Báo cáo khoa học: Gene regulation by tetracyclines Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes Christian Berens and Wolfgang Hillen pptx

REVIEW ARTICLE
Gene regulation by tetracyclines
Constraints of resistance regulation in bacteria shape TetR for application
in 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
of a family of tetracycline (tc) resistance determinants in
Gram-negative bacteria. The resistance protein TetA, a
membrane-spanning H
+
-[tcÆM]
+
antiporter, must be sen-
sitively regulated because its expression is harmful in the
absence of tc, yet it has to be expressed before the drugs’
concentration reaches cytoplasmic levels inhibitory for
protein synthesis. Consequently, TetR shows highly speci-
fic tetO binding to reduce basal expression and high affinity
to tc to ensure sensitive induction. Tc can cross biological
membranes by diffusion enabling this inducer to penetrate
the majority of cells. These regulatory and pharmacological

properties are the basis for application of TetR to selec-
tively control the expression of single genes in lower and
higher eukaryotes. TetR can be used for that purpose in
some organisms without further modifications. In mam-
mals and in a large variety of other organisms, however,
eukaryotic transcriptional activator or repressor domains
are fused to TetR to turn it into an efficient regulator.
Mechanistic understanding and the ability to engineer and
screen for mutants with specific properties allow tailoring
of the DNA recognition specificity, the response to inducer
tc and the dimerization specificity of TetR-based eukary-
otic regulators. This review provides an overview of the
TetR properties as they evolved in bacteria, the functional
modifications necessary to transform it into a convenient,
specific and efficient regulator for use in eukaryotes and
how the interplay between structure ) function studies in
bacteria and specific requirements of particular applica-
tions in eukaryotes have made it a versatile and highly
adaptable regulatory system.
Keywords: antibiotic resistance; disease models; fusion pro-
tein; inducible gene expression; ligand-binding specificity;
mammalian cell lines; protein engineering; structure–activity
relationship; Tet repressor; transgenic organism.
Properties of bacterial Tet systems
Efflux-mediated tetracycline resistance is always
regulated in Gram-negative bacteria
In Gram-negative bacteria, resistance to tetracyclines (tc)
is mediated by the TetA protein, a proton-[tcÆMg]
+
anti-

porter embedded in the cytoplasmic membrane [1,2]. Eleven
tc resistance determinants (Tet classes A–E, G, H, J, Z, 30,
and 33 [3–5]) share the organization of structural and
regulatory genes (reviewed in [6]). In enteric bacteria, the
efflux-encoding tetA genes are strictly regulated at the level
of transcription by the tc-responsive Tet repressor (TetR).
In the absence of inducer, TetR dimers bind to the operators
tetO
1
and tetO
2
, shutting down transcription of its own
gene, tetR, and of the resistance gene, tetA.Oncetchas
entered the cell, it binds TetR with high affinity as a
[tcÆMg]
+
complex [7]. This induces a conformational change
in TetR [8] resulting in dissociation from tetO [9]. The
following expression burst of TetA and TetR leads to a
rapid reduction of the cytoplasmic tc concentration [10]
which, in turn, shuts expression of both genes off again.
Expression of TetA is fine-tuned in the presence of tc so that
export overcomes the slow uptake (compare below).
Regulation of Tc resistance is optimized for tightness
and sensitivity
Regulation of tet determinants is subject to strong, opposing
selective pressures. Expression of the resistance protein
TetA is detrimental to the cell [11,12]. Overexpression of this
integral membrane protein is lethal for Escherichia coli [13],
probably due to the collapse of the membrane potential [14].

Consequently, expression of TetA must be tightly repressed
in the absence of the drug. However, when tc diffuses into
the cell the resistance protein must be expressed before the
cytoplasmic concentration of tc reaches the micromolar
level necessary to inhibit translation. This requires: (a) high
Correspondence to W.Hillen,Lehrstuhlfu
¨
r Mikrobiologie, Institut
fu
¨
r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander
Universita
¨
t, Staudtstr. 5, D-91058 Erlangen, Germany.
Fax: +49 9131 8528082, Tel.: +49 9131 8528081,
E-mail:
Abbreviations: tc, tetracycline; dox, doxycycline; atc, anhydrotetra-
cycline; tTA, tetracycline-dependent transactivator; rtTA, reverse
tetracycline-dependent transactivator; tTS, tetracycline-dependent
trans-silencer; CMV, cytomegalovirus; GFP, green fluorescent
protein.
(Received 8 April 2003, revised 14 May 2003,
accepted 15 May 2003)
Eur. J. Biochem. 270, 3109–3121 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03694.x
affinity of TetR for both tetO andtctokeepthebasal
expression level of tetA low and to ensure that its
transcription is initiated at concentrations which are still
subinhibitory for translation; (b) low affinity of the TetR–
[tcÆMg]
+

complex for DNA; and (c) high-level, but short-
term expression of TetA to initially reduce the internal
concentration of tc. A low level of TetR is important for
sensitive induction, since E.coli strains expressing high
levels of TetR need high concentrations of tc for full
induction [15]. These conflicting requirements are met by
the genetic organization of the resistance determinants
(reviewed in [6]) and by the ligand binding properties of
TetR. High sensitivity towards tetracyclines [see Fig. 1 for
the structures of tc, doxycycline (dox) and anhydro-tc (atc)]
is achieved by the remarkable binding constant of TetR for
[tcÆMg]
+
(K
a
 10
9
M
)1
), [doxÆMg]
+
(K
a
 10
10
M
)1
)or
[atcÆMg]
+

(K
a
 10
11
M
)1
) [7,9], about 10
3
)10
5
-fold higher
than the affinity of the drugs to their intracellular target, the
ribosome [16]. Binding of two molecules of [tcÆMg]
+
to a
TetR dimer diminishes repressor affinity for tetO by about
nine orders of magnitude to the unusually low background
DNA binding level of less than 10
5
M
)1
[9]. This high ratio
of specific over nonspecific DNA binding enables TetR to
bind tetO efficiently, even in larger genomes containing
competing nonspecific DNA to a much higher degree than
bacteria. Taken together, the evolutionary pressures on
tc-dependent gene regulation have led to tight repression in
the absence of tc, without compromising sensitivity of
induction, so that regulated tc resistance determinants
impose no burden on the fitness of E.coliin the absence of

the antibiotic, but still mediate high levels of resistance to tc
in its presence [12].
The structural change of TetR associated with induction
by tetracycline is known
X-ray crystal structures of free TetR [17], TetR complexed
with different tetracyclines [18–21] and with tetO [8] have
been determined at resolutions of 1.9–2.5 A
˚
, revealing
the allosteric conformational change leading to induction.
These results have been reviewed in detail [22] and have been
compared to Lac repressor [23]. Thus, they are only
summarized here (Fig. 2). The DNA reading head of TetR
(magenta) is connected to the protein core (blue) by the helix
a4 (green). Binding of [tcÆMg]
+
(yellow) to TetR unwinds
the C-terminal residues of helix a6 (light blue), which bump
into a4 and displace it. As the C terminus of a4isheldin
place by contacts to tc, the displacement leads to a
pendulum-like swing of the a4 N terminus increasing the
distance between the recognition helices by 3 A
˚
, so that they
do not fit into successive major grooves of DNA anymore
[24]. These conformational changes are consistent with
many noninducible TetR mutants [24,25], spectroscopic
analysis of TetR in vitro [26], in vivo [27] and in vitro [28]
disulfide trapping experiments. Furthermore, a movement
of a9 closes the tc binding pocket after the drug has entered

[17], and the loop between a8anda9 is also important for
induction [29–31].
Tetracycline penetrates cells by diffusion
Tetracyclines (Fig. 1) diffuse in their uncharged forms
through lipid bilayers without the aid of protein channels
[32–36]. Measuring the increase in fluorescence intensity of
tc observed upon binding to TetR [7] allows us to determine
the cytoplasmic concentration of tc and, thus, to calculate
permeation coefficients for tc uptake into liposomes
[(2.4 ± 0.6) · 10
)9
cmÆs
)1
] and whole E.coli cells
[(5.6 ± 1.9) · 10
)9
cmÆs
)1
] [36]. These translate into half-
equilibration times of 35 ± 15 min for tc to cross the
membranes and are in good agreement with the half-
equilibration time of 15 min measured for [
3
H]tc-uptake in
Bacillus subtilis [37], and the slow uptake of tc observed in
Staphylococcus aureus [38]. Tetracycline diffusion through
phospholipid membranes is, thus, slow and appears to be
the rate-limiting step of uptake into cells [36]. The previously
observed rapid uptake of tc [33,39] might rather reflect
unspecific adsorption of tc to membrane surfaces [32,36]. A

detailed model explaining the transport and accumulation
of tc across the Gram-negative cell envelope has been
presented by Nikaido and coworkers ([40,41] and references
cited therein). In the medium, as well as in the periplasm and
cytoplasm, tc is present in one uncharged and several
charged or zwitterionic species, due to its three titratable
groups (Fig. 1). The distribution between these species
depends on the pH of the respective compartment [40]. The
Fig. 1. Structures of tetracyclines used in eukaryotic gene regulation.
(A) Structure of tetracycline with the pK
a
values of the three titratable
groups. (B) Structure of doxycycline. (C) Structure of anhydrotetra-
cycline.
3110 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003
uncharged form of tc can penetrate the outer membrane
directly. But the major fraction of tc equilibrates as a
[tcÆM]
+
-complex rapidly through the outer membrane via
porins, with the Donnan potential across the outer mem-
brane leading to a two- to threefold accumulation of this
charged complex in the periplasm. Tc then diffuses passively
in its uncharged form through the cytoplasmic membrane.
Due to the pH gradient across the cytoplasmic membrane,
a larger fraction of the uncharged tc dissociates in the
cytoplasm than in the periplasm. Since equilibrium is
reached when the concentration of uncharged tc is identical
in both compartments, this results in a higher intracellular
concentration of [tcÆM]

+
, the biologically active compound.
Again, accumulation of tc is the product of this passive
equilibration across the inner membrane [40,41].
Tc-based gene regulation functions in
different setups in many eukaryotic systems
The evolved properties of TetR described above combined
with the favorable pharmacokinetics of tetracyclines and
their long record of safe use in clinical practice make the Tet
system a good candidate to fulfill the criteria that are required
for an ideal transcriptional regulator in eukaryotic cells as
given by Saez and others [42,43]. Consequently, the past
15 years have seen the broad application of tc-dependent
regulatory systems, mainly in mammalian cell culture, but to
an increasing degree in transgenic organisms like plants,
yeasts, protozoan parasites, slime molds, flies, and rodents.
These topics have been extensively reviewed [42–52]. The
following section presents an overview of the basic Tet
systems used to regulate gene expression in eukaryotes.
Gene regulation by TetR in eukaryotes
The most basic and first published application of
tc-mediated gene regulation in eukaryotes is transcriptional
repression by unmodified TetR [53]. Here, TetR most
likely acts by interfering sterically with binding of RNA
polymerase or auxiliary transcription factors [42,54]. To
achieve this, one or more tetO elements are placed in
proximity of either the TATA box or the transcriptional
start site of the respective target gene and TetR is expressed
concomitantly by a strong, constitutive promoter. Promot-
ers of all three eukaryotic RNA polymerases have been

targeted in the manner described. Unfortunately, as will
become evident in the following paragraph, the published,
successful approaches do not yet allow formation of a
simple strategy for establishing a TetR-repressed system,
although they clearly point out that the positioning of the
tetO boxes is crucial for efficient regulation.
In Leishmania donovani, an RNA polymerase I promo-
ter was brought under tc-control by placing a single tetO site
2–24 bp upstream of the transcriptional start site [55],
whereas in Trypanosoma brucei at least one tetO element
had to be inserted at a position +2 or )2relativetothe
transcription start site of an RNA polymerase I-like
promoter [56]. For RNA polymerase III-mediated tran-
scription of suppressor tRNA genes, induction factors
between two- to fivefold were observed in Saccharomyces
cerevisiae, Dictyostelium discoideum and carrot protoplasts
when tetO was introduced within 10 bp upstream of the
transcriptional start site [57–59]. A regulated version of the
human U6 snRNA promoter, also transcribed by RNA
polymerase III, was developed by replacing sequences
between the proximal sequence element and the transcrip-
tional start site with tetO [60]. Flanking the TATA-box with
two operators completely abolished transcriptional activity.
In contrast, introduction of a single tetO element affected
transcription only slightly, but led to up to 25-fold
repression in the presence of TetR. A regulated U6 snRNA
promoter with a defined expression window [61,62] would
be a very powerful tool as this promoter is used to express
the small interfering RNA [63] needed for silencing gene
Fig. 2. Structure of the TetR–[tcÆMg]

+
complex. Tet repressor is shown as a ribbon
diagram with one monomer in gray and the
other monomer color-coded as follows: The
DNA-binding region is in magenta, the helix
connecting it with the protein core is in green.
The protein core is dark blue, with the helix a6
in light blue. Tetracycline is displayed as
space-filling CPK model in yellow. For clarity,
the helices a1–a10 of one monomer are num-
beredandtheNandCterminiofbothsub-
units are indicated. The coordinates were
taken from the PDB entry 2TRT [18].
Ó FEBS 2003 Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3111
expression by RNA interference [64]. Repression of RNA
polymerase II promoters exerted by TetR is strongest in
plants [65,66], mammalian cells [67] and fungi like Schizo-
saccharomyces pombe [68,69] when multiple tet operators
are positioned within a region from 5 bp upstream to 35 bp
downstream of the TATA-element. In contrast, placement
of one to four tet operators immediately downstream of the
transcription initiation site has been shown to be most
effective in the parasitic protozoa Entamoeba histolytica
[70,71], Toxoplasma gondii [72] and Giardia lamblia [73].
Gene regulation by TetR-based transregulators
While unmodified TetR acts as a transcriptional repressor in
plants and lower eukaryotes, it can be, but not always is
efficient in mammalian cells [67,74]. A consistently func-
tional version for yeasts, flies and mammalian cell lines is
TetR fused to an eukaryotic regulatory domain, such as an

acidic activation domain (Fig. 3A; tTA or Tet-Off) [75–80]
or a repression domain (Fig. 3B; tTS) [81–83]. The trans-
activator tTA directs expression from a tc-dependent
promoter that contains seven repeats of a tetO
2
element
from the transposon Tn10. The palindromic centers of two
adjacent operators are separated by 41 bp. This element is
fused to a minimal promoter, typically derived from the
human cytomegalovirus (CMV) immediate early promoter
[75]. When both components are stably integrated into
proper chromosomal loci of mammalian cell lines, tran-
scription from the hybrid promoter is silent in the presence
of more than 10 ngÆmL
)1
dox. Removal of dox leads to
binding of tTA to tetO and subsequent activation of
transcription. Regulatory factors of up to five orders of
magnitude can be reached with sensitive reporter genes like
firefly luciferase [75]. Luciferase activity is expressed within
4 h of removal of tc and about 20% of the steady-state level
is reached after 12 h. While the use of a strong, constitutive
promoter (CMV IE, EF-1a, Ubiquitin C) is common in cell
culture applications, the use of tissue-specific promoters in
transgenic animals provides spatial control to the Tet
system, restricting expression of the Tet transregulator and,
subsequently, the transgene to the desired tissue [84,85]. In
Drosophila, usage of the Gal4-UAS system to control Tet
transregulator expression allows the generation of spatially
delimited expression patterns by simple crossing with one of

the many Gal4 driver lines available in the Drosophila
research community [86].
One concern has been the expression levels of Tet
transregulators as influenced by a potentially low mRNA
stability or efficiency of translation. This was recently
addressed by generating a synthetic coding sequence for
tetR. Potential splice donor and acceptor sites identified by
sequence analysis, several potential endonuclease cleavage
sites, and potential stable hairpin structures in the mRNA
were eliminated and human codon usage was used [87–90].
The consequence of this optimization protocol is a higher
protein level in Drosophila, HeLa and HEK293 cells.
Another concern voiced was that the CMV-derived
minimal promoter was not transcriptionally silent under all
experimental conditions [91–93]. This promoter leakiness
can be caused by promoter-dependent or integration site-
dependent effects and has been discussed in detail [94].
Promoter-dependent leakiness has been addressed by the
use of alternative minimal promoters [75,95,96]. In transient
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 transregulators
are shown as follows: the DNA reading heads are in light gray, the inducer-binding and dimerization domain is in dark gray, activation domains are
black boxes, and the silencing domain is stippled. The conformational change leading to the loss of DNA-binding activity is pictured as a light gray
box. High-level activated transcription is displayed by a bold arrow, low-level basal transcription by a dotted arrow. (A) tTA. (B) tTS. (C) rtTA.
3112 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003
than P
tet
-1, but also do not reach its maximal activation
level. Thus, the regulatory window for target gene expres-
sion is shifted and expanded due to the stronger reduction of

the basal activity. Integration site-dependent leakiness has
been attributed to enhancers located close to the integration
site of the target gene construct. Besides screening additional
clones until one harboring the desired properties is found,
the problem has been approached by insulating P
tet
-1 from
external activating signals through insertion of a chicken
lysozyme matrix attachment region just upstream of P
tet
-1
[87] or by flanking the target gene expression unit with either
chicken b-globin insulators [90] or SCS and SCS’ boundary
elements from Drosophila [86].
A different strategy was adopted by engineering a
tc-controlled trans-silencer protein [81]. Fusion of the
KRAB domain of Kox1 [97] to TetR yielded a hybrid
protein called tTS, that not only substantially repressed
basal transcription from P
tet
-1 even if the tet operators were
located 3 kbp distant from the minimal promoter, but also
efficiently down-regulated gene expression from a CMV
enhancer-driven P
tet
-1 [83]. This strategy therefore appears
to be more versatile in coping with unwanted target gene
expression than the promoter adaptation proposed above.
In addition and in contrast to tTA, the tc-dependent
silencing of complex promoters offers the unique possibility

of reversibly down-regulating the expression of cellular
genes on top of their normal regulation. The KRAB
domain is inactive in S. cerevisiae and Drosophila where it
was replaced with repression domains from the proteins
SSN6 [82], knirps, giant or dCtBP [83].
The expression of transfected genes can be rapidly
repressed in mammalian cells by epigenetic mechanisms
[98]. Although this Ôtransgene silencingÕ is not specific for
the Tet system, it is often observed for genes under tc
control due to its frequent usage as conditional expression
system. Approaches to achieve stable gene expression have
been to: (a) screen many transfected clones; (b) the use of
lentiviral vectors [99]; (c) replace the viral promoters that
direct expression of the transregulators with promoters of
human origin [100]; (d) use chromatin insulator sequences
to protect transgene expression [98]; or (e) couple transgene
expression to a selectable marker via an IRES element
[101] or by fusion of the transregulator with green
fluorescent protein (GFP) [102]. Note that in this fusion
protein GFP is connected to the DNA-binding domain of
TetR which can interfere with nonspecific DNA-binding
activity of TetR at low levels of dox (see Fig. 2A in [102]
and [78]). The few published examples make it impossible
to recommend one of the strategies for use in establishing
homogenous expression of transgenes, but silencing of
transregulator expression is not completely suppressed by
the use of lentiviral vectors [103] or insulator sequences
[101].
Modifications of the Tet transregulators
The TetR–VP16 fusion works very well in many cases, but

may not be optimal for all applications. Structure–function
studies based on powerful selection and screening systems in
E.coli [104,105] and in S. cerevisiae [88] have lead to a
profound understanding of how DNA binding, inducer
binding and dimerization function in TetR. This informa-
tion can be used to find solutions to some of the problems
and limitations that arise for Tet system applications in
eukaryotes.
Alterations of the activator domain of tTA
Especially for gene therapy, concern about a viral protein is
often voiced, as humoral as well as cellular immune
response against the VP16 protein has been found in herpes
simplex infected humans [106–108]. Thus, immune res-
ponses against transactivators containing the VP16 domain
cannot be rigorously excluded, although they have not been
observed so far in a mouse model using reverse tTA (rtTA;
Tet-On) [109]. Two solutions circumventing this concern
have been developed: (a) the VP16 domain has been
replaced by three repeats of a minimal activation domain
derived from a 12-amino acid activating patch of the VP16
protein (tTA2 [76]); and (b) a variety of human activator
domains from the acidic, glutamine-rich, serine/threonine-
rich and proline-rich functional groups were tested for their
ability to replace the VP16 domain. When fused to TetR,
only acidic activation domains were highly active [78–80].
Minor activation was observed with the serine/threonine-
rich domains from the transcription factors ITF-1, ITF-2,
and MTF-1. Transactivators with activation potentials
spanning more than three orders of magnitude have been
generated by combination of various minimal activation

domains (see above; [76]). They are attractive for combined
Ôknock-in/knock-outÕ strategies to convey tissue-specific
expression of the transactivator, while at the same time
inactivating expression of the genomic copy of the target
gene. Expression of the regulatory protein is then an
invariant function of the genomic locus and, if too high, can
lead to ÔsquelchingÕ [110]. This can be addressed by
employing a transactivator with reduced activation poten-
tial as these are tolerated in the cell at higher concentrations
[76].
Conversion of TetR to reverse TetR
Eukaryotic gene regulation by tTA shows a high dynamic
range and works consistently well, but has several practical
drawbacks. Tc has to be continually present to keep
expression of the gene of interest downregulated. Although
tc is not toxic at the levels utilized in gene regulation,
prolonged exposure to the antibiotic is not always desirable
in transgenic animals nor is it possible in gene therapy.
Furthermore, induction of target genes is mostly slow as it
requires removal of the drug from the culture or organism.
To be able to control the time point of induction more
precisely, and since organisms are more easily saturated
with an effector than depleted of it [111,112], reverse TetR
variants which bind tetO onlyinthepresenceoftcwere
searched for and found (Fig. 3C). Screening in E.coli[113]
and in S. cerevisiae [88] revealed that a small number of
mutations in TetR can lead to that phenotype [113]. Once
this was discovered, intensive screening led to rtTA alleles in
which the initial disadvantages of occasional background
expression and low sensitivity for dox were eliminated [88].

The rtTA-S2 allele was obtained by screening for reduced
background expression and rtTA-M2 was the result of
screening for higher sensitivity towards dox starting from
Ó FEBS 2003 Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3113
the alterations in rtTA-S2 that are responsible for the
reverse phenotype [88]. None of the exchanges found in
these new alleles were present in the original rtTA. The
mutations leading to the reverse phenotype are located at
the interface between the DNA reading head and the
protein core or in the last turn of helix a6 that undergoes a
conformational change upon inducer binding. Structural
analysis of the DNA-bound form of TetR has led to the
proposal that the mutations present in rtTA [113] restrict
the repressor to a noninducible conformation and lock the
DNA-binding domains in the position necessary for oper-
ator binding [8]. Taken together, the phenotype of rtTA can
be improved and designed by using appropriate screens.
Tet transregulators vary in their sensitivity towards
tetracyclinic inducers
The tTA and rtTA variants presently employed in eukary-
otic gene regulation display differential sensitivity towards
tc and its derivatives. While tTA can be induced by tc,
dox and atc [114], reverse transactivators respond only to
dox and atc [113] and tTS
G
is about twofold less sensitive to
dox than rtTA [115]. The response range of tTA to dox (0.1–
10 ngÆmL
)1
) is clearly lower and, more importantly, non-

overlapping with that of rtTA to dox (100–3000 ngÆmL
)1
)
[114], but slightly overlapping with that of the more sensitive
rtTA2
s
-M2 allele (2–200 ngÆmL
)1
) [88]. The molecular
mechanisms responsible for these different sensitivities are
presently unknown. The isolation of a tc-like antagonist for
TetR [116] and the demonstration of its activity in
transgenic plants [117] make it seem likely that alternative
inducers for TetR can be identified by screening.
The DNA binding specificity of Tet-transregulators
can be changed
Structure–function analyses of TetR–tetO interactions had
shown that only few changes (shown in Fig. 4) in the DNA
binding helix–turn–helix motif of TetR suffice to switch the
recognition specificity from the 19-base pair wild-type tetO
to variants containing symmetric exchanges of bases at
position 4 (tetO-4C [118]) and position 6 (tetO-6C [119]).
The TetR mutants were converted into the transactivators
tTA2
4C
or tTA2
6C
and minimal promoters P
tet4
and P

tet6
were constructed with the respective tetO variants [114].
DNA binding of the modified transactivators is efficient;
in transient transfections in HeLa cells, they specifically
achieve induction factors between 2000 and 8000 and are,
thus, as active as wild-type tTA2. Moreover, they are also
highly specific, as they induce the converse operator less
than twofold [114]. Modulation of the DNA-binding
specificity is not confined to tTA. Alleles specific for the
4C- [120] and 6C-tet operators [114] have been constructed
with rtTA and also regulate tc-dependent expression units
efficiently. This now leaves us with different tTA- and rtTA-
operator combinations capable of controlling gene expres-
sion tightly over a wide range of inducer concentrations.
Mastering subunit recognition of TetR
Comparison of the TetR primary structures reveals 38–90%
identical amino acids overall, but only 18% in the four-helix
bundle involved in dimerization. Detailed structural infor-
mation [19] of the dimerization interface [121] suggested that
TetR proteins from individual classes would not readily
form heterodimers. The modular architecture of TetR
allows the combination of a class B DNA-binding domain
with the inducer-binding and/or dimerization domains of
Tet repressors from other classes [121]. Fusion to the
reading head from TetR(B) increases activity of Tet
repressors from several other classes [121] and ensures tight
binding to the tetO boxes from Tn10 [122]. Class B TetR
does not form heterodimers with Tet repressors from classes
D [121], E [93,114,120], or G [115]. The fusion points can be
chosen with some flexibility; functional chimeras have been

obtained either by connecting the entire protein core from
TetR(D) or TetR(E) to a TetR(B) DNA-reading head
[114,120,121] or by replacing the four-helix bundle formed
by the helices a8anda10 from both subunits (see Fig. 2),
with the respective region from TetR(G) [115]. The resulting
transrepressors or transactivators regulate gene expression
efficiently and do not form heterodimers as demonstrated in
DNA-retardation assays [114], immunoprecipitation and
FACS analysis [115] opening up the possibility to introduce
two or more TetR-based regulatory proteins into the same
cell without having to cope with the disadvantages of
heterodimer formation [114,115].
Combinatorial Tet regulation solves special
problems and allows sophisticated
applications
The previous section has shown that DNA-binding speci-
ficity, subunit recognition and response to the inducer can
be altered in TetR. Fig. 5 gives an overview of the present
state of the Tet modules that are available for use and the
following section presents a few principles of how the
modular nature of the transregulators can be exploited to
address specific experimental requirements and open up new
applications for conditional regulation.
Expression can be switched between two alleles
of one gene
The expression of two genes or of two alleles of one gene can
be controlled in a mutually exclusive manner by combining
different dimerization domains, different operator-binding
Fig. 4. Operator specificity combinations for the Tet system. The pri-
mary structure of the TetR(B) recognition helix a3 and the flanking

loops is given in standard one-letter abbreviations. The entire sequence
of tetO
2
is shown with the palindromic center marked by an asterisk
and the base numbering shown above one operator half-side. The
exchanges in TetR and tetO are highlighted in inverse print for each
matching pair (wt, 4C, 6C).
3114 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003
specificities and by exploiting the differential sensitivity of
Tet transregulators towards tetracyclines [114]. Interference
between the two expression units is excluded by using a tTA
allele with an alternative class E or G dimerization domain
and by furnishing rtTA with a modified DNA-binding
domain that contacts the tetO-4C operator in P
tet4
speci-
fically. Expression of the wild-type allele, for example, is
placed under tTA control and represents the normal state of
the cell. A knockout situation can be generated by adding
either tc or, alternatively, atc or dox at concentrations
between 10 and 100 ngÆmL
)1
which dissociates tTA from
the promoter but does not lead to DNA binding by rtTA
[114]. Maintaining the intermediate concentration of dox
needed to shut down expression of both alleles will be
feasible in cell culture applications. In transgenic animals,
however, the necessary fine-tuning of a dox or atc concen-
tration may prove impossible suggesting instead the use of tc
to shut down tTA-dependent gene expression without

interfering with regulation by rtTA. To switch to the
expression of the mutant allele requires atc or dox concen-
trations of 1 lgÆmL
)1
or more.
Such a dual control system can provide valuable
insights into developmental and pathogenic processes.
One can imagine shutting down expression of a tumor
suppressor while inducing expression of an oncogene to
study cancerogenesis. Switching off expression of the
oncogene after tumor formation can establish whether the
respective protein is a valid target for therapeutic inter-
vention. One could also switch from a wild-type to a
mutant allele at a defined developmental state of the
organism and then return to wild-type expression at a later
stage. This type of regulatory circuit can also deliver an
additional degree of freedom to gene therapeutic strat-
egies ) one regulatory circuit may be used to control a
therapeutic gene, while the other may be exploited to serve
as a suicide switch to terminate the treatment once the
therapeutic goal has been reached or, if necessary, in case
of emergency.
One gene can be regulated stringently by conversely
acting transrepressor and transactivator
Detectable levels of transgene expression in animals or cells
in which the transactivator is not active can limit the
usefulness of any conditional expression system for mode-
ling complex biological processes or evaluating the effects of
a gene product. For the Tet system, this Ôtransgene leakageÕ
has been attributed either to basal activity of the respective

tetO-based minimal promoter used (see above; [115]); or, in
systems with rtTA, to residual binding of the reverse
transactivator to tetO in the absence of dox [123,124]. A
stringently controlled regulatory system can now be
accomplished by combining a trans-silencer with a reverse
transactivator, since heterodimer formation and concomit-
ant phenotype blurring will be prevented if the trans-silencer
is equipped with a dimerization domain from the TetR
classes E or G. Thus, both transregulators bind in a
mutually exclusive manner. Gene expression is actively
repressed in the absence of dox by the binding of tTS
E
/tTS
G
to the minimal promoter. Upon addition of dox, tTS
E
/tTS
G
dissociates from tetO, allowing the reverse transactivator to
bind and activate transcription. This setup efficiently
reduces background expression in yeast [82], in mammalian
cell lines [93,115,120] and in transgenic animals [125–127],
while affecting the maximal expression level only slightly
[128] or not at all [93].
Transgenes can be expressed in a graded
or in a binary manner
Transcriptional control has generally been assumed to
operate as a binary switch with on/off characteristics
[129,130], but several examples displaying graded changes
in gene expression have recently been published [131,132].

The manner of gene expression might well be a key factor in
programs of cell differentiation or stimulus response.
Different regulatory setups of the Tet system allow a
transgene to be expressed in one or the other manner [133–
135], enabling not only an analysis of a gene’s function, but
also of its mode of expression. When tTA and rtTA are
expressed constitutively in mammalian cells and also in
S. cerevisiae, they drive transgene expression in a dose-
dependent, graded manner [133,135]. However, when rtTA
was expressed in S. cerevisiae under conditions of positive
feedback using an autoregulatory circuit, the cell population
was clearly divided into regulator-expressing and nonex-
pressing cell pools [135]. In mammalian cells, the combined
usage of tTS
G
and rtTA also led to bimodal expression of
the GFP reporter (see Fig. 3 in [134]). Although not
formally proven, we assume that a bimodal expression
pattern will not be observed for all repressor/activator
combinations, but only for those in which the sensitivity of
tTS for the inducer is lower than that of the rtTA allele used,
as is the case for tTS
G
(compare the dose–response curve of
tTS
E
and rtTA of Fig. 4 in [93] with the one for tTS
G
and
rtTA of Fig. 2 of [134]). This will ensure that rtTA is

preloaded with inducer and ready to activate transcription
the moment the dox concentrations needed for binding to
Fig. 5. The Tet toolbox. TetR modules and regulatory domains are
displayed with the possible combinations. The different binding func-
tions of TetR were coded in different shades of gray and placed at their
approximate position in the protein, but not drawn to scale. The TetR
variants characterized were classified in the corresponding module.
The regulatory domains that can be fused to TetR are coded in dif-
ferent shades of gray according to their viral, human, insect or fungal
origin. Note that not every possible combination of modules need
result in a transregulator with acceptable regulatory properties.
Ó FEBS 2003 Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3115
tTS
G
are reached and tetO is subsequently released. In
principle, only two regulatory states are observed: either the
tetO sites are fully occupied with tTS
G
and gene expression
is shut off, or they are saturated with the rtTA variant,
resulting in full transcriptional activation. The consequence
is a binary expression pattern of the target gene. While this
setup already works with rtTA, the effect should be even
more pronounced with rtTA2
s
-M2, as its inducer response
range overlaps completely with that of tTS
G
.
Highlighting the regulatory potential

and looking into the future
The properties and the adaptability of Tet regulation as
presented in the previous sections allow its use in many
different applications. We would like to demonstrate this
enormous variability by referring to a few key studies that,
in our opinion, highlight the potential of Tet regulation.
Regulation by tetracyclines is sensitive and efficient
enough to control target gene expression in pathogenic
organisms even when they have been injected into a
mammalian host. The role of individual genes in infection
and pathogenesis can, thus, be probed and their validity as
targets for therapeutic intervention determined in an in vivo
disease model [136]. This has not only been demonstrated
for trypanosomes [137,138], but also for common human
pathogens like Staphylococcus aureus [139] and Candida
glabrata [140]. In the fungus, squalene synthase [136] and
sterol 14a-demethylase [141] were, thus, shown not to be
ideal targets for antifungal development.
The successful expression of the diphtheria toxin A
subunit by tTA/P
tet
-1 in transgenic mice has demonstrated
the stringency of regulation that can be reached with the Tet
system [142]. Although mouse lines that carried the target
transgene were obtained at an approximately 10-fold lower
frequency than normal, those that were established regula-
ted the transgene efficiently. Induction of toxin expression
led to cell death and development of cardiomyopathies.
Stringent control of transgene expression using rtTA has
also been achieved in HeLa cells for the Shiga toxin B

subunit [143], for the proapoptotic gene PUMA in SAOS-2
and H1299 cell lines [144] and, using rtTA2
s
-S2 in transgenic
mice, for Cre-recombinase [145].
The strength of a true conditional system ) the possibi-
litytoswitchgeneexpressiononandoffatleisureand
repeatedly ) represents a powerful method with which to
explore the relationship between mutant protein expression
and disease progression. This has become evident upon
studying transgenic mouse models for cancer and neuro-
logical disorders. Here, the use of tTA and rtTA to control
expression of an oncogene revealed for solid tumors
[146,147] and for leukemias [148,149] that the oncogene is
not only necessary for tumor formation but also for tumor
maintenance, suggesting pharmacological inactivation of
oncogenes as a possible therapeutic strategy for cancer. This
assumption has been substantiated by the unexpected
observation that, after having gone through one cycle of
MYC-gene expression and silencing, reactivation of the
oncogene does not lead to tumor regrowth, but rather to
apoptosis [150]. Similar effects have also been found for
neurological disorders. In a conditional model of Hunting-
ton’s disease, mice expressing a mutated huntingtin
fragment in the brain demonstrated that its continuous
supply was needed to maintain the characteristic neuro-
pathology and behavioral phenotype, raising the possibility
that the disease may be reversible by targeting the causative
agent [151].
Regulation by the Tet system has also had a significant

impact on behavioral studies. Expression of constitutively
active mutant forms of the calcium/calmodulin dependent
kinase II or calcineurin in the brain of adult mice resulted in
altered synaptic plasticity and impairments in spatial
memory storage and retrieval, but these deficits were fully
reversed when transgene expression was suppressed
[84,152]. Because expression of the transgene was limited
to the hippocampus, this structure was additionally proven
to be the site responsible for the behavioral effects. In a
different example, knockout mice lacking the serotonin 1A
receptor show increased anxiety-like behavior which could
be rescued by conditional expression, but only if the
receptor was synthesized during the early postnatal period
in the hippocampus and cortex [153].
Nevertheless, improvement and additions to the Tet
system, among them the regulatory components, are still
possible and necessary. Promoter development has not
received the same degree of attention as the transregulators.
The number of tetO elements and their spacing [154], as well
as the linker sequence separating the operators [155] have
not been optimized yet. It remains to be seen if an ÔidealÕ
minimal promoter with no intrinsic leakiness supporting
very high-level activation can be identified or designed.
Fortunately, screens for regulators with improved prop-
erties can now be performed in eukaryotic systems [88]
and, as an example, the isolation of novel Tet regulators
which recognize nonantibiotically active tetracyclines or
even nontetracyclinic inducers, would be of great benefit.
They would not only facilitate gene therapy applications
which, at the moment, can be impaired by the use of

tetracyclines in anti-infective therapy or their misuse as
growth promoting additives to animal food. If these novel
inducers are not only ecologically safe, but also easy and
nonexpensive to manufacture, the inducer–regulator pair-
ing could also be useful in insect population control using
dominant, repressible, lethal genetic systems [156,157] and
might even introduce regulation by the Tet system to crop
plants. They would add to the repertoire of transregulators
and finally, since multiple dimerization and DNA-binding
specificities are already present, allow fully independent
expression control of more than one gene by the Tet
system.
A major experimental challenge will be to express a target
gene within its physiological window, which might depend
on environmental stimuli and even change during develop-
ment, since over- or underexpression often results in altered
phenotypes [131] or pathologies. While tc-controlled expres-
sion can mimic the natural level [146], this must not always
be the case. A solution might be precise promoter targeting
by tetO elements, to minimally interfere with gene expres-
sion. This will be difficult and will require extensive
knowledge about the influence of chromatin structure on
gene expression and its sensitivity to perturbation, partic-
ularly when regulatory regions are modified [158]. But, if
successful, this approach will provide an additional degree
of freedom to manipulate gene expression, as the existing
3116 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003
transregulators can be used to activate or silence gene
expression, in addition to and independent of the promo-
ter’s natural expression pattern.

Conclusion
The Tet system is the most widely used regulatory system
for conditional gene expression at the moment. The
increasing number of: (a) cell lines stably transfected with
tTA and rtTA; (b) cell lines harboring tTA or rtTA that
have been derived from transgenic mice; and (c) transgenic
mice expressing either the transregulators via cell-type
specific promoters or a target gene under P
tet
-1 control will
greatly facilitate genetic studies by allowing combination of
the existing components instead of having to generate all cell
and mouse lines, a costly and time-consuming process.
Ongoing improvement of the existing components as well as
the continuous addition of new components to extend its
applicability have turned the Tet system into a highly
versatile and flexible regulatory system that can be adapted
to many different applications. Starting from an extensive
knowledge-base of TetR structure–activity relationships
and the strength of the genetic screening and selection
systems in both pro- and eukaryotes, the Tet system is
becoming more and more capable of modeling the sophis-
ticated regulatory setups needed [48,51] to analyze complex
and multifactor biological processes in development and
disease, thereby not only improving our understanding of
living organisms, but also revealing novel and innovative
approaches to treat maladies.
Acknowledgements
This work was supported by the Bayerische Forschungsstiftung
through their FORGEN initiative, by the Deutsche Forschungsgeme-

inschaft through SFB473 and the Fonds der Chemischen Industrie
Deutschlands. We would also like to thank Dr Anja Knott and Felix
Kuphal for critical reading of the manuscript.
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