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Eur. J. Biochem. 270, 3109–3121 (2003) FEBS 2003
Gene regulation by tetracyclinesConstraints of resistance regulation in bacteria shape TetR for applicationin eukaryotes
Christian Berens and Wolfgang Hillen
Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universita¨t Erlangen-Nu¨rnberg;Germany
The Tet repressor protein (TetR) regulates transcription
are fused to TetR to turn it into an efficient regulator.
of a family of tetracycline (tc) resistance determinants in
Mechanistic understanding and the ability to engineer and
Gram-negative bacteria. The resistance protein TetA, a
screen for mutants with specific properties allow tailoring
membrane-spanning H+-[tcÆM]+ antiporter, must be sen-
of the DNA recognition specificity, the response to inducer
sitively regulated because its expression is harmful in the
tc and the dimerization specificity of TetR-based eukary-
absence of tc, yet it has to be expressed before the drugs'
otic regulators. This review provides an overview of the
concentration reaches cytoplasmic levels inhibitory for
TetR properties as they evolved in bacteria, the functional
protein synthesis. Consequently, TetR shows highly speci-
modifications necessary to transform it into a convenient,
fic tetO binding to reduce basal expression and high affinity
specific and efficient regulator for use in eukaryotes and
to tc to ensure sensitive induction. Tc can cross biological
how the interplay between structure ) function studies in
membranes by diffusion enabling this inducer to penetrate
bacteria and specific requirements of particular applica-
the majority of cells. These regulatory and pharmacological
tions in eukaryotes have made it a versatile and highly
properties are the basis for application of TetR to selec-
adaptable regulatory system.
tively control the expression of single genes in lower and
Keywords: antibiotic resistance; disease models; fusion pro-
higher eukaryotes. TetR can be used for that purpose in
tein; inducible gene expression; ligand-binding specificity;
some organisms without further modifications. In mam-
mammalian cell lines; protein engineering; structure–activity
mals and in a large variety of other organisms, however,
relationship; Tet repressor; transgenic organism.
eukaryotic transcriptional activator or repressor domains
Properties of bacterial Tet systems
of transcription by the tc-responsive Tet repressor (TetR).
In the absence of inducer, TetR dimers bind to the operators
Efflux-mediated tetracycline resistance is always
tetO1 and tetO2, shutting down transcription of its own
regulated in Gram-negative bacteria
gene, tetR, and of the resistance gene, tetA. Once tc hasentered the cell, it binds TetR with high affinity as a
In Gram-negative bacteria, resistance to tetracyclines (tc)
[tcÆMg]+ complex [7]. This induces a conformational change
is mediated by the TetA protein, a proton-[tcÆMg]+ anti-
in TetR [8] resulting in dissociation from tetO [9]. The
porter embedded in the cytoplasmic membrane [1,2]. Eleven
following expression burst of TetA and TetR leads to a
tc resistance determinants (Tet classes A–E, G, H, J, Z, 30,
rapid reduction of the cytoplasmic tc concentration [10]
and 33 [3–5]) share the organization of structural and
which, in turn, shuts expression of both genes off again.
regulatory genes (reviewed in [6]). In enteric bacteria, the
Expression of TetA is fine-tuned in the presence of tc so that
efflux-encoding tetA genes are strictly regulated at the level
export overcomes the slow uptake (compare below).
Regulation of Tc resistance is optimized for tightness
Correspondence to W. Hillen, Lehrstuhl fu¨r Mikrobiologie, Institut
fu¨r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander
Regulation of tet determinants is subject to strong, opposing
Universita¨t, Staudtstr. 5, D-91058 Erlangen, Germany.
selective pressures. Expression of the resistance protein
Fax: +49 9131 8528082, Tel.: +49 9131 8528081,E-mail:
[email protected]
TetA is detrimental to the cell [11,12]. Overexpression of this
Abbreviations: tc, tetracycline; dox, doxycycline; atc, anhydrotetra-
integral membrane protein is lethal for Escherichia coli [13],
cycline; tTA, tetracycline-dependent transactivator; rtTA, reverse
probably due to the collapse of the membrane potential [14].
tetracycline-dependent transactivator; tTS, tetracycline-dependent
Consequently, expression of TetA must be tightly repressed
trans-silencer; CMV, cytomegalovirus; GFP, green fluorescent
in the absence of the drug. However, when tc diffuses into
the cell the resistance protein must be expressed before the
(Received 8 April 2003, revised 14 May 2003,
cytoplasmic concentration of tc reaches the micromolar
accepted 15 May 2003)
level necessary to inhibit translation. This requires: (a) high
3110 C. Berens and W. Hillen (Eur. J. Biochem. 270)
competing nonspecific DNA to a much higher degree thanbacteria. Taken together, the evolutionary pressures ontc-dependent gene regulation have led to tight repression inthe absence of tc, without compromising sensitivity ofinduction, so that regulated tc resistance determinantsimpose no burden on the fitness of E . coli in the absence ofthe antibiotic, but still mediate high levels of resistance to tcin its presence [12].
The structural change of TetR associated with inductionby tetracycline is known
X-ray crystal structures of free TetR [17], TetR complexedwith different tetracyclines [18–21] and with tetO [8] havebeen determined at resolutions of 1.9–2.5 A˚, revealingthe allosteric conformational change leading to induction.
These results have been reviewed in detail [22] and have beencompared to Lac repressor [23]. Thus, they are onlysummarized here (Fig. 2). The DNA reading head of TetR(magenta) is connected to the protein core (blue) by the helixa4 (green). Binding of [tcÆMg]+ (yellow) to TetR unwindsthe C-terminal residues of helix a6 (light blue), which bumpinto a4 and displace it. As the C terminus of a4 is held inplace by contacts to tc, the displacement leads to apendulum-like swing of the a4 N terminus increasing thedistance between the recognition helices by 3 A˚, so that theydo not fit into successive major grooves of DNA anymore[24]. These conformational changes are consistent withmany noninducible TetR mutants [24,25], spectroscopicanalysis of TetR in vitro [26], in vivo [27] and in vitro [28]disulfide trapping experiments. Furthermore, a movementof a9 closes the tc binding pocket after the drug has entered[17], and the loop between a8 and a9 is also important for
Fig. 1. Structures of tetracyclines used in eukaryotic gene regulation.
(A) Structure of tetracycline with the pKa values of the three titratablegroups. (B) Structure of doxycycline. (C) Structure of anhydrotetra-cycline.
Tetracycline penetrates cells by diffusion
Tetracyclines (Fig. 1) diffuse in their uncharged forms
affinity of TetR for both tetO and tc to keep the basal
through lipid bilayers without the aid of protein channels
expression level of tetA low and to ensure that its
[32–36]. Measuring the increase in fluorescence intensity of
transcription is initiated at concentrations which are still
tc observed upon binding to TetR [7] allows us to determine
subinhibitory for translation; (b) low affinity of the TetR–
the cytoplasmic concentration of tc and, thus, to calculate
[tcÆMg]+ complex for DNA; and (c) high-level, but short-
permeation coefficients for tc uptake into liposomes
term expression of TetA to initially reduce the internal
[(2.4 ± 0.6) · 10)9 cmÆs)1]
concentration of tc. A low level of TetR is important for
[(5.6 ± 1.9) · 10)9 cmÆs)1] [36]. These translate into half-
sensitive induction, since E . coli strains expressing high
equilibration times of 35 ± 15 min for tc to cross the
levels of TetR need high concentrations of tc for full
membranes and are in good agreement with the half-
induction [15]. These conflicting requirements are met by
equilibration time of 15 min measured for [3H]tc-uptake in
the genetic organization of the resistance determinants
Bacillus subtilis [37], and the slow uptake of tc observed in
(reviewed in [6]) and by the ligand binding properties of
Staphylococcus aureus [38]. Tetracycline diffusion through
TetR. High sensitivity towards tetracyclines [see Fig. 1 for
phospholipid membranes is, thus, slow and appears to be
the structures of tc, doxycycline (dox) and anhydro-tc (atc)]
the rate-limiting step of uptake into cells [36]. The previously
is achieved by the remarkable binding constant of TetR for
observed rapid uptake of tc [33,39] might rather reflect
), [doxÆMg]+ (Ka 1010 M ) or
unspecific adsorption of tc to membrane surfaces [32,36]. A
) [7,9], about 103)105-fold higher
detailed model explaining the transport and accumulation
than the affinity of the drugs to their intracellular target, the
of tc across the Gram-negative cell envelope has been
ribosome [16]. Binding of two molecules of [tcÆMg]+ to a
presented by Nikaido and coworkers ([40,41] and references
TetR dimer diminishes repressor affinity for tetO by about
cited therein). In the medium, as well as in the periplasm and
nine orders of magnitude to the unusually low background
cytoplasm, tc is present in one uncharged and several
DNA binding level of less than 105
[9]. This high ratio
charged or zwitterionic species, due to its three titratable
of specific over nonspecific DNA binding enables TetR to
groups (Fig. 1). The distribution between these species
bind tetO efficiently, even in larger genomes containing
depends on the pH of the respective compartment [40]. The
Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3111
Fig. 2. Structure of the TetR–[tcÆMg]+complex. Tet repressor is shown as a ribbondiagram with one monomer in gray and theother monomer color-coded as follows: TheDNA-binding region is in magenta, the helixconnecting it with the protein core is in green.
The protein core is dark blue, with the helix a6in light blue. Tetracycline is displayed asspace-filling CPK model in yellow. For clarity,the helices a1–a10 of one monomer are num-bered and the N and C termini of both sub-units are indicated. The coordinates weretaken from the PDB entry 2TRT [18].
uncharged form of tc can penetrate the outer membrane
repression by unmodified TetR [53]. Here, TetR most
directly. But the major fraction of tc equilibrates as a
likely acts by interfering sterically with binding of RNA
[tcÆM]+-complex rapidly through the outer membrane via
polymerase or auxiliary transcription factors [42,54]. To
porins, with the Donnan potential across the outer mem-
achieve this, one or more tetO elements are placed in
brane leading to a two- to threefold accumulation of this
proximity of either the TATA box or the transcriptional
charged complex in the periplasm. Tc then diffuses passively
start site of the respective target gene and TetR is expressed
in its uncharged form through the cytoplasmic membrane.
concomitantly by a strong, constitutive promoter. Promot-
Due to the pH gradient across the cytoplasmic membrane,
ers of all three eukaryotic RNA polymerases have been
a larger fraction of the uncharged tc dissociates in the
targeted in the manner described. Unfortunately, as will
cytoplasm than in the periplasm. Since equilibrium is
become evident in the following paragraph, the published,
reached when the concentration of uncharged tc is identical
successful approaches do not yet allow formation of a
in both compartments, this results in a higher intracellular
simple strategy for establishing a TetR-repressed system,
concentration of [tcÆM]+, the biologically active compound.
although they clearly point out that the positioning of the
Again, accumulation of tc is the product of this passive
tetO boxes is crucial for efficient regulation.
equilibration across the inner membrane [40,41].
In Leishmania donovani, an RNA polymerase I promo-
ter was brought under tc-control by placing a single tetO site
Tc-based gene regulation functions in
2–24 bp upstream of the transcriptional start site [55],
different setups in many eukaryotic systems
whereas in Trypanosoma brucei at least one tetO elementhad to be inserted at a position +2 or )2 relativ e to the
The evolved properties of TetR described above combined
transcription start site of an RNA polymerase I-like
with the favorable pharmacokinetics of tetracyclines and
promoter [56]. For RNA polymerase III-mediated tran-
their long record of safe use in clinical practice make the Tet
scription of suppressor tRNA genes, induction factors
system a good candidate to fulfill the criteria that are required
between two- to fivefold were observed in Saccharomyces
for an ideal transcriptional regulator in eukaryotic cells as
cerevisiae, Dictyostelium discoideum and carrot protoplasts
given by Saez and others [42,43]. Consequently, the past
when tetO was introduced within 10 bp upstream of the
15 years have seen the broad application of tc-dependent
transcriptional start site [57–59]. A regulated version of the
regulatory systems, mainly in mammalian cell culture, but to
human U6 snRNA promoter, also transcribed by RNA
an increasing degree in transgenic organisms like plants,
polymerase III, was developed by replacing sequences
yeasts, protozoan parasites, slime molds, flies, and rodents.
between the proximal sequence element and the transcrip-
These topics have been extensively reviewed [42–52]. The
tional start site with tetO [60]. Flanking the TATA-box with
following section presents an overview of the basic Tet
two operators completely abolished transcriptional activity.
systems used to regulate gene expression in eukaryotes.
In contrast, introduction of a single tetO element affectedtranscription only slightly, but led to up to 25-foldrepression in the presence of TetR. A regulated U6 snRNA
Gene regulation by TetR in eukaryotes
promoter with a defined expression window [61,62] would
The most basic and first published application of
be a very powerful tool as this promoter is used to express
tc-mediated gene regulation in eukaryotes is transcriptional
the small interfering RNA [63] needed for silencing gene
3112 C. Berens and W. Hillen (Eur. J. Biochem. 270)
expression by RNA interference [64]. Repression of RNA
magnitude can be reached with sensitive reporter genes like
polymerase II promoters exerted by TetR is strongest in
firefly luciferase [75]. Luciferase activity is expressed within
plants [65,66], mammalian cells [67] and fungi like Schizo-
4 h of removal of tc and about 20% of the steady-state level
saccharomyces pombe [68,69] when multiple tet operators
is reached after 12 h. While the use of a strong, constitutive
are positioned within a region from 5 bp upstream to 35 bp
promoter (CMV IE, EF-1a, Ubiquitin C) is common in cell
downstream of the TATA-element. In contrast, placement
culture applications, the use of tissue-specific promoters in
of one to four tet operators immediately downstream of the
transgenic animals provides spatial control to the Tet
transcription initiation site has been shown to be most
system, restricting expression of the Tet transregulator and,
effective in the parasitic protozoa Entamoeba histolytica
subsequently, the transgene to the desired tissue [84,85]. In
[70,71], Toxoplasma gondii [72] and Giardia lamblia [73].
Drosophila, usage of the Gal4-UAS system to control Tettransregulator expression allows the generation of spatiallydelimited expression patterns by simple crossing with one of
Gene regulation by TetR-based transregulators
the many Gal4 driver lines available in the Drosophila
While unmodified TetR acts as a transcriptional repressor in
research community [86].
plants and lower eukaryotes, it can be, but not always is
One concern has been the expression levels of Tet
efficient in mammalian cells [67,74]. A consistently func-
transregulators as influenced by a potentially low mRNA
tional version for yeasts, flies and mammalian cell lines is
stability or efficiency of translation. This was recently
TetR fused to an eukaryotic regulatory domain, such as an
addressed by generating a synthetic coding sequence for
acidic activation domain (Fig. 3A; tTA or Tet-Off) [75–80]
tetR. Potential splice donor and acceptor sites identified by
or a repression domain (Fig. 3B; tTS) [81–83]. The trans-
sequence analysis, several potential endonuclease cleavage
activator tTA directs expression from a tc-dependent
sites, and potential stable hairpin structures in the mRNA
promoter that contains seven repeats of a tetO2 element
were eliminated and human codon usage was used [87–90].
from the transposon Tn10. The palindromic centers of two
The consequence of this optimization protocol is a higher
adjacent operators are separated by 41 bp. This element is
protein level in Drosophila, HeLa and HEK293 cells.
fused to a minimal promoter, typically derived from the
Another concern voiced was that the CMV-derived
human cytomegalovirus (CMV) immediate early promoter
minimal promoter was not transcriptionally silent under all
[75]. When both components are stably integrated into
experimental conditions [91–93]. This promoter leakiness
proper chromosomal loci of mammalian cell lines, tran-
can be caused by promoter-dependent or integration site-
scription from the hybrid promoter is silent in the presence
dependent effects and has been discussed in detail [94].
of more than 10 ngÆmL)1 dox. Removal of dox leads to
Promoter-dependent leakiness has been addressed by the
binding of tTA to tetO and subsequent activation of
use of alternative minimal promoters [75,95,96]. In transient
transcription. Regulatory factors of up to five orders of
transfection experiments, these show lower basal activities
Fig. 3. Regulation of gene expression by Tet transregulators. The promoter proximal tetO boxes are represented by black boxes. The transregulatorsare shown as follows: the DNA reading heads are in light gray, the inducer-binding and dimerization domain is in dark gray, activation domains areblack boxes, and the silencing domain is stippled. The conformational change leading to the loss of DNA-binding activity is pictured as a light graybox. High-level activated transcription is displayed by a bold arrow, low-level basal transcription by a dotted arrow. (A) tTA. (B) tTS. (C) rtTA.
Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3113
than Ptet-1, but also do not reach its maximal activation
tion can be used to find solutions to some of the problems
level. Thus, the regulatory window for target gene expres-
and limitations that arise for Tet system applications in
sion is shifted and expanded due to the stronger reduction of
the basal activity. Integration site-dependent leakiness hasbeen attributed to enhancers located close to the integration
Alterations of the activator domain of tTA
site of the target gene construct. Besides screening additionalclones until one harboring the desired properties is found,
Especially for gene therapy, concern about a viral protein is
the problem has been approached by insulating Ptet-1 from
often voiced, as humoral as well as cellular immune
external activating signals through insertion of a chicken
response against the VP16 protein has been found in herpes
lysozyme matrix attachment region just upstream of Ptet-1
simplex infected humans [106–108]. Thus, immune res-
[87] or by flanking the target gene expression unit with either
ponses against transactivators containing the VP16 domain
chicken b-globin insulators [90] or SCS and SCS' boundary
cannot be rigorously excluded, although they have not been
elements from Drosophila [86].
observed so far in a mouse model using reverse tTA (rtTA;
A different strategy was adopted by engineering a
Tet-On) [109]. Two solutions circumventing this concern
tc-controlled trans-silencer protein [81]. Fusion of the
have been developed: (a) the VP16 domain has been
KRAB domain of Kox1 [97] to TetR yielded a hybrid
replaced by three repeats of a minimal activation domain
protein called tTS, that not only substantially repressed
derived from a 12-amino acid activating patch of the VP16
basal transcription from Ptet-1 even if the tet operators were
protein (tTA2 [76]); and (b) a variety of human activator
located 3 kbp distant from the minimal promoter, but also
domains from the acidic, glutamine-rich, serine/threonine-
efficiently down-regulated gene expression from a CMV
rich and proline-rich functional groups were tested for their
enhancer-driven Ptet-1 [83]. This strategy therefore appears
ability to replace the VP16 domain. When fused to TetR,
to be more versatile in coping with unwanted target gene
only acidic activation domains were highly active [78–80].
expression than the promoter adaptation proposed above.
Minor activation was observed with the serine/threonine-
In addition and in contrast to tTA, the tc-dependent
rich domains from the transcription factors ITF-1, ITF-2,
silencing of complex promoters offers the unique possibility
and MTF-1. Transactivators with activation potentials
of reversibly down-regulating the expression of cellular
spanning more than three orders of magnitude have been
genes on top of their normal regulation. The KRAB
generated by combination of various minimal activation
domain is inactive in S. cerevisiae and Drosophila where it
domains (see above; [76]). They are attractive for combined
was replaced with repression domains from the proteins
knock-in/knock-out strategies to convey tissue-specific
SSN6 [82], knirps, giant or dCtBP [83].
expression of the transactivator, while at the same time
The expression of transfected genes can be rapidly
inactivating expression of the genomic copy of the target
repressed in mammalian cells by epigenetic mechanisms
gene. Expression of the regulatory protein is then an
[98]. Although this transgene silencing is not specific for
invariant function of the genomic locus and, if too high, can
the Tet system, it is often observed for genes under tc
lead to squelching [110]. This can be addressed by
control due to its frequent usage as conditional expression
employing a transactivator with reduced activation poten-
system. Approaches to achieve stable gene expression have
tial as these are tolerated in the cell at higher concentrations
been to: (a) screen many transfected clones; (b) the use of
lentiviral vectors [99]; (c) replace the viral promoters thatdirect expression of the transregulators with promoters of
Conversion of TetR to reverse TetR
human origin [100]; (d) use chromatin insulator sequencesto protect transgene expression [98]; or (e) couple transgene
Eukaryotic gene regulation by tTA shows a high dynamic
expression to a selectable marker via an IRES element
range and works consistently well, but has several practical
[101] or by fusion of the transregulator with green
drawbacks. Tc has to be continually present to keep
fluorescent protein (GFP) [102]. Note that in this fusion
expression of the gene of interest downregulated. Although
protein GFP is connected to the DNA-binding domain of
tc is not toxic at the levels utilized in gene regulation,
TetR which can interfere with nonspecific DNA-binding
prolonged exposure to the antibiotic is not always desirable
activity of TetR at low levels of dox (see Fig. 2A in [102]
in transgenic animals nor is it possible in gene therapy.
and [78]). The few published examples make it impossible
Furthermore, induction of target genes is mostly slow as it
to recommend one of the strategies for use in establishing
requires removal of the drug from the culture or organism.
homogenous expression of transgenes, but silencing of
To be able to control the time point of induction more
transregulator expression is not completely suppressed by
precisely, and since organisms are more easily saturated
the use of lentiviral vectors [103] or insulator sequences
with an effector than depleted of it [111,112], reverse TetR
variants which bind tetO only in the presence of tc weresearched for and found (Fig. 3C). Screening in E . coli [113]
Modifications of the Tet transregulators
and in S. cerevisiae [88] revealed that a small number ofmutations in TetR can lead to that phenotype [113]. Once
The TetR–VP16 fusion works very well in many cases, but
this was discovered, intensive screening led to rtTA alleles in
may not be optimal for all applications. Structure–function
which the initial disadvantages of occasional background
studies based on powerful selection and screening systems in
expression and low sensitivity for dox were eliminated [88].
E . coli [104,105] and in S. cerevisiae [88] have lead to a
The rtTA-S2 allele was obtained by screening for reduced
profound understanding of how DNA binding, inducer
background expression and rtTA-M2 was the result of
binding and dimerization function in TetR. This informa-
screening for higher sensitivity towards dox starting from
3114 C. Berens and W. Hillen (Eur. J. Biochem. 270)
the alterations in rtTA-S2 that are responsible for the
DNA binding of the modified transactivators is efficient;
reverse phenotype [88]. None of the exchanges found in
in transient transfections in HeLa cells, they specifically
these new alleles were present in the original rtTA. The
achieve induction factors between 2000 and 8000 and are,
mutations leading to the reverse phenotype are located at
thus, as active as wild-type tTA2. Moreover, they are also
the interface between the DNA reading head and the
highly specific, as they induce the converse operator less
protein core or in the last turn of helix a6 that undergoes a
than twofold [114]. Modulation of the DNA-binding
conformational change upon inducer binding. Structural
specificity is not confined to tTA. Alleles specific for the
analysis of the DNA-bound form of TetR has led to the
4C- [120] and 6C-tet operators [114] have been constructed
proposal that the mutations present in rtTA [113] restrict
with rtTA and also regulate tc-dependent expression units
the repressor to a noninducible conformation and lock the
efficiently. This now leaves us with different tTA- and rtTA-
DNA-binding domains in the position necessary for oper-
operator combinations capable of controlling gene expres-
ator binding [8]. Taken together, the phenotype of rtTA can
sion tightly over a wide range of inducer concentrations.
be improved and designed by using appropriate screens.
Mastering subunit recognition of TetR
Tet transregulators vary in their sensitivity towards
Comparison of the TetR primary structures reveals 38–90%
tetracyclinic inducers
identical amino acids overall, but only 18% in the four-helix
The tTA and rtTA variants presently employed in eukary-
bundle involved in dimerization. Detailed structural infor-
otic gene regulation display differential sensitivity towards
mation [19] of the dimerization interface [121] suggested that
tc and its derivatives. While tTA can be induced by tc,
TetR proteins from individual classes would not readily
dox and atc [114], reverse transactivators respond only to
form heterodimers. The modular architecture of TetR
dox and atc [113] and tTSG is about twofold less sensitive to
allows the combination of a class B DNA-binding domain
dox than rtTA [115]. The response range of tTA to dox (0.1–
with the inducer-binding and/or dimerization domains of
10 ngÆmL)1) is clearly lower and, more importantly, non-
Tet repressors from other classes [121]. Fusion to the
overlapping with that of rtTA to dox (100–3000 ngÆmL)1)
reading head from TetR(B) increases activity of Tet
[114], but slightly overlapping with that of the more sensitive
repressors from several other classes [121] and ensures tight
rtTA2s-M2 allele (2–200 ngÆmL)1) [88]. The molecular
binding to the tetO boxes from Tn10 [122]. Class B TetR
mechanisms responsible for these different sensitivities are
does not form heterodimers with Tet repressors from classes
presently unknown. The isolation of a tc-like antagonist for
D [121], E [93,114,120], or G [115]. The fusion points can be
TetR [116] and the demonstration of its activity in
chosen with some flexibility; functional chimeras have been
transgenic plants [117] make it seem likely that alternative
obtained either by connecting the entire protein core from
inducers for TetR can be identified by screening.
TetR(D) or TetR(E) to a TetR(B) DNA-reading head[114,120,121] or by replacing the four-helix bundle formedby the helices a8 and a10 from both subunits (see Fig. 2),
The DNA binding specificity of Tet-transregulators
with the respective region from TetR(G) [115]. The resulting
transrepressors or transactivators regulate gene expression
Structure–function analyses of TetR–tetO interactions had
efficiently and do not form heterodimers as demonstrated in
shown that only few changes (shown in Fig. 4) in the DNA
DNA-retardation assays [114], immunoprecipitation and
binding helix–turn–helix motif of TetR suffice to switch the
FACS analysis [115] opening up the possibility to introduce
recognition specificity from the 19-base pair wild-type tetO
two or more TetR-based regulatory proteins into the same
to variants containing symmetric exchanges of bases at
cell without having to cope with the disadvantages of
position 4 (tetO-4C [118]) and position 6 (tetO-6C [119]).
heterodimer formation [114,115].
The TetR mutants were converted into the transactivatorstTA24C or tTA26C and minimal promoters Ptet4 and Ptet6
Combinatorial Tet regulation solves special
were constructed with the respective tetO variants [114].
problems and allows sophisticatedapplications
The previous section has shown that DNA-binding speci-ficity, subunit recognition and response to the inducer canbe altered in TetR. Fig. 5 gives an overview of the presentstate of the Tet modules that are available for use and thefollowing section presents a few principles of how themodular nature of the transregulators can be exploited toaddress specific experimental requirements and open up newapplications for conditional regulation.
Fig. 4. Operator specificity combinations for the Tet system. The pri-mary structure of the TetR(B) recognition helix a3 and the flanking
Expression can be switched between two alleles
loops is given in standard one-letter abbreviations. The entire sequence
of tetO2 is shown with the palindromic center marked by an asteriskand the base numbering shown above one operator half-side. The
The expression of two genes or of two alleles of one gene can
exchanges in TetR and tetO are highlighted in inverse print for each
be controlled in a mutually exclusive manner by combining
matching pair (wt, 4C, 6C).
different dimerization domains, different operator-binding
Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3115
therapeutic goal has been reached or, if necessary, in caseof emergency.
One gene can be regulated stringently by converselyacting transrepressor and transactivator
Detectable levels of transgene expression in animals or cellsin which the transactivator is not active can limit theusefulness of any conditional expression system for mode-ling complex biological processes or evaluating the effects ofa gene product. For the Tet system, this transgene leakagehas been attributed either to basal activity of the respectivetetO-based minimal promoter used (see above; [115]); or, insystems with rtTA, to residual binding of the reversetransactivator to tetO in the absence of dox [123,124]. Astringently controlled regulatory system can now be
Fig. 5. The Tet toolbox. TetR modules and regulatory domains are
accomplished by combining a trans-silencer with a reverse
displayed with the possible combinations. The different binding func-
transactivator, since heterodimer formation and concomit-
tions of TetR were coded in different shades of gray and placed at theirapproximate position in the protein, but not drawn to scale. The TetR
ant phenotype blurring will be prevented if the trans-silencer
variants characterized were classified in the corresponding module.
is equipped with a dimerization domain from the TetR
The regulatory domains that can be fused to TetR are coded in dif-
classes E or G. Thus, both transregulators bind in a
ferent shades of gray according to their viral, human, insect or fungal
mutually exclusive manner. Gene expression is actively
origin. Note that not every possible combination of modules need
repressed in the absence of dox by the binding of tTSE/tTSG
result in a transregulator with acceptable regulatory properties.
to the minimal promoter. Upon addition of dox, tTSE/tTSGdissociates from tetO, allowing the reverse transactivator tobind and activate transcription. This setup efficiently
specificities and by exploiting the differential sensitivity of
reduces background expression in yeast [82], in mammalian
Tet transregulators towards tetracyclines [114]. Interference
cell lines [93,115,120] and in transgenic animals [125–127],
between the two expression units is excluded by using a tTA
while affecting the maximal expression level only slightly
allele with an alternative class E or G dimerization domain
[128] or not at all [93].
and by furnishing rtTA with a modified DNA-bindingdomain that contacts the tetO-4C operator in Ptet4 speci-
Transgenes can be expressed in a graded
fically. Expression of the wild-type allele, for example, is
or in a binary manner
placed under tTA control and represents the normal state ofthe cell. A knockout situation can be generated by adding
Transcriptional control has generally been assumed to
either tc or, alternatively, atc or dox at concentrations
operate as a binary switch with on/off characteristics
between 10 and 100 ngÆmL)1 which dissociates tTA from
[129,130], but several examples displaying graded changes
the promoter but does not lead to DNA binding by rtTA
in gene expression have recently been published [131,132].
[114]. Maintaining the intermediate concentration of dox
The manner of gene expression might well be a key factor in
needed to shut down expression of both alleles will be
programs of cell differentiation or stimulus response.
feasible in cell culture applications. In transgenic animals,
Different regulatory setups of the Tet system allow a
however, the necessary fine-tuning of a dox or atc concen-
transgene to be expressed in one or the other manner [133–
tration may prove impossible suggesting instead the use of tc
135], enabling not only an analysis of a gene's function, but
to shut down tTA-dependent gene expression without
also of its mode of expression. When tTA and rtTA are
interfering with regulation by rtTA. To switch to the
expressed constitutively in mammalian cells and also in
expression of the mutant allele requires atc or dox concen-
S. cerevisiae, they drive transgene expression in a dose-
trations of 1 lgÆmL)1 or more.
dependent, graded manner [133,135]. However, when rtTA
Such a dual control system can provide valuable
was expressed in S. cerevisiae under conditions of positive
insights into developmental and pathogenic processes.
feedback using an autoregulatory circuit, the cell population
One can imagine shutting down expression of a tumor
was clearly divided into regulator-expressing and nonex-
suppressor while inducing expression of an oncogene to
pressing cell pools [135]. In mammalian cells, the combined
study cancerogenesis. Switching off expression of the
usage of tTSG and rtTA also led to bimodal expression of
oncogene after tumor formation can establish whether the
the GFP reporter (see Fig. 3 in [134]). Although not
respective protein is a valid target for therapeutic inter-
formally proven, we assume that a bimodal expression
vention. One could also switch from a wild-type to a
pattern will not be observed for all repressor/activator
mutant allele at a defined developmental state of the
combinations, but only for those in which the sensitivity of
organism and then return to wild-type expression at a later
tTS for the inducer is lower than that of the rtTA allele used,
stage. This type of regulatory circuit can also deliver an
as is the case for tTSG (compare the dose–response curve of
additional degree of freedom to gene therapeutic strat-
tTSE and rtTA of Fig. 4 in [93] with the one for tTSG and
egies ) one regulatory circuit may be used to control a
rtTA of Fig. 2 of [134]). This will ensure that rtTA is
therapeutic gene, while the other may be exploited to serve
preloaded with inducer and ready to activate transcription
as a suicide switch to terminate the treatment once the
the moment the dox concentrations needed for binding to
3116 C. Berens and W. Hillen (Eur. J. Biochem. 270)
tTSG are reached and tetO is subsequently released. In
fragment in the brain demonstrated that its continuous
principle, only two regulatory states are observed: either the
supply was needed to maintain the characteristic neuro-
tetO sites are fully occupied with tTSG and gene expression
pathology and behavioral phenotype, raising the possibility
is shut off, or they are saturated with the rtTA variant,
that the disease may be reversible by targeting the causative
resulting in full transcriptional activation. The consequence
agent [151].
is a binary expression pattern of the target gene. While this
Regulation by the Tet system has also had a significant
setup already works with rtTA, the effect should be even
impact on behavioral studies. Expression of constitutively
more pronounced with rtTA2s-M2, as its inducer response
active mutant forms of the calcium/calmodulin dependent
range overlaps completely with that of tTSG.
kinase II or calcineurin in the brain of adult mice resulted inaltered synaptic plasticity and impairments in spatial
Highlighting the regulatory potential
memory storage and retrieval, but these deficits were fully
and looking into the future
reversed when transgene expression was suppressed[84,152]. Because expression of the transgene was limited
The properties and the adaptability of Tet regulation as
to the hippocampus, this structure was additionally proven
presented in the previous sections allow its use in many
to be the site responsible for the behavioral effects. In a
different applications. We would like to demonstrate this
different example, knockout mice lacking the serotonin 1A
enormous variability by referring to a few key studies that,
receptor show increased anxiety-like behavior which could
in our opinion, highlight the potential of Tet regulation.
be rescued by conditional expression, but only if the
Regulation by tetracyclines is sensitive and efficient
receptor was synthesized during the early postnatal period
enough to control target gene expression in pathogenic
in the hippocampus and cortex [153].
organisms even when they have been injected into a
Nevertheless, improvement and additions to the Tet
mammalian host. The role of individual genes in infection
system, among them the regulatory components, are still
and pathogenesis can, thus, be probed and their validity as
possible and necessary. Promoter development has not
targets for therapeutic intervention determined in an in vivo
received the same degree of attention as the transregulators.
disease model [136]. This has not only been demonstrated
The number of tetO elements and their spacing [154], as well
for trypanosomes [137,138], but also for common human
as the linker sequence separating the operators [155] have
pathogens like Staphylococcus aureus [139] and Candida
not been optimized yet. It remains to be seen if an ideal
glabrata [140]. In the fungus, squalene synthase [136] and
minimal promoter with no intrinsic leakiness supporting
sterol 14a-demethylase [141] were, thus, shown not to be
very high-level activation can be identified or designed.
ideal targets for antifungal development.
Fortunately, screens for regulators with improved prop-
The successful expression of the diphtheria toxin A
erties can now be performed in eukaryotic systems [88]
subunit by tTA/Ptet-1 in transgenic mice has demonstrated
and, as an example, the isolation of novel Tet regulators
the stringency of regulation that can be reached with the Tet
which recognize nonantibiotically active tetracyclines or
system [142]. Although mouse lines that carried the target
even nontetracyclinic inducers, would be of great benefit.
transgene were obtained at an approximately 10-fold lower
They would not only facilitate gene therapy applications
frequency than normal, those that were established regula-
which, at the moment, can be impaired by the use of
ted the transgene efficiently. Induction of toxin expression
tetracyclines in anti-infective therapy or their misuse as
led to cell death and development of cardiomyopathies.
growth promoting additives to animal food. If these novel
Stringent control of transgene expression using rtTA has
inducers are not only ecologically safe, but also easy and
also been achieved in HeLa cells for the Shiga toxin B
nonexpensive to manufacture, the inducer–regulator pair-
subunit [143], for the proapoptotic gene PUMA in SAOS-2
ing could also be useful in insect population control using
and H1299 cell lines [144] and, using rtTA2s-S2 in transgenic
dominant, repressible, lethal genetic systems [156,157] and
mice, for Cre-recombinase [145].
might even introduce regulation by the Tet system to crop
The strength of a true conditional system ) the possibi-
plants. They would add to the repertoire of transregulators
lity to switch gene expression on and off at leisure and
and finally, since multiple dimerization and DNA-binding
repeatedly ) represents a powerful method with which to
specificities are already present, allow fully independent
explore the relationship between mutant protein expression
expression control of more than one gene by the Tet
and disease progression. This has become evident upon
studying transgenic mouse models for cancer and neuro-
A major experimental challenge will be to express a target
logical disorders. Here, the use of tTA and rtTA to control
gene within its physiological window, which might depend
expression of an oncogene revealed for solid tumors
on environmental stimuli and even change during develop-
[146,147] and for leukemias [148,149] that the oncogene is
ment, since over- or underexpression often results in altered
not only necessary for tumor formation but also for tumor
phenotypes [131] or pathologies. While tc-controlled expres-
maintenance, suggesting pharmacological inactivation of
sion can mimic the natural level [146], this must not always
oncogenes as a possible therapeutic strategy for cancer. This
be the case. A solution might be precise promoter targeting
assumption has been substantiated by the unexpected
by tetO elements, to minimally interfere with gene expres-
observation that, after having gone through one cycle of
sion. This will be difficult and will require extensive
MYC-gene expression and silencing, reactivation of the
knowledge about the influence of chromatin structure on
oncogene does not lead to tumor regrowth, but rather to
gene expression and its sensitivity to perturbation, partic-
apoptosis [150]. Similar effects have also been found for
ularly when regulatory regions are modified [158]. But, if
neurological disorders. In a conditional model of Hunting-
successful, this approach will provide an additional degree
ton's disease, mice expressing a mutated huntingtin
of freedom to manipulate gene expression, as the existing
Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3117
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