DOX-responsive tetO-hCMV promoter and theglyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter were measured by real-time PCR after ChIP with appropriateantibodies and directly compared within the same cell preparation. Cells were cultivated for 24 h in 1000 ng/ml DOX (blackbars), in the presence of 10 ng/ml DOX (hatched bars), or in DOX-free medium (white bars). Recovery of the corresponding DNAfragments is expressed as percentage of input DNA. The mean of three independent experiments and the standard deviation isshown

will provide robust, completely reversible and also DOX-adjustable control of target genes.

As a first step to developing new tet-responsivetranscriptional repressors, we fused various knownsilencing domains to the TetR (B/E) DNA-bindingand dimerization domain and tested the resultingchimeric molecules for their ability to down-regulatethe constitutively active HPRT promoter (Figure 1).From all generated heterologous proteins only theTetR (B/E) fusion to the carboxy-terminal repressiondomain of the human HDAC4 protein effectivelyrepressed transcription of the HPRT promoter in transienttransfection assays (Figure 2). However, TetR fusionsto the repression domains of MeCP2, HDAC1, HDAC3,Sin3, EED polycomb group protein and the enzymaticde novo methyltransferase domain of Dnmt3 failed toreduce transcription of the HPRT target promoter. Theseresults indicated that for developing active tet-responsiverepressors it is not sufficient to simply fuse knownrepression domains to the Tet (B/E) moiety but thatvery specific functional and structural constraints haveto be met. The observation that chimeric fusions ofknown silencing elements to heterologous DNA-bindingdomains will not necessarily result in functional repressorsis also shared by a similar report from the literature[36]. However, in the case of the tTS-H4 fusion weobserved transcriptional repression of the HPRT targetpromoter in transient co-transfection assays. Furthermore,transcriptional repression of the HPRT promoter was onlyseen in the absence of the tetracycline analogue DOX,thus directly confirming the tetracycline responsivenessof the novel repressor (Figure 3B). In addition, analogoustransient co-transfection experiments with the commonlyused tTS tet-dependent repressor suggested that, under

the experimental conditions used, the classical tTS willbe somewhat more effective. However, the differencesbetween the classical tTS and novel tTA-H4 fusionobserved in the transient transfection assays do notnecessarily reflect the absolute silencing potential of eachrepressor. As a matter of fact the observed differencesbetween the two repressors might also be explainedby general or cell-type-specific transcription/translationefficiencies or the relative stability of the specific mRNAor recombinant fusion protein in 3T3 fibroblasts. For thisreason it was important to, in addition, determine thetTA-H4 silencing potential using stable clones. Indeed,when tTS-H4 was permanently expressed in HRL9 HeLacells, several clones reached 100% repression of thehCMV target promoter (shown in Figure 4D for the HRL9-H4/102 stable cell line). This result demonstrated that ifadequate levels of protein are provided, tTS-H4 will besuitable for completely extinguishing the transcription ofthe commonly used hCMV target promoter.

Next the tTS-H4 repressor was subjected to anadditional series of basic quality controls. To ensurehigh and graded response levels of therapeutic genesin vivo, it is of great advantage to combine tet-responsiveactivators with antagonistic repressors [14,37,38]. Forthis reason we tested the compatibility of tTS-H4 withthe previously established rtTA TET-ON system [12].Our experimental data provides direct evidence thatsimultaneous expression of tTS-H4 with the widely usedrtTA transactivator does not disturb proper regulationof a standard tetO-hCMV regulatory element. Indeed,we found that tTS-H4 completely repressed the basictranscriptional activity of the constitutively active hCMVpromoter and the tTS-H4 also did not interfere withhigh transcriptional rtTA-mediated transgene induction



(Figure 4). Moreover, quantitative analysis of luciferaseactivity at different levels of DOX (no DOX, low and highDOX) resulted in graded DOX-dependent transcriptionalactivity of the hCMV promoter (Figures 4D–4F). Thisresult directly implies that co-ordinate tTS-H4/rtTA-mediated regulation is possible and that transgeneexpression can be graded between complete repressionand high level induction. To our knowledge, tTS-H4 is thesecond tet-controllable prototypic repressor reported toallow the co-ordinated control of target genes over severalorders of magnitude without background. Furthermore,tTS-H4 expression did not affect the known inductionkinetics of the rtTA transactivator [14,32]: thus allowing aswitch to maximal luciferase expression within 10 h post-induction (Figure 5A) and to reverse gene expressionto background levels within 30 h (Figure 5B). The factthat the time interval needed for extinguishing luciferaseactivity is significantly longer than the time requiredto completely induce luciferase activity is in part to beexplained by the half-life time of the luciferase proteinwhich approximates about 3 h in mammalian cells [39].If precisely timed on/off kinetics are essential for theexperiment the induction and repression times should bedetermined for each setup.

For many transgenic and future gene therapeuticapplications it might be essential to completely extinguishand subsequently re-induce transgene expression atvarious selected time points. For this reason wedetermined whether the tTS-H4 repressor will allowfor repeated cycles of gene silencing with subsequentcomplete restoration of transgene expression. Usingthe stable HRL9-H4/102 cell line, we showed thatthe tTS-H4/rtTA system can be repetitively switchedwithout compromising the range of target gene regulation(Figure 6A). In addition, one possible problem of tet-responsive regulatory elements is that they might becomerefractory to transcriptional activation after long periodsof transcriptional silencing [40]. For this reason, weevaluated the effect of long-term tTS-H4 repression.The experiments conducted here clearly showed thatlong-term transcriptional repression of the standardDOX-responsive hCMV promoter element did not haveany adverse effects on the inducibility of the targetpromoter (Figure 6B). Furthermore, the observed DOX-dependent complete reversal of the long-term repressedhCMV promoter to moderate and high activity levelsprovides direct evidence that tTS-H4 acts as a flexibleand completely reversible gene switch. These resultssuggest that the novel tTS-H4 repressor will be acandidate for conditionally controlling and fine-tuninggene expression in transgenic mice and future genetherapeutic applications.

Although the classic tTS repressor [14] has been widelyused for gene switching in tissue culture and transgenicanimals, to our knowledge it is not known whethertTS-mediated gene silencing is linked to epigeneticmodifications of the target promoter. Here we havetested the tTS-H4 repressor for its ability to induce

post-transcriptional histone deacetylation. Since the tTS-H4 fusion contained the carboxy-terminal region of theHDAC4 protein (Figure 2, amino acids 530–1084) whichharbours a canonical histone deacetylase motif, it wasto be expected that tTS-H4 repression might involveepigenetic modification of the targeted promoter region.To assess whether tTS-H4 silencing is associated withdeacetylation, we conducted ChIP assays. The observeddifferences between the hyper-activated and the tTS-H4-repressed hCMV promoter in terms of histone H4 andH3 acetylation (Figure 7) directly suggest that tTS-H4transcriptional repression is, at least in part, linked toreversible epigenetic modification of core histones. Thispossible mode of action is also in line with the fact thathypoacetylation of histones will induce a less-permissivechromatin state associated with transcriptional repression(reviewed by Turner [41]).

In summary, our results demonstrate the compatibilityof the tTS-H4 repressor with the commonly used rtTAtransactivator system. Our studies show that antagonizingrtTA induction with tTS-H4 repression allows for highand graded levels of target gene expression withoutbackground activity. In addition, the combined tTS-H4/rtTA system was completely reversible and had fastinduction kinetics thus permitting the precise adjustmentof transgene expression levels during selected timewindows. Finally, the ChIP experiments reported herepoint towards a possible silencing mechanism involvingepigenetic histone deacetylation of the promoter region.Given its functional properties, the tTS-H4 repressorappears to be a potential candidate for future genetherapeutic strategies and represents an alternative tothe classical tTS system. However, before consideringany gene therapeutic applications with tet-responsiverepressors, more basic research is needed to evaluatethe specific advantages and drawbacks of each individualregulator. Important issues to be addressed will includeimmunogenicity, toxicity, tissue or target gene-specificregulatory differences, and the possible induction ofunwanted or dangerous side effects.

Acknowledgements

We would like to acknowledge Dr. T. Kouzarides for providingthe hHDAC4 and HDAC1 cDNA, Dr. F. Lizcano for the HDAC3cDNA, Dr. E. Li for the Dnmt3a cDNA, Dr. A. Otte for theEED cDNA, Dr. A. Bird for the MeCP2 cDNA, and Dr. D. Ayerfor the Sin3A cDNA. Our thanks go also to Hermann Bujardfor making available the HRL9 HeLa cell line and WolfgangHillen for sending the TetR-(B/E)-KRAB plasmid. This work wassupported by grants from the Deutsche Forschungsgemeinschaft(BO 1635/3-1), the European Union (QL67-CT-199-00335), andthe Mildred Scheel Foundation for Cancer Research (10-1982-Bo I).

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Documents

Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system