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INTRODUCTION |
The study of transcriptional regulation makes extensive use of
artificial reporter constructs that contain several binding sites for a
particular protein and are introduced into cells by transfection. This
useful approach has two limitations. (i) Bona fide targets
of eukaryotic transcription factors are assembled into chromatin,
whereas nonreplicating episomes are not (1). (ii) The primary sequence
of eukaryotic promoters is seldom a reiteration of binding sites for
one protein. With only a few exceptions (2, 3), we are far from
understanding how the regulatory behavior of a eukaryotic promoter
emerges from the union of nonhistone protein-binding sites that it
contains. In general, naturally occurring promoters studied in their
native chromosomal context, e.g. the mouse mammary tumor
virus long terminal repeat (MMTV
LTR)1 (4-6), the
Drosophila hsp26 gene (7-9), and the human pS2 (10, 11) and
cathepsin D (12) promoters, offer a far more intricate picture of
transcriptional regulation than assays with synthetic reporter
constructs placed on nonreplicating episomes (13).
The Xenopus oocyte offers an attractive experimental system
for studies of transcription; evolutionarily molded to contain sufficient histone protein and chromatin assembly machinery for several
thousand somatic cells, it efficiently assembles microinjected DNA into
physiologically spaced chromatin (14), which offers an excellent
opportunity to investigate functional interactions between various
regulatory factors and chromatin templates in vivo (15-20).
The power of this model system is illustrated by data from our
laboratory on action by the thyroid hormone receptor (TR); this
extensively studied member of the nuclear hormone receptor (NHR)
superfamily is exquisitely sensitive to nanomolar quantities of its
ligand, thyroid hormone (T3) (21, 22). TR and
T3-driven metamorphosis is a central phenomenon in anuran
ontogeny (23, 24), and the frog oocyte contains vast stores of
auxiliary components, i.e. corepressor and coactivators
(25), to allow for transcriptional regulation by TR. By using the
oocyte system, we showed that unliganded TR represses transcription via
a chromatin-dependent pathway (20) that involves the
targeting of a complex between the corepressor N-CoR and histone
deacetylase 3 (19), and an auxiliary pathway of an undetermined
mechanistic foundation (19), whereas in the presence of ligand, TR
targets chromatin disruption (26) and the histone acetyltransferase
CBP/p300 to activate transcription (27).
Significant progress in revealing the biochemical accomplices of the
NHRs in effecting transcriptional control (25, 28) is in striking
contrast to the paucity of structural data regarding their action on
chromatin templates. In agreement with earlier chromatin-wide studies
(29), our in vitro data showed that unliganded TR can engage
a response element on the surface of a histone octamer (30). How TR,
and other class II NHRs, accesses its binding sites within chromatin
in vivo is unknown; we had earlier shown that TR can bind to
a fully chromatinized, replicatively inert template in vivo
(31), but whether such access is contingent on DNA assembly into a
specific nucleoprotein architecture in vivo remains
unresolved both for the promoter we studied and for every other class
II NHR-responsive gene.
Because the regulatory properties of NHRs are significantly affected by
the DNA sequence to which they bind (32-35), we studied TR signaling
on one of its naturally occurring target sites in the
Xenopus genome, the promoter for the TR
A gene (36). TR effects a >300-fold range of regulation on its own promoter (31), and
this enables an autoregulatory loop that coordinates titers of hormone
and receptor during Xenopus development (24). In the present
study, we set out to map the structural determinants of primary DNA
sequence of the TR
A gene promoter and of its assembly into chromatin
that allows for such robust synergy with TR.
Our data reveal an upstream enhancer in the TR
A gene locus that
contains several unusual TREs and is required for full range regulation
of this gene by T3. We describe an array of translationally positioned nucleosomes over the promoter and enhancer in
vivo that accommodates at least 4 TR/RXR heterodimer molecules
both in linker DNA between adjacent nucleosomes and on the surface of
histone octamers. In contradiction with commonly held notions that
chromatin prevents access to DNA by regulatory factors, we show that
chromatin assembly potentiates both binding to and action by TR/RXR on
the enhancer. We hypothesize that structural distortion of the
DNA assembled into a nucleosomal fiber creates a template that has a
higher affinity for TR than naked DNA would be, and we suggest that
chromatin disruption by liganded TR enables a novel mechanism of
attenuating response to hormone by causing receptor release from the template.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs for Transcription Analysis and in Vitro
mRNA Synthesis--
Plasmid pTR
A (31) used as a reporter
construct contains a fragment of the Xenopus
laevis TR
A gene (36) (positions
1335 to +313 relative
to the major transcription start site) subcloned into pBluescript II
KS(
). The pTR
A-(
site 1) construct was generated by replacing
the TRE at +276 with a unique NsiI recognition site using a
combination of published techniques (39-41); pTR
A was denatured
with NaOH as described (39) and used in a polymerase chain reaction
with Pwo DNA polymerase (Roche Molecular Biochemicals) and the
following primers (the NsiI site is underlined; these primers anneal immediately outside the TRE at +276): forward primer, 5'-AAAATGCATGCCCAGCGCCCTGGTGCACGATCAG-3', and reverse
primer, 5'-AAAATGCATGCCTAGGGAGGACGAACAAGGATAG-3'.
Polymerase chain reaction settings were as follows: 94 °C (2 min)
10 cycles of 94 °C (15 s), 62 °C (30 s), 72 °C (3 min)
20
cycles with the same parameters, except 5 s added at each cycle
extension step. This reaction generated a spurious 2.2-kb and a
full-length 5-kb product that contained the entire starting plasmid
with the 14 bp of the TRE replaced with the NsiI site; the
5-kb product was gel-purified, digested to completion with NsiI, recircularized with a Rapid Ligation kit (Roche
Molecular Biochemicals), and transformed into E. coli
following standard procedures. Clones were sequenced to confirm
successful TRE replacement. Plasmid pTR
A-
enhancer (gift of Dr.
Dmitry Guschin) contains a fragment of the TR
A locus (positions
154 to +313) subcloned into pCR2.1 (Invitrogen; Carlsbad, CA). Data
identical to those shown here were obtained with a plasmid in which a
more parsimonious deletion (a 350-bp NdeI-FseI
fragment, positions
800 to
450]) eliminated just the TRE sequences
from the TR
A enhancer (data not shown). Clones of TR and RXR were
described previously (19, 31); these plasmids were linearized with
EcoRI and in vitro synthesis of capped mRNA
for microinjection was performed with an mMessage Machine kit (Ambion)
exactly as described (19).
Preparation, Microinjection, and Maintenance of Oocytes and
Primer Extension--
Oocytes were prepared and injected with mRNA
and DNA exactly as described (19, 31). Total RNA was extracted using
RNAzol (Tel-Test; Friendswood, TX) and assayed by primer extension with SuperScript reverse transcriptase (Life Technologies, Inc.) as described (19, 31). The reaction contained labeled primers that
detected both mRNA synthesized from the reporter construct as well
as an RNA stored in the oocyte that served as a loading control during
analysis on 6% sequencing urea-polyacrylamide gel electrophoresis (19,
31).
Mapping of DNase I Hypersensitive Sites in
Chromatin--
Oocytes were injected with 5 ng of RXR
mRNA and
5 ng of TR mRNA; following incubation for 10 h, 1 ng of
double-stranded pTR
A DNA was injected. After 16 h of
incubation, the oocytes were rinsed in MBSH medium, and 20 oocytes per
experimental sample were homogenized in 10 mM Hepes, pH
7.6, 25 mM KCl, 5 mM MgCl2, 5%
glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT (42). The homogenate was placed in a 37 °C water
bath for 1 min, and DNase I (DPRF grade; Worthington) was then added to
110-132 Worthington units/ml. Aliquots of 100 µl were removed from
each sample at 1 and 3 min following addition of the enzyme and mixed
vigorously with an equal volume of stop buffer (20 mM Tris,
pH 7.4, 200 mM NaCl, 2 mM EDTA, 2% SDS).
Proteinase K (Roche Molecular Biochemicals) was added to 700 µg/ml,
and after incubation overnight at 37 °C, the DNA was extracted twice
with an equal volume of phenol/chloroform/isoamyl alcohol (50:48:2,
v/v/v), once with chloroform, precipitated with sodium acetate, and 0.7 volumes of isopropyl alcohol for 1 h on ice, and resuspended in TE
buffer. RNA was removed by treatment with 100 µg/ml RNase A (Roche
Molecular Biochemicals), and the DNA was digested to completion with
EcoRI, followed by brief treatment with proteinase K,
phenol/chloroform extraction, and precipitation with isopropyl alcohol
as described above. The DNA was resuspended in TE buffer and
fractionated (1 oocyte equivalent per sample) on a 2% high resolution
blend (Amresco; Solon, OH) agarose gel in 1× TAE. Southern blotting
with random prime-labeled probes was performed by transferring the DNA
to 0.2 µm pore size Nytran Plus nylon membranes (Schleicher & Schuell), hybridizing, and washing as per the manufacturer's
instructions. The final wash was performed in 0.2× SSC, 0.5% SDS,
65 °C. In Figs. 1C and 4A, the probe used for
Southern blotting hybridized to the 3'-most portion of the TR
A gene
construct and in Fig. 4B to the 5'-most portion. Filters
were exposed to PhosphorImager screens or to x-ray film as described above.
Electromobility Shift Assays (EMSA)--
Proteins for EMSA were
prepared by in vitro translation in a coupled
transcription-translation system exactly as described (19). Equal
volumes of reticulocyte lysate containing TR and RXR were incubated in
binding buffer (43) in the presence of 1-2 µg
poly(dI-dC)-poly(dI-dC) (Amersham Pharmacia Biotech) for 10 min on ice;
5 or 10 pmol of unlabeled competitor double-stranded oligonucleotide
and 0.2 pmol 32P-labeled double-stranded oligonucleotide
probe were then added, and the incubation was continued for an
additional 35 min at room temperature prior to electrophoresis on a 5%
polyacrylamide gel electrophoresis in 0.5× TBE at room temperature,
drying, and autoradiography. All oligonucleotides used in the study
were designed to have identical 4-bp 5' overhangs and to yield 28-bp
fragments once the overhangs were filled in. The malic enzyme TRE (44)
has the sequence GGGTTAggggAGGACA.
Mapping of MPE Cleavage Sites in Vivo--
A large group
of oocytes was injected with 0.5 ng of single-stranded pTR
A DNA
(gift of Dr. Trevor Collingwood); after 8 h, 5 ng each
Xenopus TR
and RXR
mRNA was injected. After
culture for 16 h in the presence or absence of 100 nM
T3, chromatin was treated with methidiumpropyl EDTA
as described (45, 46), with the following modifications: 7 oocytes were
homogenized in 350 µl of room temperature 0.3 M sucrose,
10 mM Tris-Cl, pH 7.5, 60 mM KCl, 15 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride,
1 mM DTT, 0.15 mM spermidine, 0.5 mM spermine. Methidiumpropyl EDTA (Sigma) was diluted to a
final concentration of 5 mM in water and stored at
20 °C in single-use aliquots. Immediately prior to use, one such
aliquot was diluted with water to a final concentration of 1.25 mM and mixed with an equal volume of 1.25 mM
ferrous ammonium sulfate
(Fe(NH4)2(SO4)2 (Sigma)
prepared from a fresh 0.5 M aqueous stock. DTT was added to
the MPE-Fe mixture to a final concentration of 10 mM from a
1 M stock prepared immediately prior to use. Freshly diluted hydrogen peroxide (Fisher) was added to the oocyte homogenate to 4 mM final, followed by the addition of 37.5 µl of the
MPE-Fe-DTT reagent (final MPE concentration, 62.5 µM).
The reaction was allowed to proceed at room temperature for 3 or 6 min
and was stopped by addition of bathophenanthroline disulfonate (Fluka)
to 5 mM final and 1/4th volume of stop buffer (2.5% SDS,
50 mM EDTA, 50 mM Tris-Cl, pH 7.5). DNA was
purified by treatment with proteinase K and phenol extraction and
analyzed by Southern blotting as described above.
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RESULTS |
The TR
A Promoter Contains Multiple in Vivo TR-binding
Sites--
An outline of the experiment is shown in Fig.
1A; we first microinjected
mRNAs for TR and RXR into the oocyte cytoplasm, and following
receptor synthesis, we injected reporter DNA, the Xenopus TR
A promoter (36), into the nucleus. After an incubation period to
allow chromatin assembly (14), we measured transcriptional activity of
the reporter by primer extension. Because the oocyte lacks endogenous
TR (31), high levels of basal transcription driven by the reporter were
insensitive to T3 in the absence of added TR (Fig.
1B, lanes 3 and 4). In contrast, when TR/RXR was introduced into the oocyte, the promoter was very efficiently silenced
(Fig. 1B, compare lane 5 and lanes 3 and 4), and addition of T3 to the medium
activated transcription above basal levels (Fig. 1B, compare
lanes 3 and 4 with lane 6). These data
agree with published observations from our laboratory (31).

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Fig. 1.
TR-dependent regulation of
transcription and chromatin structure in the oocyte. A,
outline of the experiment. B, uninjected oocytes
(lanes 1 and 2), oocytes injected with
double-stranded pTR A DNA (lanes 3 and 4), or
with TR/RXR mRNA and pTR A DNA (lane 5 and
6) were cultured in the presence of T3 (where
indicated), followed by RNA extraction and primer extension (see
"Experimental Procedures"). Histone H4 mRNA that is stored in
the oocyte was used as a loading control; transcriptional activity
(txn) of the TR A gene promoter (upper panel)
was measured in each sample by normalizing against H4 mRNA levels
(lower panel) in that sample and defining the average of
transcriptional activity in lanes 3 and 4 (i.e. in the absence of injected mRNA) as 1 unit.
C, oocytes left uninjected (lanes 2-5) or
injected with TR/RXR mRNA (lanes 6-9) were cultured in
the presence of T3 where indicated and treated with DNase
I, and DNA was purified and analyzed by indirect end labeling (see
"Experimental Procedures"). The arrow represents the
TR A gene, and the zigzag designates the receptor- and
hormone-dependent disruption of chromatin upstream of the
transcription start site. Lane "6 light" is a lighter
exposure of lane 6, and the positions of the
TR-dependent footprints (relative to +1) are shown to the
right of the gel.
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Like other NHRs (4), liganded TR targets disruption of histone-DNA
contacts to the immediate vicinity of its bona fide binding
sites in chromatin (26). We used DNase I footprinting coupled with
indirect end labeling (47, 48) to map cis acting elements
that allow the TR
A promoter to respond to receptor and hormone. As
expected (30), TR-dependent binding and
TR/T3-dependent disruption was observed over
the previously characterized (49) TR response element (TRE) located at
+276 relative to the transcription start site (open ellipse
in Fig. 1C, compare lanes 2-5 with lanes 6-9).
In the presence of TR and T3, we reproducibly observed a
disruption of histone-DNA contacts ~500 bp upstream of the
transcription start site (zigzag between lanes 5 and 6 in Fig. 1C). In the absence of TR, this
stretch of chromatin did not respond to T3 (compare lanes 2 and 3 with lanes 4 and
5 in Fig. 1C), indicating such disruption to be
TR-dependent. Importantly, identical induction of this
DNase I-hypersensitive site by T3 was observed in the presence of
-amanitin (data not shown), which abrogates all RNA polymerase II-dependent transcription in the oocyte,
indicating such chromatin disruption to be a direct, rather than a
secondary, consequence of TR-T3 action.
Within this ~300-bp DNA segment, at least 3 receptor footprints were
reproducibly visualized both in the presence (open circles in Fig. 1C, compare lane "6 light" with
lanes 4 and 5) and absence (lanes 8 and 9) of hormone, indicating that unliganded TR can access
its binding sites within unremodeled chromatin. In multiple experiments
(data not shown), even in the absence of T3 we observed three TR-dependent,
-amanitin-insensitive footprints in
this area. We therefore conclude that in addition to the previously characterized TRE downstream of the transcription start site (49), the
TR
A promoter contains an upstream TR- and T3-responsive
segment of chromatin that can be bound by TR in vivo via 3 target sites. Furthermore, although we cannot formally exclude the
possibility that chromatin structure alterations we describe (Fig.
1C) are due to secondary effects of unliganded TR, our data
argue very strongly against this notion.
Noncanonical TREs in the TR
A Promoter--
The DNA sequence of
the upstream stretch of the TR
A promoter bound by TR in
vivo (Fig. 1C) is shown in Fig.
2A, and it contains three
matches to a consensus TR response element (22). We used indirect end
labeling with custom size markers and nucleotide resolution DNase I
footprinting analysis to confirm that the footprints observed in
vivo (Fig. 1C) coincide with these TREs (data not shown). The TRE located at
515 matched the canonical response element
arrangement of a direct repeat of two hexameric half-sites spaced by 4 bp ("DR4"), whereas the TRE at
690 had a half-site spacing of 3 bp ("DR3"), and the TRE at
780 of 2 bp ("DR2"). The primary
sequences of the half-sites diverged from the canonical "AGG(T/A)CA" consensus in precise agreement with in
vitro data on TR/RXR binding to DNA (43, 50) (see
"Discussion").

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Fig. 2.
Multiple TR-binding sites in the
TR A gene locus. A, sequence of
the TR A enhancer; location of partial sequence matches to TRE
consensus and restriction sites used for custom markers in Fig.
4B and 5E are indicated. B, in
vitro synthesized TR and RXR were incubated with a
32P-labeled double-stranded oligonucleotide probe
corresponding to the Xenopus TR A promoter exon B TRE
(site 1). In lanes 2-15, a 25- or 50-fold molar excess
(even- and odd-numbered lanes, respectively) of
the indicated unlabeled double-stranded oligonucleotide was included in
the reaction. The mal.enz. TRE competitor represents
the well characterized TRE from the malic enzyme promoter (44); the
mut. TRE is an oligonucleotide of identical length with
point mutations in residues critical for binding by TR/RXR (50), and
non-sp. is an oligonucleotide of identical length of random
sequence. The reaction mixes were resolved by EMSA and the
bound and free bands quantitated via
PhosphorImager analysis. To quantify relative competitor potency, the
intensity of the shifted band (TR/RXR-DNA) derived from
receptor binding in the absence of competitor (lane 1) was
set to 100%, and the signal obtained in the presence of the various
competitors was expressed as a fraction thereof (shown below
each lane as % bound).
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We used an electromobility shift assay (EMSA) to confirm that these DNA
sequences can bind TR/RXR. As shown in Fig. 2B, a 25- or
50-fold molar excess of these putative TREs competed with a canonical
response element for binding by TR (lanes 5-11). Consistent with their divergence from a 4-bp half-site spacing, elements 2 and 3 bound TR/RXR with lower affinity than element 4 (compare lanes
8-11 with lanes 6 and 7) or a well defined
TRE from the human genome (lanes 4 and 5). A
"TRE" with point mutations in all DNA residues crucial for binding
(50) (lanes 12 and 13) or a DNA segment of equal
length but random sequence (lanes 14 and 15) both
failed to compete for TR/RXR binding (compare with lane 1).
We obtained identical results in experiments using all these TREs in
labeled form rather than as competitors (data not shown).
We conclude that the putative upstream enhancer of the TR
A gene
contains 3 binding sites for TR/RXR, 2 of which have altered half-site
spacing relative to a canonical TRE (51). We show that these unusual
TREs can bind TR/RXR in vitro in a sequence-specific manner,
albeit with reduced affinity relative to canonical TREs.
The TR
A Enhancer Functions in Vivo to Augment Response to
T3--
We used in vivo transcription assays to
determine if the putative enhancer has a role in mediating response to
T3. We hypothesized that its function may be to expand the
range of regulation effected by hormone. If that were indeed the case,
then a construct containing site 1 (Fig. 2A) as its sole TRE
would be expected to exhibit a less prominent response to ligand than
the wild-type promoter. We compared the full-length promoter to a
truncated version in which all 3 enhancer TREs were deleted. In
multiple experiments we reproducibly found that the
enhancer
construct was repressed by TR as efficiently as the wild-type promoter
(compare lanes 2 and 5 in Fig.
3A). This indicated that TR
binding to site 1 is sufficient to allow full repression by unliganded
TR/RXR on this promoter. In contrast, activation by liganded TR/RXR was reproducibly impaired by deleting the enhancer (compare lanes 3 and 6 in Fig. 3A). These observations
confirmed our hypothesis that the enhancer functions to augment
transcriptional activation driven by liganded TR/RXR.

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Fig. 3.
The TR A enhancer
functions in vivo to augment regulation by
T3. A, oocytes were injected with TR/RXR
mRNA (lanes 2-6), followed by injection of pTR A
(lanes 1-3) or pTR A-( enhancer) (lanes
4-6) DNA, and culture in the presence or absence of
T3 as indicated. Transcriptional activity of the TR A
promoter was measured as described in Fig. 1. A summary of data from 4 independent experiments is shown in the graph on the right;
the S.E. for TR + T3 samples in lanes 3 (wild-type promoter) and 6 (enhancer deletion) were
10.8 ± 2.4 and 2.7 ± 0.5, respectively. B,
oocytes were injected with increasing quantities of TR/RXR mRNA as
indicated, followed by injection of pTR A (lanes 1-7) or
pTR A-( site 1) (lanes 8-14). Following culture in the
presence or absence of T3, transcriptional activity of the
TR A promoter was measured as described in Fig. 1. A summary of data
from 4 independent experiments is shown on the graph below (the S.E.
for lane 12 is 2.45).
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If the enhancer TREs are bona fide low affinity binding
sites for TR, as indicated by our EMSA data (Fig. 2B), then
a deletion of the highest affinity (i.e. site 1) TRE from
the full-length TR
A promoter would be expected to impair the
resulting response of the construct to low concentrations of TR/RXR,
whereas a normal range of regulation by T3 in the presence
of high quantities of the receptor should be observed. We microinjected
several TR/RXR mRNA concentrations into the oocyte, and we used
EMSA to confirm that the quantities of mRNA we inject fall within
the range of a linear translational response (data not
shown). We then injected the wild-type TR
A promoter, and a construct
in which the major TRE at +276 was replaced by an NsiI site
(pTR
A-(
site 1)) into oocytes, and we evaluated their
transcriptional competence when challenged with a range of TR amounts
and in the presence and absence of T3.
In agreement with our hypothesis that the putative enhancer can confer
response to hormone, we reproducibly found that at intermediate and
high concentrations of TR, the
site 1 construct responded to
T3 in a manner identical to that of the wild-type promoter
(Fig. 3B, compare lanes 4-7 with lanes
11-14). In addition, we reproducibly observed that at low
concentrations of TR/RXR, the construct containing only the lower
affinity enhancer TREs was impaired relative to the wild-type promoter
in repression by unliganded TR (compare lane 2 with
lane 9) and also moderately impaired in activation by
liganded TR (compare lane 3 with lane 10). These
data support our hypothesis that the enhancer contains low affinity
in vivo binding sites for TR.
We conclude that the multiple TREs found upstream of the TR
A
promoter transcription start site form a bona fide enhancer for TR/RXR action in vivo; we show that the enhancer
augments the range of regulation affected by T3 (Fig.
3A) and that it can mediate response to T3 when
sufficient quantities of receptor are present in the system (Fig.
3B, lanes 11-14).
A TR-bound Array of Translationally Positioned Nucleosomes within
the Enhancer--
The TREs within the TR
A enhancer diverge from
consensus and yet bind TR in vivo (Fig. 1C) and
respond to T3 when bound (Fig. 3). We hypothesized that
some feature of in vivo nucleoprotein organization of this
DNA stretch potentiates TR action on the enhancer. Three NHR-controlled
promoters offer examples of such potentiation; these are GR regulation
of the MMTV LTR (4-6), and estrogen receptor (ER) regulation of
the pS2 (10, 11) and the vitellogenin B1 (52) promoters. In all these
cases, nonrandom histone octamer positions relative to the DNA within the target locus have been implicated in augmenting response to hormone.
We investigated directly whether the TR
A enhancer forms a specific
chromatin organization in vivo by using the chemical agent methidiumpropyl-EDTA-iron(II) (MPE); it exhibits a marked preference for cleaving linker DNA while displaying negligible primary sequence bias (45, 46, 53). In contrast to MPE action on naked DNA, which
generated a homogeneous smear (Fig.
4A, lanes 2 and 3), chromatin templates exhibited a marked and reproducible nonrandom sensitivity to MPE; over multiple experiments we consistently found
that, even in the absence of TR, pronounced sites of cleavage by MPE
spaced by ~180 bp were observed in chromatin (lanes 4 and 5). In the presence of unliganded TR no significant
alteration was observed (compare lanes 4 and 5 with lanes 6 and 7), whereas addition
of T3 lead to a localized destruction of this regular chromatin array (lanes 8 and 9; zigzag
bar next to autoradiograph). Identical disruption was observed in
the presence of
-amanitin (data not shown).

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Fig. 4.
The TR A promoter and
enhancer are bound into an array of translationally positioned
nucleosomes in vivo. A, oocytes were
injected with single-stranded pTR A DNA (lanes 4-9),
followed by injection TR/RXR mRNA (lanes 6-9), and
culture in the presence or absence of T3 as indicated.
Oocytes were treated with MPE (see "Experimental Procedures"), and
MPE cleavage sites in chromatin were revealed by indirect end labeling
exactly as in Fig. 1C. Lanes 2 and 3
contain sample of naked pTR A DNA treated with MPE. Lane
10 contains custom size markers. A schematic representation of the
nucleosomal organization of the locus (ellipses) relative to
the transcription start site (arrow), TRE location
(filled circles), and
TR/T3-dependent chromatin disruption
(zigzag) is included to the right of the
autoradiograph. B, MPE-treated chromatin was analyzed by
indirect end labeling with a probe that hybridizes to the upstream end
of the TR A gene. Custom size markers in lane 10 reveal
the location of the enhancer TREs (see Fig. 2A for location
of restriction enzyme recognition sites). Lane "7 dark"
represents a longer exposure of the sample in lane 7. The
schematic is annotated as above. C, a summary of the
relative location of histone octamer positions and TR-binding sites in
the TR A promoter/enhancer in vivo.
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These data indicate that the TR
A promoter is assembled into an array
of "translationally" positioned nucleosomes in vivo (lanes 4 and 5; octamer position shown
schematically to the right), that unliganded TR can bind to
this promoter without disturbing this array (lanes 5 and
6; approximate location of TR footprints shown as
filled circles), and that in the presence of ligand, TR
destroys this positioned array over ~4 nucleosomes, while leaving other nucleosomes in this area intact. We note, however, that whereas
these data reveal the predominant positions of histone octamers over
this locus relative to the DNA, they cannot be used to determine
nucleosome translational frames to nucleotide precision nor used as
evidence for identical nucleosome positions over every template in the
nucleus; thus, the existence of a frequency-biased distribution of
multiple frames (54, 55) is possible.
To obtain a more precise estimate of the location of the new TREs we
described in the enhancer relative to predominant nucleosome positions,
we used custom size markers (4) that migrate at the exact location of
the TR footprints (these are prepared by digesting genomic DNA with a
restriction enzyme with a cleavage site close to the location of the
TRE, see Fig. 2A). A sample of MPE-treated chromatin in the
presence and absence of TR was analyzed by indirect end labeling along
such markers (shown in lane 10). This analysis revealed that
site 4 (the TRE at position
515 located over the SmaI site
(Fig. 2A)) is in a linker region between two nucleosomes and
that site 3 (the TRE at position
690) found just upstream of the
EarI site (Fig. 2A) is also in a linker region,
175 bp away (Fig. 4B, lanes 6 and 7, and also
"7 dark" which represents a longer exposure of the
sample in lane 7). Thus, two molecules of TR/RXR bound to
sites 3 and 4 straddle linker DNA entering and exiting the same
translationally positioned histone octamer. In contrast, site 2 (the
TRE at position
780 immediately downstream of the EagI
site) is only 90 bp away from linker DNA and is, therefore, located on
the surface of a histone octamer.
We reproducibly found an area of TR-independent MPE hypersensitivity
immediately upstream of site 2; the resulting stretch of DNA resistant
to MPE was ~110 bp in length, i.e. less than 1 nucleosome
(see stretch of chromatin between EagI and ApaI
sites in Fig. 4B, lanes 4-7). We do not understand the
structural basis of this nucleoprotein assembly, and we have
provisionally represented the adjacent DNA as a remodeled nucleosome
(dotted ellipse); it is possible that factors endogenous to
the oocyte exert some unilateral (i.e. from the upstream
side only) disruption of histone-DNA contacts in the area.
Our data on nucleosome and TRE position in the area are summarized in
Fig. 4C; we show that nucleosomes in this area exhibit a
nonrandom distribution relative to the DNA and that the TR
A enhancer
is assembled into 3 translationally positioned nucleosomes, with 2 molecules of TR/RXR bound to linker DNA, and a 3rd molecule occupying
the surface of a nucleosome-like particle.
TR Binding and Action on the TR
A Enhancer Is Potentiated by
Chromatin Assembly--
The nonrandom chromatin infrastructure over
the enhancer (Fig. 4) suggested that its dynamic assembly might affect
the ability of TR/RXR to act via this stretch of DNA. The
Xenopus oocyte offers a unique opportunity to investigate
this issue directly; the assembly of microinjected double-stranded DNA
into chromatin occurs gradually over the course of several hours (14).
For example, as shown in Fig.
5B, 1 h after injection
into the oocyte nucleus, pTR
A DNA is highly sensitive to
micrococcal nuclease, and nucleosomal density is low
(lanes 2 and 3); after 3 h nucleosome
density increases, but the bulk of the signal is derived from mono- and
dinucleosomal particles, and the signal from a "seminucleosome"
(0.5n), a chromatin assembly intermediate (56, 57), is
conspicuous. By later time points (lanes 11 and
12), chromatin assumes a more mature configuration, and
signal from assembly intermediates is undetectable.

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Fig. 5.
Chromatin assembly potentiates binding to and
action by TR/RXR on the TR A enhancer.
A, schematic representation of the experiment. B,
kinetics of assembly of double-stranded pTR A DNA following injection
into oocytes were monitored by micrococcal nuclease cleavage and
Southern blotting exactly as described (19). The location of various
cleavage intermediates and a scan of the indicated lanes is included to
the right of the autoradiograph. C, levels of TR
in the oocyte during the experiment shown in D were
monitored by extracting protein from uninjected oocytes (lane
1) or oocytes injected with TR at the indicated time points, and
Western blotting with an antibody against Xenopus TR (20)
was performed as described (19). The location of size markers is
indicated to the left; the question mark
designates a large polypeptide that cross-reacts with the antibody and
serves as a loading control. D, oocytes were injected with
TR/RXR mRNA as indicated. Double-stranded pTR A or
pTR A-( site 1) DNA was then injected as indicated, and oocytes
were processed for primer extension 2.5 (lanes 1-6) or
16 h (lanes 7-12) following DNA injection.
Transcriptional activity was measured as described in Fig. 1.
E, oocytes left uninjected with mRNA (where indicated)
or injected with TR/RXR mRNA were injected with double-stranded
pTR A and treated with DNase I exactly as described in Fig.
1C at 2.5 (lanes 1-7) and 16 h (lanes
8-10) following DNA injection. TR/RXR footprints were revealed by
indirect end labeling using custom size markers that designate the
location of TR footprints exactly as in Fig. 2B. A scan of
the indicated lanes is included to the right of the
autoradiograph. The position of the enhancer is indicated by the
shaded bar, and the position of the
TR/RXR-dependent footprints is indicated by the black
ovals.
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This interesting property of the oocyte, i.e. a kinetics of
chromatin assembly that is markedly delayed relative to the 30-min replication-coupled pathway (58), allows for the experimental arrangement shown in Fig. 5A; oocytes are injected with
mRNA and incubated for a set length of time to allow receptor
synthesis. DNA is then injected, and oocytes are cultured in the
presence or absence of T3; groups of oocytes are removed at
specific time points following DNA injection, and transcription is
measured (or chromatin structure is assayed, see below). Thus, the
regulatory competence of TR/RXR can be examined on templates that are
in various states of chromatin assembly.
In such experiments, we reproducibly found that the full-length
TR
A promoter was regulated by T3 with equal efficiency
at all states of template chromatinization (Fig. 5D, compare
lanes 1-3 with lanes 7-9). In contrast, a
construct that contained only the TR
A enhancer (i.e. no
site 1) was robustly responsive to TR when DNA was fully chromatinized
(lanes 10-12) but impaired in response to hormone at early
time points (lanes 4-6), when DNA is in immature, partly
assembled chromatin (Fig. 5B, lanes 2 and 3 and
5 and 6). We reproducibly found that at early
time points following DNA injection, the
site 1 construct could not be repressed by unliganded TR/RXR (Fig. 5D, compare
lane 4 with lane 5). In addition, on immature
chromatin templates we reproducibly observed lower transcriptional
activation by liganded TR on the
site 1 construct as compared with
the wild-type promoter (Fig. 5D, compare lanes 4 and 6 with lanes 1 and 3).
The data shown in Fig. 5D had a trivial explanation; we had
earlier shown that the
site 1 construct is impaired in responding to
TR and T3 at low TR levels (Fig. 3B, lanes
8-10). It was possible that the difference we saw in TR action on
the two reporter constructs (Fig. 5D, lanes 1-3
versus lanes 4-6) was due to low TR levels at
the early time point (lanes 1-6) and an increase by the
late time point (lanes 7-12). To address this issue, we
examined the TR content of the oocyte during the course of the
experiment by Western blotting (Fig. 5C); the antibody used
recognized a ~240-kDa polypeptide (Fig. 5C, ?) in oocytes
that were not injected with TR mRNA (Fig. 5C, lane 1),
which served as a loading control. Such analysis showed that TR levels
did not change in the oocyte over the course of the experiment (Fig.
5C, compare lanes 2 and 3; also
compare TR levels in each lane to loading control), indicating that the
difference in regulation of the
site 1 construct by TR at various
time points (Fig. 5D, lanes 4-6
versus 10-12) was not due to a change in TR
concentration over the course of the experiment.
The impaired ability of TR to act via response elements bound into
immature chromatin (Fig. 5D, lanes 4-6) suggested that chromatin assembly might potentiate the physical interaction of TR/RXR
with the enhancer. To investigate this issue, we performed genomic
footprinting experiments identical to those shown in Fig. 1C, except we assayed TR/RXR binding to chromatin at various
time points following DNA microinjection (the experimental outline is
shown in Fig. 5A). As shown in Fig. 5E, at an
early time point, no detectable change in chromatin architecture over
the enhancer could be detected in the presence of TR/RXR (compare
stretch of chromatin designated by the shaded bar, the
enhancer, in lanes 2-4 with lanes 5-7).
PhosphorImager-based comparison of samples containing and lacking TR
yielded identical scans (scans of lanes 3 and 6 in Fig. 5E are shown to the right of the gel).
In contrast, after 16 h of chromatin assembly, 3 footprints were
observed over this area in the presence (lane 9) but not in
the absence (lane 8) of TR. A PhosphorImager scan of the
sample is shown to the right of the gel (lane 9),
and the location of the footprints, which was coincident with custom
markers (lane 11) that designate the position of the TREs
(Fig. 2A), is indicated by shaded ovals.
We conclude that action of TR/RXR on the enhancer element we have
described is potentiated by assembly of the target DNA into chromatin
(Fig. 5D). We show that the capacity of unliganded TR to
bind the unusual TREs in the TR
A enhancer is regulated by the extent
of template chromatinization, such that more mature chromatin templates
are bound to a greater extent than those in the process of assembly
(Fig. 5E). Importantly, this change in the functional
properties of TR (Fig. 5D) and in its ability to bind the
enhancer (Fig. 5E) occurs without any detectable alteration of TR protein levels (Fig. 5C).
 |
DISCUSSION |
The thyroid hormone receptor can regulate transcription in
vitro on naked DNA templates, i.e. in the absence of
any chromatin (59, 60). In the nucleus, however, the receptor
establishes a poorly understood functional relationship with the
chromatin infrastructure of its target promoters (21, 22). The present study describes a positive role for a structurally mature chromatin template in allowing maximal response to TR and T3; we show
that a naturally occurring TR-regulated gene contains an upstream
enhancer bound into an array of translationally positioned nucleosomes in vivo (Fig. 4), and we demonstrate that this nucleoprotein
entity potentiates binding by TR/RXR to multiple nonconsensus TREs
located in the enhancer (Figs. 1C, 2A and
5E), thereby expanding the range of regulation effected by
the liganded NHR (Figs. 3A and 5D). To the best
of our knowledge, these are the first data in the literature that a
non-viral NHR target gene presents itself to the receptor in the form
of a specifically structured chromatin template that is required for
full range regulation by hormone.
The previously described (49) TR-binding site in the TR
A promoter
("site 1" at +276) is sufficient to make it responsive to
T3 (Fig. 3A). We found, however, that its action
cannot account for the entirety of T3 response (Fig. 3),
and we present evidence that additional regulation is imparted by a
segment of DNA located at positions
800 to
500. This element fits
several criteria for an enhancer; it is bound by TR in vivo
(Fig. 1C); it contains 3 sequence elements that can be bound
by TR in vitro (Fig. 2B); it is sufficient to
enable response to TR (Fig. 3B) and allows liganded TR to
drive higher levels of transcription than can be achieved via a single
TRE (Fig. 3A). Our data agree with findings of an earlier
study (61), in which EMSA and transient transfection assays suggested
the existence of TR-responsive elements upstream of the TR
A
transcription start site.
Our footprinting (Fig. 1C), EMSA (Fig. 2B), and
functional (Fig. 3A) data reveal the existence of 3 unusual
TREs in the TR
A gene enhancer. The primary sequence of these
response elements diverges from the established AGG(A/T)CA consensus;
for example, the TR-bound right half-site (51) of element 4 is GGGTTA
and of element 3 is GTGTCA. Both class I (62, 63) and class II NHRs
(50) are known to tolerate variation in primary DNA sequence and
half-site spacing of their target sites. The naturally occurring TREs
in the TR
A gene locus conform to findings from several in vitro studies (43, 50, 64); for example an "A-G" transition at
position 1 in the AGGTCA half-site was shown to enhance TR/RXR binding
by ~40%, whereas a "C-T" transition in position 5 reduced it to
~60% of wild type (50). The resulting half-site, GGGTTA, is found in
the TR
A enhancer and efficiently binds TR/RXR in vivo
(Fig. 1C) while exhibiting only a moderate reduction in
affinity in vitro (Fig. 2B). The 1.9-kb TR
A
promoter fragment contains ~20 sequences that are partial
(mismatch = 3 nucleotides) matches to the TRE "consensus"
(data not shown), but only 3 are bound by TR in vivo (Fig.
1C); comparison of the remaining 17 to data presented by
Judelson and Privalsky (50) indicates that the mismatches would be
expected to significantly impair receptor binding. Our in
vivo data thus fully support the notion that TR exhibits a
restricted, rule-based promiscuity in DNA binding.
Our data indicate that binding and action by TR on the enhancer are
potentiated by chromatin assembly (Fig. 5), a surprising finding
considering that nucleosomes impede binding to DNA by most nonhistone
factors (66, 67) with only a few exceptions (65). In the promoter we
study, the translational frame of nucleosome occupancy is
receptor-independent (Fig. 4A), and sites 3 and 4 are
presented to the receptor in linker DNA surrounding the same nucleosome
(Fig. 4C). It is possible, therefore, that the spatial proximity of DNA entering and exiting the histone core creates a
"static loop" (7, 8, 10, 11, 52) that enables an interaction
between two TR/RXR heterodimers. It is also possible that the unwinding
and distortion of the DNA helix complexed with histones (68)
potentiates binding by TR/RXR to site 2 (a motif of two direct repeats
spaced by 2 bp) by increasing the distance between the two half-sites
to a level more compatible with heterodimerization on the DNA of the
two NHRs (which normally require a 4-bp spacer (51)).
Naturally occurring promoters frequently contain multiple binding sites
for a particular regulator (69, 70), and many NHR-responsive genes are
controlled via arrays of receptor response elements; for example, the
MMTV LTR contains at least 6 GR response elements (71), and the
androgen receptor (AR)-regulated prostate-specific antigen
enhancer includes 4 AR response elements (72). Such promoter
structure is thought to enable a sigmoidal response curve to a rise in
regulator titer (69, 70, 73, 74). The TR
A promoter conforms to this
paradigm, having evolved under selective pressure to coordinate ligand
and receptor levels and allow for autoregulation by hormone of the
titer for its own receptor during metamorphosis (75, 76). In addition,
our data illuminate a novel role for chromatin assembly and primary
promoter sequence in a general phenomenon, enabling a correlation
between titers of a given NHR and its cognate ligand (76), which has
been observed for GR (77-79), ER (80), as well as the
androgen (81, 82), ecdysone (83), and retinoic acid (84) receptors.
Early in development, when TR levels are low, regulation of the TR
A
gene likely occurs via the site 1 TRE, which has the highest
affinity for TR (this is sufficient for repression (Fig. 3A)
and moderate activation (Fig. 3B)). As hormone and receptor titers rise during ontogeny, binding by TR to the lower affinity enhancer TREs (Fig. 2B) is predicted to lead to graded
transcriptional activation above basal levels (Fig. 3, A and
B). Thus, one possible developmental function of the
enhancer may be to mediate the positive, feed-forward branch of the
autoregulatory loop between T3 and TR; the placement of
lower affinity TREs in the enhancer ensures that it begins to function
only when TR titer is sufficiently high.
An important component of an autoregulatory circuit is the attenuation
of response to signal which, in the case of TR and T3, is
partially effected by proteolysis of liganded TR (19, 85). We suggest
that the unique structure of the TR
A enhancer may also contribute to
such attenuation; binding by unliganded TR/RXR to the enhancer is
potentiated by assembly of the DNA into a mature array of
translationally positioned nucleosomes (Fig. 5E) and
liganded TR disrupts this array (Figs. 1C and
4A), thereby creating a lower affinity template for itself.
Thus, TR-targeted chromatin disruption may lead to receptor release
from the enhancer and a lowering of transcription initiation rates
(Fig. 3A). In vivo, non-NHR transcriptional
regulators such as Swi5p (86), and such NHRs as GR (71, 87) and ER
(12), bind chromatin only transiently, and it is quite possible,
although unproven, that the large ATP-dependent chromatin
remodeling complexes they target are partly responsible for such
"hit-and-run" action. In our model system this issue can be studied
with in vitro assembled chromatin templates that have been
shown to recapitulate certain aspects of gene regulation by NHRs (37,
71, 88).