J. steroid Biochem. Vol. 27, No. l-3. pp. Y-14, 1987 0022-473 1/X7 $3.00 + 0.

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Printed in Great Britain. All rights reserved Copyright @ 1987 Pergamon Journals Ltd

Proceedings of the VII International Congress on Hormonal Steroids (Madrid. Spain, lYX6)

GENE REGULATION BY STEROID HORMONES

M. BEATO, J. ARNEMANN, G. CHALEPAKIS, E. SLATER

and T. WILLMANN
Institut fiir Molekularbiologie und Tumorforschung der Philipps-Universitlt, Emil-Mannkopff-Str. 1,
D-3550 Marburg, Federal Republic of Germany

Summary-The location, orientation, and structure of the hormone regulatory elements (HRE) in nine
hormonally modulated genes is described. Based on analysis of the contact points between the
glucocorticoid receptor (GR) and the DNA double helix within the HREs, a model for the interaction is
proposed in which a dimer of the receptor in head-to-head orientation binds to the inverted symmetry
element of the HRE. The relationship between the regulatory elements for glucocorticoids and
progesterone in the long terminal repeat region (LTR) of mouse mammary tumor virus (MMTV), and in
the promoter region of the chicken lysozyme gene, indicates that the recognition mechanism for both
receptors is similar but not identical. Curiously, the hormone ligand is not an absolute requirement for the
GR to bind its HRE, though it influences the kinetics of the interaction. Other possible functions of the
hormone in uiuo are discussed, as well as the molecular mechanism responsible for transcriptional
regulation after receptor binding to the HRE.

INTRODUCTION teraction with the same receptor protein modulates

Steroid hormones modulate gene activity in a tissue-
and gene-specific manner, which means that a parti-
cular hormone can induce or repress different sets of
genes depending on the cell type upon which the
hormone is acting. A typical example of this is the
differential effect of estrogens in the chick oviduct
and in the liver. In the oviduct a set of genes for
egg-white proteins including ovalbumin, lysozyme,
ovomucoid, and conalbumin are induced by
estrogens [l], whereas in the liver vitellogenin
and lipoproteins are expressed following estra-
diol administration [2]. Transferrin, the hepatic
equivalent to conalbumin, is expressed constitutively
in the liver and no influence of estrogen treatment is
observed [3]. An example of differential gene reg-
ulation by steroid hormones in mammals is the
regulation of uteroglobin gene expression in
different rabbit tissues [4]. In the uterus the protein is
induced mainly by progesterone, whereas in the
oviduct estrogens are the main inducers, and in the
lung glucocorticoids enhance uteroglobin synthesis.
There are also indications that androgens can induce
uteroglobin expression in the male genital tract [S].
In addition to these tissue-specific genes there are,
however, genes like the metallothionein genes that
are expressed in many different cell types and are
regulated by glucocorticoids in all cells that have the
appropriate receptors [6].
All these various effects of steroid hormones are
mediated by specific proteins called receptors, which
appear to be very similar in every tissue. In fact, the
genes for the receptors of glucocorticoids, estrogens
and progesterone have been cloned and appear to be
single copy genes [7-91. This means that there is
probably only one receptor for each particular hor-
mone in one organism, and therefore, one has to
assume that the same hormone acting through in-
different genetic programs in different cells. Thus,
additional tissue- and gene-specific factors have to
be involved in hormonal regulation of gene expres-
sion in order to account for the observed cell and
gene specificity of the hormonal response.
On. the other hand, different steroid hormones
acting upon the same cell type in some cases are able
to regulate the same set of genes. So, for instance,
during secondary stimulation of the chick oviduct all
four classes of the steroid hormones, estrogens,
progestins, androgens and glucocorticoids, are able
to induce the expression of egg-white proteins [lo].
Thus, the oviduct cells show relatively little hor-
monal specificity in terms of egg-white protein gene
expression. In other systems, however, only one or
two particular hormones are active. For example,
uteroglobin can be induced in the rabbit endo-
metrium mainly by progesterone and to a lesser
extent by estrogens, but not by glucocorticoids,
although there is a glucocorticoid receptor in
endometrial cells [4]. Since the responsive cells ap-
pear to be an homogenous population one is forced
again to postulate the existence of hormone-specific
additional factors that will determine responsiveness
to a particular hormone. These factors could either
act at the level of the receptor, by modification of the
protein, or/and at the level of the gene, by providing
a particular array of the gene in chromatin.
This brief introduction makes clear that hor-
monal regulation implies a wide spectrum of tissue,
gene and hormone specificities and that we are
dealing with a complex regulatory network involving
several factors. We know almost nothing about the
molecular basis of this complexity, but there are now
well-defined model systems and practical strategies
that could be followed in trying to uncover all these
very complicated regulatory pathways.

9
10 M. BEATO et al.

In this paper we will first summarize what we are recognized preferentially by each of the hormone
know about the interaction of the steroid hormone receptors may differ considerable ([ 171, see below).
receptor with DNA regulatory elements and then Some of the above-mentioned properties of hor-
illustrate some of the systems that we are using to monally regulatory elements are reminiscent of the
study differential gene regulation by glucocorticoids behaviour of so-called enhancer elements, suggest-
and progestins. ing that the HREs act as hormone-dependent en-
hancers [18]. Since the genes we have studied are
REGULATORY ELEMENTS IN HORMONALLY from several different species, and no species barrier
MODULATED GENES has been detected in gene transfer experiments, we
A summary of the location and orientation of can conclude that there is a great conservation of
hormonally regulatory elements (HREs) in nine HREs through evolution.
genes is shown in Fig. 1. The location varies between
2.6 kb upstream (uteroglobin gene) and 0.1 kb
MODEL FOR RECEPTOR BINDING TO DNA
downstream of the initiation of transcription (growth
hormone gene), and both orientations are found. In A comparison of the nucleotide sequence of 22
most of the genes studied, the elements are present in binding sites for the glucocorticoid receptor that
more than one copy, although the contribution of have been mapped so far, allows to derive a 15mer
each individual element to the hormonal response is consensus sequence (Fig. 2). This sequence exhibits
very variable [l l-141. In the case of mouse mam- an imperfect inverted symmetry centered around
mary tumour virus (MMTV), the human metallo- base pair number 8. The right half of the consensus
thionin II, (hMT HA), the chicken lysozyme gene sequence containing the hexanucleotide motif S-
(chLYS), and the rabbit uteroglobin gene (rUG), the TGTYCT-3’ is very well preserved, suggesting the
same or overlapping DNA regions are recognized by interaction of a receptor molecule with this part of
the glucocorticoid and progesterone receptors, and the sequence as the first step in receptor binding to
the HREs can mediate induction by both hor- DNA. The type of contact detected by methylation
mones ([13, 15, 161 and unpublished observa- protection studies indicates that the receptor ap-
tions). Nevertheless, the nucleotide sequences that proaches the DNA double helix through the major

-600 -500 - 400 - 300 - 200 - 100 C!P l 100

I 0 8 I I I I
GENES

WV- LTR

hYT- IA

PG GP 1
ch LYS - P 1
- _

Fig. 1. Location and orientation of hormone regulatory elements (HRE) in: MMTV-LTR-long
terminal repeat region of mouse mammary tumor virus[l9]; hMTII,-human metallothionein IIA
gene [12]; chLYS-chicken lysozyme gene [13]; hGHI-human growth hormone gene I [33];
rUG-rabbit uteroglobin gene [ 161; Mr-DNA-mouse ribosomal RNA gene [20]; RTO-rat tryp-
tophan oxygenase gene (Danesch, Gloss, Schiitz and Renkawitz, unpublished); MSV-Moloney murine
sarcoma virus [14]; RTAT-rat tyrosine aminotransferase gene (Gloss, Schmid and Schultz, unpub-
lished). The genes are aligned according to the main site for transcription initiation ‘CAP”. The scale on
the top refers to the distance from “CAP” given in nucleotides. The HREs are indicated by boxes. The
horizontal arrows show the orientation of the hexanucleotide motive S-TGTYCT-3’. “GP” indicates
sites that are recognized by both the glucocorticoid and the progesterone receptors.
 Gene regulation by steroid hormones 11

COfiPARISON OF BINDING SITES FOR THE GLUCOCORTICOID RECEPTOR

(A) 10 12 14
123456789 11 13 15
. . . . * * * . . ., . . . . .
nTV I ~TTACAAACT~TTCT
. ,.
HMT IIa I $~TA~ACTGT~TCCT
. ^
RUG I T:TTCACTCT;TTCT
,. .
RUG II CGGACACGGAETCCT
^ I
tlRib CTGACACGCTkTCCT
* ^
RUG III TGTCAGTCTTaTTCT
,.
flTV IIa GGTATCAAATZTTCT
,.
DSC7 GATTTGATCTETTCT
.
CLYS I ~ACAACTGTA~AACA
^ ,.
CLYS 11 AATTCCTCTaTGGCT
^ ^
rTAT I CGTACAGGATiTTCT

rTAT 11 GGACTTGTTTETTCT

(B)

HGH I GGCACAATGTGTCCT
CVIT II GGATCAATGTGTTCT
rT0 I TGCACAGCGAGTTCT
rT0 II CTTTCATGATGTCCT
nsv I GGGACCATCTGTTCT
nsv II TGTTCCATCTGTTCT
nsv 111 CTGTCTCTCTGTTCC
cov I TTTTCTGCCTGTTCT
cov 11 GTCACGTCTTGTTCT
H?lTIIa II CCTCCCTCCTGTCCT

123456789 11 13 15

CONSENSUS: cc;ncniNN;i;;c;’

(22 SITES)

PROHIBITED BASES: ac-GG----CCCAGG

9AaaAa
t99 c

Fig. 2. Nucleotide sequence in the centers of 22 binding sites for the glucocorticoid receptor. In addition
to the nucleotide sequences of the HREs depicted in Fig. 1, the following sites have been included: (A)
DSC7, a binding site for the glucocorticoid receptor (GR) found in the promoter region of an ecdysone
responsive gene of Drosophila [20]. (B) CVITII, a binding site for the GR overlapping the estrogene
responsive element of the chicken vitellogenen II gene [20]. COVI and COVII are two binding sites for
the GR found in the promoter region of the chicken ovalbumin gene (unpublished). All the sequences are
aligned according to the hexanucleotide motif. The numbers below each nucleotide in the consensus
indicate the frequency with nucleotide in the consensus indicate the frequency with which this particular
base is found at this position. Prohibited bases: Capital letters are used when the indicated base is never
found at a particular position. Some letters when the indicated base is found only once.

groove [ 191. The left half of the consensus sequence MOLECULAR MECHANISM OF TRANSCRIPTIONAL
is less well conserved in different binding sites, ACTIVATION

suggesting that a weaker interaction of another How could binding of the receptor to the HRE
receptor molecule with this part of the sequence influence the transcriptional efficiency of adjacent
could be stabilized by protein-protein interaction promoters? In cases in which the HRE is located
with the already bound receptor molecule (Fig. 3). relatively close to other essential elements of the
The simplest model, therefore, would be to postulate promoter such as the TATA-box, it is conceivable
a dimer in head-to-head orientation as the binding that a direct protein-protein interaction between the
form of the receptor. Since the distance between the receptor and transcription factors could mediate the
centers of the two blocks of contact points is about modulation of transcriptional efficiency by the hor-
9-10 base pairs, the two receptor monomers would mone. A model of this kind has been postulated to
be on the same face of the double helix in two explain the transcriptional enhancement of the SV40
subsequent turns of the helix [ 19,201. This model is early promoter by its natural enhancer element [21].
also supported by the results of binding experiments We also have preliminary evidence indicating that in
using the gel retardation technique and synthetic the mouse ribosomal promoter binding of the
oligonucleotides (Chalepakis, unpublished), and glucorticoid receptor to the region immediately ad-
would account for the orientation-independent jacent to the initiation of transcription results in a
action of HREs. 3-5fold enhancement of transcriptional efficiency
12 M. BEATO et al.

L-J
/ /
1 GGTACANNNTGTYCT I / TATA
n

Fig. 3. Model for glucocorticoid receptor binding to the HRE. The receptor is represented as a three-
domain protein with the hormone ligand (H) bound to the steroid binding domain. In the first step of
the binding reaction a monomer of the receptor binds to the conserved hexanucleotide motif and
stabilizes the binding of a second receptor molecule to the less well conserved half of the consensus
sequence. Binding of the receptor dimer influences, through unknown mechanisms, the efficiency with
which transcription factors (E) utilize an adjacent promoter.

in vitro (Schoenberg, Scheidereit, Grummt and ROLE OF THE HORMONE ON RECEPTOR

Beato, unpublished). In hormonally modulated BINDING TO DNA
genes transcribed by polymerase II we only have According to the classical concepts on steroid
circumstantial evidence for the involvement of pro- hormone action, binding of the steroid to the recep-
tein-protein interactions in the case of the MMTV tor in cytoplasm leads to a change in the structure of
(see below). the hormone-receptor complex that enhances its
Another possible mechanism of transcriptional affinity for chromatin and DNA[22]. Thus, the
regulation could be mediated by changes in secon- hormone is the actual trigger of the conformational
dary structure of the DNA in relevant promoter changes that enable the receptor molecule to inter-
sequences following receptor binding to the HRE. In act with the HRE. We have investigated this ques-
principle, these changes in structure could be pro- tion using pure cytosol as the source of receptor and
pagated in both directions along the DNA double analyzing the binding of the receptor in the presence
helix and this would explain why the HREs can act in and in the absence of hormone. We found that the
both orientations and relatively independently of the receptor free of any hormone can be activated at
distance from the initiation of transcription. If this 25°C to a form that interacts specifically with the
model holds true, binding of the receptor to the HRE same nucleotide sequence within the MMTV-HRE
should influence the topology of close circular as detected with the purified hormone receptor
plasmids. In fact, we have found that binding of the complex (Willmann and Beato, unpublished). In fact,
glucocorticoid receptor to the regulatory element of kinetic measurements show that the steroid-free
MMTV introduces topological changes in close receptor dissociates much more slowly from the
circular relaxed DNA molecules, leading to the DNA than the steroid-loaded receptor. These results
introduction of positive supercoiling (Carballo and show that, at least in vitro, the hormone is not
Beato, unpublished). This topological effect cor- required for the receptor to adopt the conformation
relates with the specific binding of the receptor and that permits its binding to the HRE. What could
can be partially eliminated if a part of the HRE is then be the function of the hormone in viva? One
deleted from the plasmid. We still do not know the possibility is that the receptor in viuo is asso-
relationship between the topological changes that ciated with some other protein factors that
follow receptor binding and the regulation of tran- prevent its translocation into the nucleus and the
scription, but we have observed that the proges- subsequent interaction with the DNA. This type of
terone induction of an adjacent promoter by the interaction with other factors could have been lost or
MMTV-HRE is highly dependent on the topology of weakened during the preparation of the cytosol. In
the transfected DNA in transient expression this model the function of the hormone in viuo would
experiments (unpublished observation). However, be to dissociate the receptor from these modulating
even after transfection of linear DNA fragments a factors in order to allow the DNA binding domain of
residual hormonal response is observed, suggesting a the receptor molecule to interact with the chromatin.
minor contribution of protein-protein interaction of Another possibility is that binding of the hormone to
the inductin mechanism. the receptor leads indeed to a conformational
 Gene regulation by steroid hormones 13

change of the receptor molecule, not in terms of a the homology of the DNA binding domains will
DNA binding region being exposed but rather by extend to the receptors for other steroid hormones.
exposing a so-called nuclear translocation signal,
similar to those described recently for the T-antigens CONCLUSIONS AND PERSPECTIVES
of SV40 and polyoma [23,24]. Still another pos-
After the establishment of the existence of HREs
sibility, which takes into account the kinetic
as the target for receptor binding to the DNA of
differences mentioned above, would be that in the
hormonally regulated genes, and after the confirma-
absence of the hormone the receptor binds to DNA
tion of the need for these regulatory elements in gene
but because the dissociation rate is too slow the
transfer experiments by mutational analysis, the
receptor does not find the appropriate HREs. Thus
main questions still open for our present understand-
in the absence of the hormone the receptor would be
ing of the mechanism of gene regulation by steroid
associated unspecifically to the DNA in chromatin.
hormones concern the elucidation of the gene, hor-
After binding the hormone, the complex would
mone and tissue specificity of the response. In that
acquire kinetic properties that allow the receptor to
context, many physiologically significant questions
find the regulatory sequences and activate the cor-
have to be answered. So, for instance, the function of
responding genes.
the hormone ligand is still unclear, as we know that
the hormone itself is not an absolute requirement for
the receptor to adopt the DNA binding confor-
DIFFERENTIAL GENE REGULATION BY
GLUCOCORTICOIDS AND PROGESTERONE mation. The study of the interaction of the receptor
with other cytoplasmatic or nuclear factors will be
As mentioned above, several of the HREs are able
one of the fields that may provide answers to some of
to bind the receptors for glucocorticoids and pro-
the open questions. A part of this study will be the
gesterone and respond to both hormones in vim. In
elucidation of the interaction between the receptor
the case of MMTV, gene transfer experiments in
and the 90-kDa heat-shock protein that has been
human mammary tumour cells show that the main
shown to be associated with the non-activated forms
inducing steroid is progesterone [25]. In fact, in the
of the receptor in cytosol[27-291. Another very
cell line T47D, which has constitutively high levels
important aspect will be to study the influence of
of progesterone receptor and of glucocorticoid
receptor phosphorylation on the different functions
receptor, no glucocorticoid induction of MMTV
of receptor protein, including steroid binding,
carrying plasmids was observed [23]. Mutational
activation, binding to DNA, and modulation of
analysis of the MMTV HRE shows that the in-
transcriptional efficiency. As the receptors are
dividual binding sites for the receptor exhibit a
phosphoproteins that exhibit several phosphoryl-
different functional relevance in respect to gluco-
ation sites, a complex pattern of modulations
corticoid as compared to progesterone induction.
through specific phosphorylation could be
Therefore, although the two receptors are interac-
envisaged [30-321.
ting with the same region of the DNA they may be
The final goals of all these studies will be to
recognizing different features of the sequence, or at
develop a cell-free transcriptional assay in which the
least utilizing the same nucleotide sequence in
modulation of transcription by the receptor protein
different ways.
could be mimic, in order to analyze the functional
A similar situation pertains for the interaction of
interactions of the receptor with different factors in
glucocorticoid and the progesterone receptor with
vitro. The availability of cloned receptor cDNA
the regulatory element in the chicken lysozyme
opens the way for a mutational analysis of the protein
promoter [17]. In this case, both proteins bind to a
and for the fine mapping of functional domains. The
similar region of the DNA but the length of the
developments in these and related fields during the
footprints obtained with each of them is different,
last few years have placed gene modulation by
and the progesterone receptor protects different
steroid hormones in the center of attention of those
guanines against methylation by dimethyl sulfate as
interested in understanding eukaryotic regulation.
compared with the glucocorticoid receptor [24].
A comparison of the amino acid sequence of the
glucocorticoid and progesterone receptor as derived
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