BIOLOGY
OF REPRODUCTION
54-69
40,
(1989)
Steroid Action
Roles of Regulatory
Possible
T. C. SPELSBERG,2
on Gene Expression:
Genes and Nuclear Acceptor
Sites’
C. RORIES,
J. J. REJMAN,
A. GOLDBERGER,
C. K. LAU, D. S. COLVARD,
and G. WISEMAN
Department
Mayo
of Biochemistry
Clinic
and
Mayo
and
Graduate
Rochester,
Molecular
School
Minnesota
K. FINK,
Biology
of Medicine
55905
ABSTRACT
There
The
has
been
structures
regulated
factors
and
genes,
i.e.
and function
of
steroid
regulation
a novel
sion of
of
in
the
steroid
steroid-altered
sites,
acceptor
code
for
progress
including
in the
acceptor
exciting
functions
understanding
receptor
response
of gene
sites,
for
nuclear
acceptor
gene
expression.
sites
have
been
suggested.
nuclear
regulatory
proteins
are
receptors
steroid
roles
A very rapid
has recently
the
mechanism
domains
currently
transcription
steroid
sites for
Possible
steroid
action
model
utilizing
regulatory
genes
(early
genes)
of
protein
elements,
regulation
nuclear-binding
these
of
the
various
being
are just
are
action
been
defined.
investigation.
is crucial
for
DNA-binding,
of steroids
described.
action
as the
elucidated.
being
under
receptors
of specific,
of
as well
of
various
steroid
The
roles
The
The
hormones.
domains
of steroid-
of
nature
transcription
of
the
composition,
understanding
chromatin
the mechanism
proteins
in
on the expression
of proto-oncogenes
Using
this information,
we have
The
latter
would
occur
via binding
of these
regulatory
elements
neighboring
these
late genes.
This model
many
laboratories
on the action
of steorid
hormones
INTRODUCTION
comprehensive
reviews
of the
of
these
that
proposed
“regulatory
genes”
whereby
the steroids
would
rapidly
alter
the
whose
protein
products
would
return
to the nucleus
to regulate
pression
of structural
genes
(late genes).
transcription
factors)
to the steroid-regulatory
many
of the characteristics
reported
from
sion.
nuclear
location,
expresthe ex-
proteins
(e.g.
would
explain
on gene
expres-
general
area
of steroid
This paper,
outlining
the plenary
lecture
presented
at the 1988
SSRB
meetings,
presents
an update
on
the mechanism
of action
of ovarian
steroids,
estrogen
(E) and progesterone
(P), on gene
expression.
It is
action
on gene expression,
the readers
are referred
to
the following
references:
Katzene!!enbogan
(1980),
Moudgil
(1987),
Rories
and Spelsberg
(1989),
Roy
and Clark
(1987),
Spelsberg
and Kumar
(1987),
and
directed
Thrall
at the
action.
utilizing
followed
novice
to the
field
of steroid
hormone
Brief
reviews
of various
aspects
of the field,
recent
publications,
are presented.
This
is.
by a review
of research
in this laboratory
on
et a!. (1978).
Mechanism
Hormones
of Action
of Steroid
on Gene Expression
1) the rapid
steroid
regulation
of proto-oncogene
(c-myc)
expression,
2) the possible
role of regulatory
genes
in the action
of steroid
hormones,
and 3) the
Figure
action
on
1 outlines
the
gene expression.
circulate
in
chromatin
with
blood
binding
proteins
(BP).
The free steroids
diffuse
in and out of a!! cells (Step
1 of Fig. 1), but
are retained
only
in target
cells via complexes
with
intranuclear
binding
proteins
termed
receptors
(R,
Step
2 of Fig. 1). These
receptors
are steroidand
DNA
authors
acceptor
complexes.
have
cited
omitted
action.
some
steps
‘This
Grants
2
work
of
was
involving
outlining
steroid
supported
HDO7 108 and
Reprint
requests.
acceptor
Due
to
space
only
example
important
Models
lated
sites
CA90441,
areas
the
action
in part
and
steroid
known
and
by NIH
the
limitations,
references,
of
are
Mayo
protein-
hormone
the
depicted.
Grant
the
and
HD9140-P1,
For
specumore
tissue-specific
1980)
with
the
a
blood,
genera!
Ovarian
both
(Thrall
et al.,
concentration
free
1978;
of
model
steroid
and
of steroid
hormones
as complexes
Katzenel!enbogan,
10,000
to 60,000
molecules
per cell. Each
receptor
binds
its respective
steroid
with
high
affinity,
displaying
equilibrium
dissociation
constant
(KD)
in the range
of 10
to
10b0
M. Once
bound
by the steroid
hormones,
the
Training
Foundation.
54
STEROID
ACTION
STEROID
ON
GENE
ACTION
55
EXPRESSION
PATHWAY
Taraet cell
S
steroid
=
#{149}
R (receptor)
2
1
‘5’
S
4mm
S - BP
#{149}
“SR”
6
S
4
SR
-
Acceptor
3
‘.
Pre-m
5mm
site
#{149}
SR
:hr
ANA
Gene
1-4hr
‘
m
RNA
Protein
“
Modified
*.
protein
49
PhysiologIcal
effects
6 -24 hr
FIG.
1. Model
outlining
receptor
termed
molecules
“activation”
to
with
bind
the
general
undergo
(Step
high
affinity
mechanism
of action
hormones
in target
change
them
target
to
on
and
specific
sites
sites
per cell available
with
an even
larger
the
sites (Step
4 of
events
occur
as
into an animal.
10,000
nuclear
for binding
number
of
steroid
acceptor
tissues.
Steroids,
a conformational
3), which
allows
chromatin,
termed
nuclear
“acceptor”
Fig.
1) (Thrall
et a!., 1978).
These
early
as 5 mm after
steroid
injection
It is estimated
that there
are 4000
to
acceptor
receptors,
of steroid
therefore,
cells
by
proteins
by
organism,
of an
(Rories
The
binding
of
steroid
post-transcriptional
affecting
the
levels
receptor
results
in
5 of Fig.
alteration
steps
are
the
major
of steady-state-specific
the
1).
of
factors
RNAs
(Steps
6 and 7 of Fig. 1) and proteins
(Steps
8 and 9
of Fig.
1) (Katzenellenbogan,
1980;
Thrall
et a!.,
1978;
Moudgil,
1987).
As depicted
in Figure
1, the
steroid-induced
changes
in messenger
RNA
(mRNA)
levels
of most
structural
genes
occurs
at 1-6
h after
steroid
injection.
The
subsequent
physiological
effects
of steroids
in cells are observed
by 12- 24 h
after
steroid
treatment.
steroid
action
pathway
later in this review.
Specific
are
described
aspects
in more
of
this
detail
and
the receptor
first
nuclear
alterations
been
tories,
Receptor
1989).
to
these
event
in gene
mRNAs
represent
which
represents
the receptor
species
for any
to be the same in different
it is speculated
Spe!sberg,
by the
authors
binding
sites (acceptor
tissues
probably
play
responses
to steroids
Further,
the
binding
a
of
acceptor
sites appears
to be the
preceding
the
steroid-induced
transcription.
Therefore,
the
of the initial
nuclear
binding
sites,
sites,
is extremely
important
and has
an area of intense
investigation
as described
in more
detail
Structure
of both
of
gene regulatory
proteins
of receptor
molecules
are
in the nuclear
different
target
tissue-specific
characterization
i.e. “acceptor”
physiology
receptors
of steroids,
tissues
Spelsberg,
to nuclear
acceptor
sites
of gene
transcription
(Step
instances,
a steroid-induced
binding
levels
steroid
intracellular
The activity
signal.
Since
are thought
that differences
sites)
between
role
in these
1985).
the
the
the
The
the external
given
steroid
sites that
appear
to be “masked”
(i.e., not available
for binding
receptors)
in the intact
chromatin
(Thral!
et a!., 1978;
Spelsberg
et al., 1983;
Martin-Dani
and
complexes
alteration
In some
controlling
in cells.
one of the first
to be identified.
triggered
regulate
and
Our
knowledge
of
ovarian
steroid
receptors
the cloning,
sequencing,
cDNAs
encoding
the
from
chicken
(Kaufmann
later
in many
laborain this review.
Function
the
structure
and function
of
has grown
significantly
with
and
functional
analysis
of
progesterone
et a!.,
1980;
receptors
Conneely
(PR)
et
SPELSBERG
56
a!.,
1986)
the
from
and
cDNAs
human
chicken
rabbit
(Loosfelt
encoding
(Greene
the
et a!.,
(Krust
et
et a!.,
a!.,
1986),
and frog
lines the possible
steroid
receptor
(Weiler
functions
proteins.
et
acid
of
ER
1987),
sequences
species
residues
among
binding)
species,
domain
domain,
In addition
binding
different
The
are
marked
amino
thought
DNA-binding
elements
are called
of
their
“fingers”
lIlA (TFIIIA),
(Klug
and
Rhodes,
role
with
et
Figure
domains
of the
different
al.,
2 outof the
amino
animal
of amino
acid
highly
conserved
the
other
domains
the
high
C-terminal
(steroid-
homology.
evolutionary
conserva-
family
(species)
of receptor,
the
to a lesser
extent
the
steroid-
acid residues
of the
to fold
into two
structures.
in
of
to
DNA-binding
adjacent
pro-
These
DNA-binding
similarity
binding
factor
A
(White
as
(ER)
1987),
are
highly
conserved
between
of steroid
receptors
(Green
et a!.,
truding
1988).
the
domains
classes
1987).
domain
and
very
a single
and
as well
(Green
et a!., 1987).
Two
domains,
the
central
show
to
tion within
DNA-binding
from
“blocks”
ER are
variability
conserved
(DNA-binding)
mouse
a!., 1987).
of various
Alignment
several
of the
warm-blooded
showing
relative
of
these
highly
tion
the
shows
that
(domains)
1986),
estrogen
receptors
1986;
Green
et a!.,
structural
“fingers”
the
in recogni-
zinc-stabilized
DNA-
Xenopus
laevis
transcription
which
contains
multiple
fingers
1987;
DNA
Evans
binding
and
for
DOMAINS
Ho!lenberg,
“zinc
finger”
OF STEROID
ET AL.
domains
However,
has been
substantiated
such “DNA-binding”
broadly
applied,
has
been
also
interactions
since
for several
proteins.
functions
may be too
this
shown
to play
(Giedroc
et a!.,
type
a role
Franke!
Frankel
such
fingers
Although
whereas
steroid
receptors
the role of these structures
by current
zinc-stabilized
binding
of the
proteins
Recent
involved
studies
1988).
utilize
SR to transcription
results
in the activation
to involve
a change
in
activation
tightly
bind
et
1978;
a!.,
1987d).
which
leads
of
to induction
two.
DNA
or acceptor
receptors
are localized
nucleus
(Jensen,
1987;
The binding
by steroids
of receptors,
the structure
et
is known
receptors
with
Pabo,
sites of SR.
evidence
that
which
of the
appears
receptor
due to the phosphorylation
(Kanury
and
Fox,
1987).
Spelsberg
little
understanding
steroid
receptors
sites
acceptor
strong
results
in the ability
to specific
nuclear
Little
steroid
contain
just
in binding
factors
in the nuclear
have provided
possibly
molecule
and
Also,
DNA-binding
multiple
(5-9)
zinc
data,
it is, therefore,
possible
fingers
could
be used in the
unoccupied
ovarian
steroid
predominantly
in the
cell
Welshons
and Jordan,
1987).
molecule,
receptor
of structure
in protein-protein
1987;
1988;
proteins,
is supported
that these
et a!.,
as TFIIIA,
general
of the
acceptor
a!.,
1983;
about
the
are activated.
how
their
and/or
repression
receptor
to
sites (Thrall
Spelsberg
et a!.,
mechanism
We also
the interaction
specific
nuclear
of the
The
by
have
of active
acceptor
of transcrip-
RECEPTORS
NH.
COOH
11(B)
1(A)
Regulatory domain
Hypervariable;
attenuates
determines
transcription
transcription;
gene-specific
FIG.
2. Domains
of Steroid
Receptors.
The general
functions
and conservation
of
Green
et at., 1986,
1987;
Krust
et al., 1986;
DNA-binding
domain
Highly conserved
among all steroid
receptors; includes
transcriptional
control
This scheme
outlines
each of these
domains
Loosfelt
et at., 1986;
Ill (C)
IV (D)
“Hinge domain’S
Steroid-binding domain
Highly conserved
For binding
other proteins
and may involve
negative control
of transcription
among animals
within same
receptor species
the 4 general
domains
(l-lV
or A-D)
of the
is listed
at the bottom
of the figure
(Kaufmann
Weiler
et al., 1987;
Adler
et al., 1988).
steroid
et al.,
receptor
protein
molecule.
1980;
Conneely
et at., 1986;
STEROID
tion
of
genes.
The
receptor
tact with
the DNA
during
this process.
DNA
binds
binding
domain
to specific
DNA
domain”
proteins
sion
et a!.,
purposes
of steroid
sequences,
receptor
molecules
and 2) the “hinge
of
brevity,
examples
steroid
action
of transcription
on
are
of
binds
to other
of gene expres-
to recent
steroid
Katzenellenbogan
(1980),
mention
different
only
types
reviews
of this
Thral!
Tata
et
al.
et a!. (1986),
of
1979;
reported
gene
1980).
Steroids
expression
at
of transcription
and/or
post-transcriptional
the
latter
including
effects
on mRNA
mRNA
stabilization,
protein
processing/secretion.
The
depend
on the steroid-target
Early
genes
wide
versus
are
late
gene
regulated
variation
at
in lag
depicted
response”
in Figure
3, the “early”
genes
show
an immediate
after
been
level
protein
to
Role
gene
bind
5 mm
et a!.,
1988)
early-induced
Gorski,
cancer
after
(see
genes
1970),
the
cells (Brown
progesterone
also
of
pS2
et
by
1-6
h after
when
factors
minutes
time
in
in
steroid
response
vitellogen-
of
90
mm
or
(Baker
example
occurs
and
in the
avian
is a 1- to 3-h lag period
between
or estrogen
bind
to nuclear
and
lysozyme
1976;
Schutz
at specific
tion
within
shown
changes
steroid
liver
administration
Another
of transcription
expression.
genes
a lag
genes
class
which
and the increase
in the mRNA
levels
for the egg white
proteins
ovalbumin,
et a!.,
sites
cells
(Harris
et
et al., 1978).
factors
Transcription
in or near
are exposed
in steroid
factors
several
have
sites
1975;
regulation
appear
to
steroid-regulated
to steroids.
(proteins)
at specific
a!.,
of
Two
been
within
transcrip-
shown
to bind
the glucocorti-
coid/progesterone-sensitive
promoter
of the mouse
mammary
tumor
virus-long
terminal
repeat
(MMTVLTR)
when
transcription
by this promoter
is induced
with glucocorticoid
(Cordingley
et al., 1987a).
These
particular
factors
appear
to be present
before
hor-
There
steroid
in rate
Spelsberg,
is a
adminis-
of transcrip1989).
As
or more
change
Steroid
Steroid
induced
events
I
later
in this
review).
rat uterus
(DeAngelo
gene of MCF-7
a!., 1984),
the
human
c-myc
(Fink
The
and
breast
gene in
binding
Nudear
bound
I
I
to receptor
regulation
n.jcl
/mRNA
/(LATE
receptor
itr
“primary
in steady
administration
j Steroid
/j
state
mRNA
levels
that
can be measured
within
15
mm after steroid
administration.
There
are only a few
examples
of early genes:
the c-myc
proto-oncogene
in
the avian
oviduct
shows
a decrease
in mRNA
levels
within
estrogen
acceptor
sites,
genes
coding
of
As
significant
only
displays
where
there
progesterone
appears
in question.
periods.
between
tration
and measurable
changes
tion of many
genes
(Rories
and
which
oviduct,
the time
have
the
and
levels
the
and
in transcription
estrogen
admin-
One example
of late
induction
of Xenopus
mRNA,
ovomucoid,
Palmiter
changes
after
mm
“secondary-control.”
late genes
show
(1978),
Moudgil
rapid
15
bulk of the known
structural
by steroids
represent
a second
genes,
called
“late”
genes,
steady-state
more
et a!.,
1987),
and
et a!., 1987; Travers
Steroid-responsive
different
times
be under
3, these
administration.
is the estradiol
in
(Dubik
(Murphy
show
within
particu-
events,
processing,
synthesis,
site of action
tissue system
responses.
1987)
levels
1978).
and tissue
growth
and therefore
must
the expression
of a multitude
of genes
(Thrall
et al., 1978;
O’Malley
et a!.,
Katzenel!enbogan,
to regulate
Knowler,
or mRNA
Shapiro,
(1987),
Roy and Clark
(1987),
Spe!sburg
and Kumar
(1987),
Rories
and Spelsberg
(1989).
Although
glucocorticoids
have
been
reported
to alter
the levels
of
only
a few protein
species
in certain
target
cells in
culture
(Ivarie
and O’Farre!,
1978),
the action
of the
sex steroids
(estrogens
and androgens)
induce
cell
differentiation
certainly
affect
in target
cells
57
cancer
cells
in rat uterus
mRNA
as well as the role
further
information,
action:
breast
N-myc
may
Figure
will
the
gene expression
factors.
For
referred
aspect
we
of
EXPRESSION
istration.
In contrast,
the
that are regulated
of steroid-responsive
of Gene Expression:
Factors
representative
readers
con-
1988).
Steroid
Regulation
Role of Transcription
For
makes
ON GENE
with other
proteins
suggest
that
1) the
of steroid
receptor
molecules
in the process
of regulation
(Adler
!ar
presumably
and possibly
Recent
data
ACTION
?
. Chromatin
stnactural
I
/
change
/
of
synthesis
GENE)
/
Steroid
regulation
or
-
#{149}
Regulator
gene/protein
activity
(EARLYGENE)J/
o
[3H] Stoid
i
events
Used
I
01
/
synthesis
/
I
/
8
2
Time (hours)
FIG.
3. Timing
of the
sion:
early
vs. late genes.
Spelsberg
et at. (1987d).
/
3
after steroid
in the steroid
with
permission
injection
action
on gene
of the publisher
expresfrom
58
SPELSBERG
mone
LTR
stimulation,
DNA,
until
vitellogenin
gene,
base pair
stimulate
the
the
but
do
steroid
a factor
not bind
is added.
was
shown
element
in the third
transcription
of the
absence
of
estrogen
DNA
in
somehow
domain
receptor
(Jost
treated
liver.
the
factor
1987).
The
to the
although
study,
to
in
1987).
to bind
factor
but
the
et
to the
is active
not
chicken
al.,
gene promoter
with
reporter
containing
Interestingly,
et a!.,
liver,
(Renkawitz
chicken
ovalbumin
were
transfected
cell lines
receptors.
rooster
In a recent
promoter
to a 14
factor
appears
to be
and the addition
of
et a!.,
estrogen-stimulated
gene
to bind
activates
(Jost
MMTVchicken
intron
and appears
gene
in vitro,
even
This intron-binding
transcription
present
in the unstimulated
cells,
estrogen
to the
In the
in un-
lysozyme
1982)
and
the
(Tora
et al., 1988)
genes
into
different
glucocorticoid
and progesterone
these
genes
only
responded
steroid
in certain
cell
all cell types
contained
types
the
but not
receptors
others,
(Tora
et a!.,
1988).
These
studies
implicate
a role
for
tissue-specific
regulatory
proteins
in the mediation
of
steroid
action
on gene
expression.
The
N-terminal
domain
of the receptors
apparently
play
a role in
this
tissue-specific
action.
ET
AL.
as transcription
essing.
Lastly,
proteins
could
factors,
as depicted
themselves
that
directly
transcription
regulate
chromatin
as transcription
roles,
the
transciption
A Role
data
forA
Using
not
Regulatory
Hormone
model
bind
the
gene
two
steroid-
tude
of genes via its protein
product.
This simplified
model
explains
some
of the riddles
related
to steroid
regulation
of gene
expression,
such
as the delayed
responses
of
receptor
binding.
recognition
many
of
responses
the
proposal
structural
It
the
in
of the
that
should
genes
be
distinct
early
midge
salivary
steroid
some
after
mentioned
and
glands
ecdysone
of the
late
early
had
genes
steroid
that
the
transcripfollowing
led earlier
code
for
Action:
Gene
the above
information
together
with
other
discussed
here, this laboratory
has developed
a different
would
or
the
receptors.
The regulation
of structural
(“late”
gene) transcription
would
occur
one
or more
hours
after
steroid
entrance
into the cell. Each steroid-controlled
regulatory
gene
could
regulate
the expression
of a multi-
tional
of Steroid
structure
factors.
In
“
to
Model
factors
even RNA
proc4, the regulatory
nuclear
factors
response
elements
(SRE)
residing
either
in the
5’
flanking
domains
or within
the
steroid-regulated
structural
gene itself (late genes).
Thus,
in this model,
the SREs
residing
near or within
“late
genes
would
function
as secondary
SREs
that
bind
transcription
factors
and possibly,
but not necessarily,
the steroid
administration
A Different
or possibly
in Figure
represent
(see
Fig.
4)
for
ovarian
steroid
POSSIBLE
MECHANISM
OF ACTION OF STEROIDS
ON GENE TRANSCRIPTION
action
(Spelsberg
et a!., 1983,
1987d;
Rories
and
Spelsberg,
1989).
The
proposed
model
invokes
the
following
steps:
beginning
at the top
left
side of
Figure
4 and following
the arrows,
steroid
hormones
enter
target
cells,
bind
resulting
complex
binds
These
sites are located
even
within
the
binding
somehow
transcription
of
after
target
to
their
to the
in the
receptors,
and
the
nuclear
acceptor
sites.
5’ flanking
domain
or
regulatory
gene
(early
gene).
This
results
in the
regulation
of the
the regulatory
gene within
minutes
the steroid
enters
the target
cell. In any given
cell, at any given
time,
there
may
be a few
dozen
of these
regulatory
genes
responding
to the
steroid.
Each
of the regulatory
genes
codes
for a
regulatory
protein
that
would
alter
the expression
of a distinct
set of genes.
The
regulatory
proteins
could
have
various
functions.
Enzymatic
regulatory
proteins
could
modify
nuclear
factors
which
in turn
could
regulate
chromatin
structure,
gene
transcription
FIG. 4. Model
for the role of regulatory
genes
in the steroid-induced
regulation
of structural
gene
expression.
In this model,
the chromatin
acceptor
sites,
composed
of specific
acceptor
proteins
bound
to specific
DNA
sequences,
would
neighbor
regulatory
genes.
The regutaotry
genes
represent
“early”
steroid-regulated
genes,
and the structural
genes
represent
the
“late”
steroid-regulated
genes.
The
regulatory
protein
(coded
by the regulatory
gene)
could
act as a transcription
factor
(TF)
and bind to steroid
response
elements
at the 5’ flanking
domains
of the
structural
genes
or could
be an enzyme
which
regulates
TF activity,
chromatin
structure
or even
mRNA
processing.
(Reprinted
with
permission
of the publisher
from
Spelsberg
et al., 1987d.)
STEROID
regulatory
(or
required
for
and Romball,
A
steroid
transcription)
recent
studies
in this
as well
coids)
(data
after
injection
for a nuclear
regulate
replication
1986;
the
not
gene
genes
“regulatory
factors
laboratory
on
are
(Clever
rapid
action
proges-
reduce
the
c-myc
involved
et al.,
et
al.,
(hsp-70)
and
1986)
and
to
negatively
the
I
gene
(Kaddurah-Daouk
steroid-myc
gene
Figure
4 and thus
system
could
et
a!.,
of c-myc
in the steroid
exists.
In any case, this
fits
the
well serve
model
shown
as an example
in
of
a rapid
steroid-controlled
“regulatory”
gene
(i.e.,
early
gene)
whose
protein
product
acts either
as a
transcription
factor
or as an activator
of transcription
which
in
genes
turn
(i.e.,
late
regulate
the
expression
of
genes).
mRNA
gene codes
to positively
in the initiation
1982;
Kelly
and
metallothionein
structural
with
a maximal
within
20 mm
of the steroid.
The c-myc
protein
that is now thought
59
EXPRESSION
factors
on c-myc
(Rories
et a!., unpublished
data).
5, progesterone
(and glucocortishown)
GENE
1987).
Thus,
a possible
role
regulation
of cell proliferation
expression
in
Spe!sberg
et a!.,
in the avian
oviduct
70%
to 90%
reduction
genes
(Gonda
ON
gene”
in
comes
from
the
proto-oncogene
et a!., 1988,
mm
Studzinski
protein
which
late
as of glucocorticoid
in the avian
oviduct
As shown
in Figure
levels
by 5
decrease
of
of the
1964).
example
of a
of transcription
terone
action
on c-myc
the avian
oviduct
(Fink
1987d),
factors,
transcription
1966; Clever,
possible
regulation
ACTION
(Binding
Sites)
The
nuclear
Sites
for
the
berg
et
a!.,
Steroid
binding
steroid-induced
(Thrall
et a!.,
shock
regulate
Acceptor
steroid
receptors
sent
the
nuclear
of DNA
Siebenlist,
heat
Nuclear
Receptors
sites
(acceptor
sites)
are important
because
they
event
immediately
preceding
alteration
of
gene
1978;
Katzenellenbogan,
1983;
Rories
and
for
reprethe
transcription
1980;
Spels-
Spelsberg,
1989).
It
c-myc and a-tubulln mRN4 Levels In Avian Oviduct
and Spleen In Response to Progesterone
and Vehicle
0
4
U
C
.
.
z
E
0
TIME (hours)
FIG.
5. Densitometric
try analyses
effect
of a
tin mRNA
vehicle
on
percentage
mission
of
analysis
of mRNA
levels
in avian
oviduct
and
spleen
after
administration
of autoradiographs
of RNA
gel blots show
the effects
of two doses
(1.0mg
and 250
1 mg dose of progesterone
on a-tubulin
mRNA
levels
in diethylstilbestrol-stimulated
levels
after
1.0 mg of progesterone
represent
the average
of two separate
experiments
c-myc
mRNA
levels
in the avian
oviduct
and of progesterone
on c-myc
mRNA
levels
of the c-myc
mRNA
level of the control
value obtained
at time zero.
(The data and
the publisher.)
of progesterone
or vehicle.
Scanning
Mg) of progesterone
on c-myc
mRNA
chick
oviducts.
The values
ofr the c-tnyc
involving
different
groups
of animals.
in the spleen
areincluded.
The data are
figure
are from
Fink et at., 1988,
and
densitomelevels and the
and a-tubuThe effects
of
plotted
as the
used with per-
SPELSBERG
60
TABLE
steroid
1. General
properties
hormone
receptors.
1.
2.
3.
4.
5.
6.
7.
Require
an
intact,
of
activated
the
chromatin
receptor
acceptor
complexed
sites
with
for
two
Figure
are three
receptors
of
approaches.
chromatin
and
the
the
binding
to specific
steroid-regulated
DNA
These
acceptor
SREs.
One
of
sequences
structural
approach
that
reside
genes (Payvar
et
a!.,
1982;
Bai!ley
et
al.,
1983,
et
a!.,
1983;
Miller
et
al.,
1984;
al.,
1985;
Renkawitz
et
Cordingley
et
a!.,
a!.,
1984;
von
1987a;
der
specific
sequences.
to the
SREs
possibly
denatured
DNA
footprinting
It is possible
be
required
binding
of steroid
in vitro
and
in vivo
to
that
when
other
achieve
receptors
of the
nuclear
acceptor
sites
and
some
of their
1988).
studies
of gene
properties.
See
text
for
complexes.
data fail to
binding
bound
factors
Further
details.
nonacdisplay
to these
(proteins)
specific
to these
conditions.
DNA acceptor sites (SRE)
1. Receptor-dependent
(nonactivated/
denatured)
2. Not saturable or high affinity?
3. Not receptor-specific?
4. Composition:
DNA
5. - >10
to 10 consensus sites/cell
6. Sites required for steroid action
on gene transcription
classes
a!.,
alone.
Further,
receptors
still
Chromatin
acceptor
sites
(May be same as matrix sites)
1. Receptor-dependent
2. Saturable high affinity
3. Receptorand tissue-specific
4. Composition:
Protein and DNA
5.
10.000-20,000
sItes/cell
6. In vivo vs. in vitro competition
three
et
transfection
regulation
receptor
published,
high-affinity
matrix sites
Receptor-dependent
2. Saturable,
high affinity
3. Receptor- and tissue-specific
4. ComposItion:
Protein and DNA
5.
10,000-20,000
sItes/cell
6. In vivo vs. in vitro competition
the
et
However,
as proposed
earlier
in this
may be involved
in binding
transcripother
regulatory
proteins
in addition
instead
of, the
steroid
analyses
of the current,
demonstrate
specific,
of receptors
tivated
and
might
SREs
and
Ahe
Waterman
1.
outlining
in or near
et al., 1981,
Compton
Nuclear
6. Model
the
directly
Chandler
IN VITRO CLASSES
OF NUCLEAR
ACCEPTOR
SITES FOR STEROID
RECEPTOR
FIG.
involves
receptors
1986;
to, or
Careful
absolutely
classes
of nuclear
acceptor
that have been identified
by
outlined
in
the nuclear
1983;
transcription.
review,
the
tion factors
and has
1 outlines
are
sites,
steroid
These
SREs have been
shown
by
to be essential
for
the
steroid
some
general
properties
of the nuclear
acceptor
sites
for steroid
receptors
as identified
from in vivo studies
as well as in vitro studies
using intact
nuclei/chromatin.
To date,
there
sites for steroid
sites,
analyses
appears
that an interaction
between
steorid
hormone
receptors
with
nuclear
acceptor
sites is required
for
steroid
regulation
of gene
expression.
Thus
the
elucidation
of the constituants
of the acceptor
sites
important
Table
different
6 as the
matrix
bound
steroid.
Bind steroid
receptor
complexes
saturably
and with high affinity.
Display
a receptor
specificity
(i.e.,
each class of steroid
receptors
binds
its own class of acceptor
sites).
Appear
to be highly
conserved
among
animals.
Appear
to be tissue-specific
(1’).
Between
io and 3 X IO total chromatin
acceptor
sites per cell
have
been
reported
for
a variety
of systems
with
over
half
“masked”
in (i.e., 4000
to 10,000
open per cell).
Over one-half
of the total chromatin
acceptor
sites are ‘masked”
in intact
chromatin
or nuclei
by other
chromatin
proteins.
for steroid
receptors
is extremely
been an area of intense
investigation.
ET AL.
saturable
sequences
discussion
under
of
STEROID
these
important
sequences
is not
to space
limitations
and to the
nuclear
acceptor
sites described
The
other
classes
of acceptor
and
nuclear
matrix
sites,
approach.
These
acceptor
variety
of
laboratories,
1978;
steroid-target
including
Goldberger
1983,
1984,
1985;
Toyoda
(Ruh
Ross
rack,
et a!.,
et
and
were
sites
the
other
chromatin
found
by another
were
identified
in a
by a variety
(Thrall
et
1987a;
Spe!sberg
Martin-Dani
and
1985;
et
Spelsberg,
due
Hora
1988);
Ruh
Wilson,
a!.,
and
1988;
Lopez
Pavlik
a!.,
a!.,
1985;
the
of
structures
et a!.,
1986;
used
binding
nuclear
As
sites,
nuclear
DeBoer
et
et
studies
specific
native
coworkers
1982;
(Bar1982;
Cushing
Franke!
Cobb
of
or
and
and
approach
steroid
of
whole
1984;
For
analyses
performed
with
sites
these
of
two
steroid
in vivo
classes
sites
the
receptor
and then
nuclear
properties
Ruh
et
analyses
of
receptors
including
acceptor
bindings
in cell-free
Cobb
and
same
class
The
Chroma
matrix
(Barrack,
al., 1981;
Spelsberg
et al.,
a!., 1986b;Kirsch
the
gesterone
terized,
oped
l987abcd;
to
Toyoda
sites.
the
This
chemical
cell-free
avian
oviduct
chromatin
tin, termed
nucleoacidic
separate
laboratories.
1985;
nature
binding
of
Gold-
of
of
First,
between
binding
endogenous
and
specific
most
nuclear
the
nuclear
binding
examined
by
tissue
systems
in Figure
7,
by
Roth
to
chromadisplayed
importantly,
the in vivo
assay
were
steroid-target
As shown
these
[3H]PR
and to dehistonized
protein
(or NAP),
expected
sites.
competition
the
cell-free
a!.,
in the avian
oviduct
was characa cell-free
binding
assay
was devel-
characterize
acceptor
et
1987a;
Hora
et a!., 1986b;
Gold1988).
The binding
of [3 H] pro-
in vivo
and then
sites,
were
binding
1983;
eta!.,
Leavitt,
and
two
different
and
co-
Avian
sites
tin Acceptor
Oviduct
and
may
(Hora
fashacceptor
show
many
et a!.,
1982;
1985;
Hora
et
eta!.,
1987;
even
et a!.,
represent
chromatin
to specific
the
1978;
Katzenellenbogan,
to DNA,
possibly
et a!., 1976;
Thrall
1980;
J Steroid
A
Spelsberg
for Nuclear Acceptor
SItes:
In Vivo and hi Vitro
BindIng
& Coworkers,
1976
Blochem.
7:1091
Chick
Chick Uvir
or OvkMact
Roth & Coworkers,
Endocrlnol
115:836
B
Ovlduct-Progtsron
Chick
Oviduct
1984
-Estra
Chick
Liver
C.)
ii
1!
I
Vehicle
2mg
p
2mg
et a!.,
Vehicle
P
SUBSTANCE
PR
bound
(Pikier
Speleberg
1986b).
Sites
acceptor
proteins
DNA
sequences
Receptors
Between
the
These
in a
Kaufmann
Spelsberg,
Cushing
et a!.,
1986;Alexander
1987)
of acceptor
sites
1980;
Ruh
and
of Steroid
Comptltlon
to
the
initially
assays
components.
steroid
receptors
acceptor
Binding
the
The authors’
laboratory
has studied
the chromatin
acceptor
sites for the avian
oviduct
PR for a number
of years.
These
sites
appear
to consist
of specific
et a!.,
et a!., 1986,
and Spe!sberg,
1987).
high-affinity,
and receptor-dependent
depicted
in Figure
6, the chromatin
and
similar
1980;
berger
berger
Senior,
Leavitt,
steroid
nuclei,
of
intact
nuclei
and
nulcear
have
been
shown
to bind
saturable,
ion. As
1984,
et
chromatin
and matrix.
outlined
in Figure
6, two
classes
of acceptor
termed
the chromatin
acceptor
sites
and the
matrix
acceptor
sites,
evolved
from
these
studies.
1983,
acceptor
1986b;
61
a!.,
1987),
and
others
(Klyzsejkoet a!., 1976;
Hamana
and Iwai,
1978;
Lopez,
1978;
Wang,
1978;
Colvard
and
1985;
These
EXPRESSION
properties
Spelsberg,
a!.,
GENE
et
1984,
1986;
of
a!.,
et a!.,
et a!., 1981,
1987;
Ruh
and Spelsberg,
and Ruh,
1984);
Barrack
and coworkers
1980,
1983,
1987;
Barrack
and Coffey,
Alexander
Stefanowicz
Perry
and
for
sites,
1982,
a!.,
here
on the
cell systems
this laboratory
l987abcd;
Goldberger
presented
emphasis
below.
ON
ACTION
3Oug
E2
INJECTED
FIG. 7. Competition
between
in vivo and in vitro nuclear
binding
of
steroid
receptors.
Unlabeled
estrogen
(E2,
30 g
per animal)
and
progesterone
(P, 2 mg per animal)
were given
to mature
rats (Panel
B)
or to estrogen-treated
chicks
(s.c.)
(Panel
A), respectively.
After
a short
period
of incubation,
the nuclei
of the chick
oviducts
and rat uteri
were
isolated
and
subjected
to a cell-free
nuclear
binding
assay
using
[‘H]labeled
estrogen
receptor
or [‘I-Il -labeled
progesterone
receptor
isolated
from
the same
respective
tissue.
The data are presented
as the percentage of control
values
(i.e., binding
to the nuclei
from
cells or animals
with no prior exposure
to the nonradiolabeled
steroid).
In Panel A, the
dotted
bar represents
values
of a second
experiment.
(These
data were
obtained
[Panel
A]
with
permission
of the
publishers
and
Chuknyiska
et al.,
1984
[Panel
from
B].
Spelsberg
et al.,
1987d
62
SPELSBERG
workers
(Chuknyiska
et a!.,
coworkers
(1987d)
partial
competition
nuclear
(KD
(Pikier
al.,
sites
1983)
with
bound
berg,
per
1976;
1987)
were
and
measured
8, a saturable,
similar
levels
of
found.
It has
chromatin
et a!.,
1983).
acceptor
sites
1976;
Thrall
et
As depicted
H] PR
was
complex
but
plexes.
Therefore,
also
not
with
these
as found
other
steroid-target
of binding
to isolated
molecules
certain
tissue
systems.
in vivo.
(Spelset a!.,
that
inactive
human
of
PR
As shown
and
in the
not
in
found
In
the
PR becomes
molting
they
bind
the
to the nuclear
expression
(Boyd
been
et
any
event,
found
intact
nucleoacidic
protein-DNA
in pure
DNA.
during
have also
(Spelsberg
In
are
same
oviduct
this
case,
and bind
gene
pure
Spelsberg
and
Halberg,
1984;
Hora
et al., 1986a).
et a!., 1988).
sites
for PR
The next
component
in Pane!
alter
receptors
the
as measured
with
or
avian
receptors
tissues
chromatin
or in
posed
of chromatin
binding
the
using
displayed
seen
states,
1979;
et al.,
Colvard
acceptor
com-
and
sites
chromatin
not
inactive.
In
fail to activate
steroid
malignant
assays,
year,
was
season),
sites
Spelsberg,
Boyd-Leinen
display
oviduct
(winter
acceptor
Webster
et a!.,
cell-free
pattern
temporarily
steroid
but
the
ER
This
physiological
period
with
the
to these
sites
acceptor
binding
patterns
bound
binding
the
cell-free
binding
Spelsberg
et
shown
avian
8, the
the
acceptor
in the
chromatin
throughout
nonradiolabeled
in
in vivo
the
8 (Panel
2), the binding
with
nonradiolabeled
nuclear
these
isolated
(Pikler
et al., 1976;
al., 1978;
Spelsberg
receptor-specificity
assays
been
8,
high-affinity
in vivo
Spelsberg
be activated
and
to display
specific
in Figure
competed
Figure
avian
oviduct
specifically
bound
highly
purified
preparations
of the PRs in the cell-free
binding
assays
(Hora
et al., 1986b).
Lastly,
as shown
in Panel
4 of
a
to
in
receptor
cell nucleus
as determined
Spelsberg
and Cox,
1976;
must
ligand
a steroid
those
3 of
and
at least
binding
M), receptor-dependent
Webster
et al., 1976;
receptor
steroid
[3
Spelsberg
AL.
Figure
assays.
in Figure
iO
to 10-10
et a!., 1976;
-
and
were
able
to display
between
the in vivo
acceptor
cell-free
binding
As outlined
1984)
ET
and
1980;
These
identified
in
al., 1987b;
these
nuclear
in nuclei
or
protein
(NAP)
comcomplexes,
but are
DNA.
goal
(or
was to attempt
components)
to determine
of
chromatin
which
were
PROPERTIES OF THE CELL-FREE BINDING OF THE
AVIAN OVIDUCT PR TO NUCLEAR ACCEPTOR SITES
A.
SATURABLE,
HIGH
AFFINITY
=
10119,
ACTIVATED
RECEPTORDEPENDENT.
B.
RECEPTOR
C.
SAI’.#{128}
SPECIFIC
D.
MIMICS
(ER vs.
SPECIFIC
IN
BINDING
VIVO
PR).
WITh
BINDING
HIGHLY
WITh
PURIFIED
REGARD
TO
RECEPTOR.
NONFUNCTIONAL
RECEPTOR.
0
C
0
C.)
0
I
C,
z
0
z
uJ
o
50
a.
FIG.
permission
8. Outline
of the
of the publisher
100
0
p1 PR
properties
of
from
Spelsberg
the
cell-free
nuclear
et al., 1983.)
200
400
0
50
p1 PR
pu ER
binding
of the
100
avian
oviduct
progesterone
SepDecMar.Mt
PR Isolation
receptor
Date
to oviduct
chromatin.
(Used
with
STEROID
responsible
sites.
for
One
specific
approach
sequentially
intact
oviduct
This
is
histones
these
was
outlined
in
(chromosomal
chromatin,
the
the
specific
PR
binding.
9, removing
the
bulk
specific
examine
various
on the
results
the
chromatin,
moderate
levels
dine
hydrocholoride)
chromatin
Figure
9.
protein
from
chromatin
Spelsberg,
residual
nonhistone
PR
“masked”
by the
proteins
1989).
chromatin
acceptor
1985;
Spelsberg
unmasked
PR
chromatin
receptor
of
from
sites.
increase
in
in Figure
proteins
binding.
Studies
in intact
chromatin
removal
of a certain
(for
a review,
see
In the avian oviduct-PR
fraction,
after removal
the nucleoacidic
consists
of the
protein
display
the
specificities,
same
and
binding
patterns
Rories
system,
of the
be
of
and
the
CP-1
protein
fraction,
DNA
and tightly
(Martin-Dani
et a!.,
1983).
acceptor
sites
in
a
have
re-
and can
fraction
and
The
the
to be the
same
1980;
Spelberg
et
Spe!sberg,
1985).
contains
the
a!.,
al.,
Spelsberg,
masked
and
avian
oviduct
affinities,
steroid
of binding
the
-
CP-2
binding,
16
0.4
0
ise
0
75
150
225
[]un
in
et a!.,
Halberg,
et
The
require
the
receptor,
ence in binding
inactive
receptor.
and
PR
there
seems
to
be
to
1984;
little
differversus
Spels-
et a!.,
1984).
of a purified
(enriched
to the pure
DNA
Scatchard
analyses
to the NAP
with
acceptor
this
genomic
Toyoda
genomic
active
1979;
preparain the PR
isolated
from
shows
hightwo apparent
classes
of acceptor
sites. Scatchard
analyses
binding
to DNA
displays
a horizontal
line,
no high-affinity
sites (Hora
et a!., 1986b).
chromatin
by reannealing
et
1985),
to pure
Boyd-Leinen
a titration
NAP fraction
acceptor
sites)
and
this
complex.
The
affinity
PR binding
al.,
contain
the
the specific
the biologically
and Spe!sberg,
1980;
shows
to the
binding
et
in level of binding
but also
the
DNA
binding
does
between
(Boyd
Halberg,
10
PR
Thra!!
Spelsberg
Toyoda
reduced
Although
cell-free
1976;
1980;
activity).
binding
DNA
is not only
is nonsaturable.
pure
the
PR
of the PR
and thus,
sites can then be reconstisame fraction
of chromatin
hen
DNA
et a!., 1985;
(Spelsberg,
Goldberger
sites
(Spelsberg
et a!., 1984).
sites display
a receptor-dependent,
PR
nonfunctional
Halberg,
1980;
reconstituted
FIG. 9. Outline
of the isolation
of the chromatin
acceptor
proteins.
This figure
outlines
past work
on the dissociation
of proteins
from
the
chromatin
and the resulting
effects
on progesterone
receptor
(PR) binding
using
the
cell-free
nuclear
binding
assay.
The
removal
of the
CP-i
proteins
(histones)
results
in only
a mild
increase
in PR binding.
An
unmasking
of sites is achieved
by the removal
of CP-2 fraction
of proteins,
which
represent
-80%
of the nonhistone
proteins.
The binding
to the residual
nucleoacidic
protein
fraction
(NAP)
displays
all of the
properties
of native
PR binding
as does
intact
chromatin.
The removal
of the CP-3 fraction
from
of specific
binding.
(Data
Spelsberg
et al, 1987d.)
from
fraction
appears
to
site “activity”
(i.e.,
saturable
DNA
1987abc;
removed
generate
the CP-3
acceptor
acceptor
stituted
7 M GdnHCI
CP.3
1984,
bound
which
et a!.,
et a!.,
1986,
1987a;
Goldberger
and Spelsberg,
1988).
These
reconstituted
sites
display
the
same
specific
PR
binding
properties
as the
native
(unreconstituted)
1.2
08
NAP
is
to
and
Martin-
tightly
(CP-3),
Therefore,
chromatin
proteins
1983,
PR
CPM/mg
ONAx
1987;
binding
(Webster
and
1983,
tuted
(Histones)
is lost
Spelsberg
appear
Spelsberg
1983,
(NAP)
specific
thus
1978;
al.,
proteins,”
the
assays
The
4 M GdnHCI
and
et a!.,
chromatin
1978;
Figure
tion
of
2.0
Dehistonizedchrornatin
(Thrall
“acceptor
DNA,
binding
berg
Chromatin
NaCI
sites
residual
genomic
from
receptors,
Halberg,
Dani and
the
from
of systems
most
steroid
63
EXPRESSION
As shown
in Figure
9, when
the
nonhistone
chromatin
protein
fraction
of the
or CP-i)
a slight
GENE
functional/nonfunctional
termed
the
CP-2
fraction,
using
of chaotropic
salts (e.g.,
4 M guaniresults
in a marked
increase
in
and CP-2,
is termed
or NAP.
This NAP
bound
effects
as shown
of laboratories
using a variety
that
the acceptor
sites
for
ceptors
are
“unmasked”
the
The
removal
fraction-i
in only
of the
acceptor
components
PR acceptor
However,
high-affinity
variety
shown
to
removing
chromatin
ON
ACTION
the NAP to obtain
pure DNA
used
with permission
of the
results
in a loss
publisher
from
CP-3
binding
and
sites
reannealled
are
to the
specific
DNA sequences
a!.,
1984;
Toyoua
et
Spelsberg,
Ruh
and
reconstitution
sites
method
in
little
PR preparation
Spelsberg
et
1988).
coworkers
of
mammalian
was utilized
or no
reconspecific,
binding
when
is used
(Spelsberg
al., 1984).
Further,
dependent
hen
The
DNA
on
and
the
seem
amount
systems.
to monitor
of
to require
in the hen DNA (Spe!sberg
1
f’
1 ..11_
ai.,
1785;
lioIuuerger
Similar
results
(Ross
and
the
estrogen
a
and
the
.
et
an
were
reported
by
Ruh,
1984)
for the
receptor
acceptor
The
the
reconstitution
PR “acceptor
64
SPELSBERG
Analysis
of the Binding
of Purified
ET
AL.
PR (Component
I) to Nucloase
Treated
NAP
0
I
Moles
of E3HPR
added
(x
Bound
1012)
C3HPR
(M x
102)
FIG.
10. Titration
of binding
of nuclease-treated
nucleoacidic
protein
fraction
(NAPf)
with
purified
progesterone
receptor
(PR).
NAP
was
digested
with DNase
I for 5 mm and the NAPf
was prepared
for binding
as described
elsewhere
(Hora et al., 1986b).
Highly
purified
PR was prepared
and the PR binding
to the NAPf
was performed.
In Panel A, fragments
from
a 5-mm digest
of NAP
(.) or pure DNA
(0)
were incubated
with
0.01-2.33
pmoles
of the [3 HI-labeled
PR. Each
point
represents
the average
± SEM
of duplicate
samples.
In Panel B, the NAPf
binding
data from
Panel
A (.) are resolved
by Scatchard
analyses
into
two classes
of binding
components
with KD,
‘-1.81
X i0#{176} M and KD2,
1.79 X 10-0
M. Projection
of the data
(o) by the method
of Rosenthal
reveals
a sepcific
binding
component
with
KD = 8.49
X 10”
M. (Data
used with permission
of
the publisher
from
Hora et al., 1986b.)
activity”
tion
of
(i.e., acceptor
avian
oviduct
chromatofocusing,
sylapatite,
procedure
protein)
during
the fractionachromatin
proteins
using
as well
as molecular
and
hydrophobic
to purify
the
sieve,
hydro-
chromatographies.
“acceptor
activity”
A
over
100,000-fold
been
Table
to
developed
2 outlines
acceptor
tein,
a
apparent
homogeneity
(Go!dberger
and
the
chemical
protein.
10 kDa
Using
species
2. General
in the avian
properties
oviduct.
of
acceptor
proteins
for
progesterone
PR
binding
Property
Value
Evidence
This
shown
Proteinaceous
Yes
i. Activity
destroyed
by proteases
but
and
Simple
or conjugated
Side chain
modification
Molecular
weight
Isoelectric
point
DNase
2. Purified
contains
or DNA
3. lsopycnic
fugation
A nonconjugated
protein
Unknown
10 kD;
5kD
Density
pH6; pH7.0
possibly
more
1.24 g/cc
Steroid
receptor
specificity
Tissue
specificity
Species
specificity
Unknown
Unknown
Unknown
RNase,
(?)
i.
2.
1.
2.
or
activity
no KNA
centri-
Molecular
sieve
SDS-PAGE
Isoelectric
focusing
Chromatofocusing
Isopycnic
centrifugation
J.,
marked
Gosse,
results).
to
hen
species
N-terminal
showing
has
Other
species
studies
protein
chromatin
together
generate
PR to
protein
(Goldberger
with
the fact
the
acceptor
proteins
appear
to
biological
“acceptor
against
a reconstituted
complex
were capable
but not the ER, binding
acceptor
sites
in oviduct
et al., 1987a).
These
results
that
this 10 kDa protein
can
a receptor-dependent,
hen genomic
DNA,
in
A., Rejman,
unpublished
support
a possible
as a PR candidate
(“native”)
DNA.
recently
been
sequence
with an
a slightly
acidic
p1
of acceptor
protein.”
Antibodies
prepared
10 kDa acceptor
protein-DNA
of inhibiting
the specific
PR,
undissociated
1988).
of this
genomic
hydrophobicity
(Go!dberger,
B.,
and
Spe!sberg,
T. C.,
exist.
Additional
role
for this
to
Spelsberg,
properties
and Spelsberg,
1988).
This
co-fractionates
with
the
i.e.,
it generates
specific
complexed
10
kDa
protein
to have a unique
acid composition
amino
not
when
recently
antibodies
against
this prowas
detected
by Western
immunoblots
(Goldberger
10
kDa
protein
species
PR
acceptor
activity,
TABLE
receptor
has
saturable
binding
supports
a role
of
sites
for
the
PR
in
of
this
avian
STEROID
TABLE
3.
General
acceptor
proteins
conclusions
for
the
of
avian
the
oviduct
chromatin
ACTION
acceptor
progesterone
ON
sites
receptor
and
(PR).
1. Several
lines
of evidence
support
that
the PR binding
to chromatin acceptor
sites in vitro
are similar
to those
in vivo.
2. The chromatin
acceptor
sites
for PR in the avian
oviduct
appear
to represent
complexes
of specific
acceptor
proteins
and DNA.
3. PR binding
analyses
and antibody
studies
suggest
these
acceptor
sites
are evolutionarily
conserved,
are receptor-specific,
and are
largely
masked
in intact
chromatin.
4. More
than
a dozen
other
laboratories
studying
a variety
of
other
steroid-target
tissue
systems
in different
animals
have
reported
similar
properties
for the nuclear
acceptor
sites.
5. One candidate
10 kDa acceptor
protein
for the avian oviduct
PR
has been
purified
and another
6 kDa-acceptor
protein
has been
identified
and antibodies
against
them
have been prepared.
6. The
N-terminal
AA
sequence
of the
10 kDa
acceptor
protein
species
is unique
but
distantly
related
to several
transcription
factors.
7. The acceptor
protein
appears
to require
specific
DNA sequence
to
generate
specific
PR binding.
GENE
EXPRESSION
Wang,
liver,
1978),
mouse
nuclei.
However,
further
analyses
of
of this 10 kDa protein
to generate
specific
binding
on
the
general
acceptor
hen DNA
properties
protein
are
of
and
the
required.
this
dependent,
binding
oviduct
papers
found
Chroma
tin Acceptor
ed
for Other
Steroid
Receptors
Other
Animal
Species
It is important
examined
that
intact
similar
NAP
that
(whole)
stitution
protein
of
these
fractions
above
for
the
Ruh,
1984).
bound
description
tissue
sites
and
avian
sites
has
of
acceptor
sites
the
Possible
Roles
to DNA
similar
DNA
many
1 and
it was
consist-
were masked
reported
the
oviduct-PR
Tables
using
systems,
sites
tightly
using
genomic
Therefore,
in
binding
as
and
in the
recon-
nonhistone
described
system
(Ross
of
properties
the
2 can
be
for
other
applied
and
to
the
steroid-target
systems.
Models
sites.
of the
for
Chromatin
Acceptor
summary,
studies
Proteins
with
PR
chromatin
acceptor
sites indicate
that (a) these
isolated
acceptor
proteins
may
be structural
components
of the
chromatin
acceptor
sites
for steroid
receptor,
(b) the recon-
other
nuclei,
preparations
proteins
many
of the acceptor
chromatin.
One group
acceptor
in
to note
intact
in rat
cells
as described
above
for the
In many
of the above
listed
other
steroid-animal
chromatin
acceptor
of nonhistone
3 outlines
candidate
Sites
receptors
leukemia
high-affinity
assays,
system.
involving
that
the
In
Related
glucocorticoid
and
human
saturable,
cell-free
PR-avian
Table
kDa
10
chromatiri
the
PR
and for
mammary,
(Hamana
and Iwai,
1978;
Ruh
et a!., 1987),
and in
human
breast
cancer
cell line (Sun et a!., 1983).
All
of these
chromatin
acceptor
sites display
a receptor-
described
oviduct
ability
65
laboratories
have
chromatin,
and
as described
above
for
the
avian
stituted
sites
ated)
sites,
conserved
closely
resemble
the
native
and (c) the PR acceptor
during
evolution.
These
(undissoci-
sites appear
to be
PRov
acceptor
oviduct
PR for the specific
binding
of a variety
other
steroid
receptors
in other
target
systems.
Examples
of specific
chromatin
acceptor
sites for PR
proteins
generate
are
protein
species)
in the generation
of the PR acceptor
sites on the DNA is unknown.
Figure
11 outlines
several
possibilities
for the role
of acceptor
proteins.
In Model
A, the
PR
binds
directly
to the acceptor
protein,
either
using
the Zn
fingers
in the
DNA
binding
domain
or the hinge
of
the
and
the
sheep
hamster
Chromatin
estrogen
oviduct
1984),
hypothalamus
uterus
acceptor
receptors
(Ruh
calf
and
uteri
(Perry
(Cobb
and
and
Lopez,
Leavitt,
1978)
1987).
sites
that
specifically
have
been
found
in the
Spe!sberg,
(Ruh
et
al.,
1982;
DeBoer
1981;
Ross
bind
avian
et
and
a!.,
Ruh,
1984;
Cushing
et a!., 1985),
rat uteri
(Chuknyiska
et
al., 1985),
mouse
uteri
(Pavlik
et a!., 1986),
shark
testes
(Ruh
et al., 1986),
human
breast
cancer
cell
lines
(Sun
et a!., 1983;
Frankel
and Senior,
1986),
rodent
malignant
breast
tissue
(Shyamala
et a!.,
1986;
Klinge
et al., 1987),
and in mouse
and sheep
brain
(Perry
and Lopez,
1978;
Lopez
et a!., 1985).
Further,
specific
chromatin
acceptor
sites for androgen
and
receptors
Sertoli
have been reported
cells (Klyzsejko-Stefanowicz
for
rat
prostate
et al.,
cells
1976;
must
be
the specific
candidate
this
comp!exed
with
hen
DNA
PRov
binding.
The function
lOkDa
(and
domain
of the receptor
PR
binds
directly
to
altered
by the acceptor
binding
Mode!
domain
C, the
protein
DNA
nately,
may
and
possibly
other
to
of
acceptor
molecules.
In Mode!
B, the
the DNA
whose
structure
is
proteins,
again using the DNA
of the
PR may
receptor
bind
molecule.
Lastly,
in
both
to the acceptor
(possibly
via the hinge
domain)
and to the
(possibly
via the DNA
binding
domain).
Altertwo receptor
species
of the avian
oviduct
PR
be involved
the
A species
with
B species
binding
to the
binding
DNA.
to the
protein
66
SPELSBERG
Potential
Molecular
ET
AL.
Models for Role of Acceptor
Steroid Receptors
Protein
in Binding
Model
A
Model
Steroid
(R.c.ptor
BInding
to
Acceptor
Protein)
= Acceptor
=
Receptor
ConIex
S-A
(Receptor
Adlolnh’sg
B
Bhid*sg
to DNA
Acceptor
Protein)
Protein
DNA
,SR
Receptor
Subunits
S
SR
jiw
Model
C:
Subwiit
Hypothesis
FIG.
11. Models
of the chromatin
acceptor
sites for please
define
(PRov):
role(s)
of the acceptor
protein
(AP).
Model
A shows
the steroid
receptor (SR)
binding
to the AP, which
in turn is bound
to the DNA.
Model
B shows
the SR binding
directly
to the DNA whose
structure
is perturbed
by the AP to bind
the SR. Model
C describes
a dual progesterone
receptor
(PR)-binding
site, i.e. to both
acceptor
and DNA or with
the possible
participation
of two subunits
of the avian oviduct
PR with the B species
binding
the Al’ and the A species
binding
the DNA.
(Data
used with permission
of the publisher
from Spelsberg
et al., 1987d.)
CONCLUSIONS
Rapid
steroid
advances
in
receptors
advances
existence
the
have
structure
been
described
briefly
and
role
of
genes”
in
expression
the
has
model,
the
which
subsequently
made.
and
function
These
have
in this review.
early-regulated
steroid
regulation
of
also
been
discussed.
regulatory
genes
code
regulate
for
the
The possible
“regulatory
structural
In this
nuclear
for
steroid
of the
expression
receptors
mechanism
of
is
of
steroid
regulation
of gene
expression.
little is known
about
these
sites.
Since
conserved
surely
steroids
and
over evolution,
are
also
conserved.
their
the
matrix
acceptor
sites for steroid
receptors
appear
to
be the same,
or at least have similar
properties.
Their
relationship
to the other
class of acceptor
sites,
i.e.,
the
SREs
the
of the
of gene
of the
nuclear
for
of
that
each
in
steroid
5’
is
flanking
unknown.
domains
The
of
steroid-
composition
of
ACKNOWLEDGMENTS
present,
highly
sites
the
genes,
the chromatin/matrix
acceptor
sites is unknown.
A
possible
role of a 10 kDa chromatin
protein
in the
chromatin
acceptor
sites of the avian
oviduct
PR has
been described
briefly.
Further
studies
of this protein
are needed
to support
it as a candidate
“acceptor
protein.”
The
are
acceptor
has
its own
nuclear
acceptor
site.
Several
of nuclear
acceptor
sites for steroid
receptors
been
reported
to date.
The
chromatin
and
regulated
crucial
action
At
receptors
nuclear
It appears
gene
cascade
proteins,
structural
genes.
This
model
explains
many
known
characteristics
of steroid
regulation
expression
in target
tissue.
The
elucidation
composition,
location,
and
function
of the
acceptor
sites
the
understanding
of
been
species
classes
have
authors
thank
Ms.
Jacquelyn
Keller
for
typing
the
manuscript.
REFERENCES
Adler
S, Waterman
ML,
mediated
inhibition
He
X,
of
Rosenfeld
rat prolactin
MG,
1988.
Steroid
gene
expression
receptordoes
not
STEROID
require
the
receptor
Alexander
RB, Greene
nuclear
matrix:
tor
Bailley
antibody.
A, Atger
uteroglobin
DNA-binding
GL,
direct
Barrack
ER,
demonstration
domain.
Cell
ON
ACTION
52:685-95
M,
with
1983.
The rabbit
the progesterone
receptor.
j Biol Chem
258:10384-89
Bailley
A, LePage
C, Rauch
M, Milgrom
E, 1986.
Sequence
specific
DNA
binding
of the progesterone
receptor
to the uteroglobin
gene:
effects
of hormone,
antihormone,
and receptor
phosphorylation.
EMBO
J 5:3235-41
Baker
HJ,
Shapiro
DJ,
1978.
Rapid
accumulation
of vitellogenin
messenger
RNA
during
secondary
estrogen
stimulation
of Xenopus
laevis.
J Biol Chem 253:4521-24
Barrack
ER, 1983.
The nuclear
matrix
of the prostate
contains
acceptor
sites for androgen
receptors.
Endocrinology
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