Involvement of the Transcription Factor IID Protein Complex in Gene Activation by the N-Terminal Transactivation Domain of the Glucocorticoid Receptor in Vitro
Jacqueline Ford,
Iain J. McEwan,
Anthony P. H. Wright and
Jan-Åke Gustafsson
Department of Biosciences at Novum, Karolinska Institute,
S-141 57 Huddinge, Sweden
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ABSTRACT
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HeLa cell nuclear extracts were used to study the
mechanism of activation of RNA polymerase II-mediated transcription by
the N-terminal transactivation domain (
1) of the glucocorticoid
receptor in vitro. When fused to the Gal4 DNA-binding
domain, the
1 domain activated transcription approximately 9-fold in
HeLa nuclear extracts. Using heat treatment to inactivate transcription
factor IID (TFIID) in the extract, it was shown that the addition of
purified TFIID complex, but not the TATA-binding protein alone, was
sufficient to restore this level of activation. The
1 domain was
shown to interact directly with the TFIID complex. This interaction was
markedly reduced by a mutation in the
1 domain that reduces its
activity. Furthermore, the interaction was specific for the TFIID
complex, since no interaction was seen with TFIIIB, an analogous
protein complex involved in RNA polymerase III transcription. The
1
domain was further shown to interact with the TATA-binding protein
subunit of the TFIID complex. These results suggest a mechanism by
which the GR
1 domain might contribute to gene activation by
recruitment of the TFIID complex to target promoters.
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INTRODUCTION
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The glucocorticoid receptor (GR) is a member of the nuclear
receptor superfamily of transcription factors that function by
modulating the activity of target genes (for reviews see Refs. 1 and
2). The amino terminus of GR contains a domain that is essential for
full transactivation activity (3, 4, 5, 6) and constitutes amino acids
77262 of the human GR (hGR) (4). When fused to a DNA binding domain,
this domain (
1) is constitutively active for transcription both
in vivo in yeast (7) and in vitro in yeast
extracts (8). The
1 domain has been further dissected, and a single
core region containing transactivation activity has been localized
(
1 core, residues 187244 of hGR). Particular segments within this
region have a propensity for
-helical conformation, and hydrophobic
amino acids that would form patches on these putative
-helices
appear to be important for the molecular mechanism by which
1
transactivates (9, 10, 11).
Stimulation of eukaryotic transcription by RNA polymerase II requires
removal or restructuring of chromatin and the ordered assembly of a
preinitiation complex at the promoter of regulated genes (12). The
preinitiation complex contains basal transcription factors, RNA
polymerase II (see Refs. 13 and 14), and additional accessory factors
such as coactivators. In vivo, in yeast and mammalian cells,
it has been suggested that this complex may be largely preassembled in
the form of a holoenzyme (15, 16, 17).
Transcriptional activators may function at one or more steps in the
processes of transcriptional initiation and elongation. However, one
important way in which many activators are thought to operate is by
interacting with component(s) of the transcription machinery to recruit
them to the promoter and so promote or stabilize the binding of the
general transcription factors to the DNA (see Refs. 12 and 18).
Interactions may occur directly between basal transcription factors and
the activator and/or may occur between the activator and coactivators
or adaptors, which form a bridge between the activator and the basal
factors (for review, see 19 .
There is a growing number of reports concerning interactions between
nuclear receptors and coactivator proteins, and for some, but not all,
direct interactions have been shown with GR. Thus the mouse protein
GRIP1 (20) and the human proteins GRIP170 (21) and RAP46 (22) interact
directly with GR and may act as coactivators. GR transactivation can
also be modestly stimulated by SRC-1 (23), which possibly occurs in a
tertiary complex involving CBP (CREB binding protein) and GR (24).
Additional proteins that can coactivate with GR include hbrm (a human
homolog of the yeast SWI/SNF proteins), which is thought to be involved
in chromatin structure modulation (25), and the yeast proteins SPT3 and
RSP5 (26). A factor RAF has also been isolated that potentiates DNA
binding by GR (27).
In contrast to the identification of these potential coactivators
for GR, there are still no reported interactions between GR and
specific basal transcription factors. In addition, no factors are
reported to bind directly to the
1 domain of GR despite the fact
that 1) it is constitutively active when fused to a DNA-binding domain
(DBD) and therefore must make sufficient interactions to activate
transcription; 2) it is the domain of GR that contributes most to the
receptors transactivation activity (4), and 3) studies on GR (and
specifically
1) suggest that it can act by recruitment and
stabilization of the preinitiation complex at the promoter (28, 29, 30),
possibly via some direct interaction(s) with component(s) of the basal
transcription machinery (8).
In recent years, much attention has been focused on interactions
between activators and TFIID. TFIID is a multiprotein complex
containing TATA box-binding protein (TBP) and tightly associated
factors termed TBP-associated factors (TAFs). TBP is a target for many
activators (for examples, see Refs. 31 and 32), but the TAFs are
necessary for activation in vitro and are also direct
targets for many transactivators (for reviews, see Refs. 3335). In
this study we investigate whether TFIID might play a role in gene
activation by the GR
1 domain.
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RESULTS
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TFIID Is Required for Activation by
1 in HeLa Nuclear
Extracts
We wished to determine whether
1 requires TFIID for activation
in vitro. It has been reported previously that
1, fused
to a heterologous DBD, activates transcription in vitro in
yeast nuclear extracts (8). In this study we used a HeLa system because
it has been reported that TBP and TFIID are heat labile in HeLa
extracts (36). We constructed plasmids for the expression and
purification of amino acids 1100 of Gal4 and a fusion protein in
which
1 is fused to Gal(1100). The Gal4 sequence is preceded by a
histidine tag, which simplified purification of the proteins (Fig. 1A
). In vitro transcription
experiments were performed in HeLa nuclear extracts with template
pG5HMC2AT (37) in which five Gal4-binding sites
are situated upstream of the HIV TATA box. A titration of Gal4-
1
protein gave a dose-dependent increase in transcription products that
required the presence of the
1 activation domain (Fig. 1B
). The
addition of 8 pmol Gal4-
1 consistently produced 7- to 10-fold
activation in our experiments (see Fig. 2B
, lanes 1 and 2).

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Figure 2. Purified TFIID Efficiently Restores 1-Dependent
Activation to Heat-Treated HeLa Nuclear Extracts
A, Western blot using an anti-TBP antibody and showing the
relative intensities of TBP contained in 1 U crude TFIID (from 1
M P11 fraction; labeled 1 M P11), 1 U
affinity-purified TFIID (labeled TFIID), 1 U rhTBP, and 10 U rhTBP. B,
In vitro transcription from HeLa nuclear extracts. Extracts
were heated at 46 C or 47.5 C as shown. After cooling they were either
not supplemented or supplemented with 1 U TFIID obtained from the 1
M P11 fraction of HeLa nuclear extracts or with 1 U
affinity-purified TFIID or with 1 or 10 U of rhTBP. Template DNA,
nucleotide triphosphates, and Gal(1100)- 1 (8 pmol, where
indicated) were then added, and the transcription reactions were
allowed to proceed. Transcripts from the Gal responsive template
pG5HMC2AT in the presence (+) or absence (-)
of Gal(1100)- 1 (8 pmol), under different supplementation
conditions, are marked (G). Fold induction (+ 1/- 1) mediated by
the addition of Gal(1100)- 1 is shown for the different
supplementation conditions. Experiments have also been performed using
a basal template as an internal standard for the level of
1-dependent activation in reconstitution reactions (data not shown).
The levels of activation under the different reconstitution conditions
in these experiments were very similar to those shown for parallel
reactions, ± 1.
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To test the functional requirement of
1 for TBP or TFIID, HeLa
nuclear extracts were heat treated, recooled, and then supplemented
with either recombinant human TBP (rhTBP) or TFIID. TFIID was either
supplied as the crude 1 M KCl phosphocellulose (1
M P11) fraction, or alternatively it was immunoaffinity
purified from the 1 M P11 fraction of LTR
3 cells, which
express HA-tagged TBP (38). The relative amounts of TBP contained in
the crude 1 M fraction and affinity-purified material were
estimated relative to rhTBP by semiquantitative Western blotting using
an antibody against hTBP (data not shown). Approximately equal molar
amounts of TFIID and rhTBP (Fig. 2A
; arbitrarily given the value of 1 U
and approximately equal to 2.5 ng TBP) were used to supplement the
heat-treated nuclear extracts. In addition, 10-fold higher amounts of
rhTBP were used (Fig. 2A
). Thus, in Fig. 2B
,
1 activated
transcription in untreated HeLa nuclear extracts by a factor of 9-fold
(Fig. 2B
, lanes 12). After the crude HeLa nuclear extracts had been
heat-treated in a 46 C water bath,
1-dependent transactivation was
lost, consistent with the destruction of an essential component of the
transcription machinery (Fig. 2B
, lane 3). Transcriptional activity was
restored by addition of both TFIID and rhTBP, but only TFIID restored
1-dependent stimulation of transcription to the starting level (Fig. 2B
, lanes 47). rhTBP restored basal transcription but had very little
effect on
1-activated levels (Fig. 2B
, lanes 811). At 47.5 C the
distinction between TFIID and TBP is even clearer such that even a
10-fold excess of TBP does not restore detectable transcription levels
while TFIID still reconstitutes
1-dependent activated transcription
(Fig. 2B
, lanes 1216). The level of reconstituted transcription at
47.5 C is somewhat lower than at 46 C, and it is possible that the
higher temperature affects the activity of other transcriptional
components.
1 Interacts with TFIID Complexes Present in Cell Extracts
We next wished to determine whether or not
1 would interact
physically with TFIID. As a control, we wanted to test the ability of
1 to discriminate between TFIID and TFIIIB (the analogous complex
specific for transcription by RNA polymerase III). Using yeast strains
that express epitope-tagged TAFs (39), we prepared cell extracts
containing either hemagglutinin (HA)-tagged yTAFII130 or
Brf1 (a polymerase III-specific TAF). When these were incubated with
the glutathione-S-transferase (GST) activation domain
constructs (Fig. 3A
), both wild type
GST-
1 and wild type GST-
1core clearly interacted with TFIID as
indicated by coprecipitation of yTAFII130 (Fig. 3B
). The
interactions were severely weakened when reduced activity mutant (H1A)
1 or
1core fusion proteins were used (Fig. 3B
). In the H1A mutant
proteins, five hydrophobic residues in
1core are substituted by
alanine, causing reduced transactivation activity in yeast and
mammalian cells (11). Under identical conditions, negligible
interaction was observed with TFIIIB (Brf1, Fig. 3C
). Thus, the
interaction was selective for the TFIID complex over TFIIIB and could
be disrupted by reduced activity mutations within
1core.
We next wanted to investigate the interaction of
1 with mammalian
complexes, and thus HeLa nuclear extracts were prepared. To partially
separate different complexes, the nuclear extracts were fractionated on
phosphocellulose (P11) (40), and 0.3 M and 1 M
KCl fractions were collected. The 0.3 M fraction contains
TFIIIB, but also additional complexes that have been reported to
include B-TFIID (41) and a subpopulation of TFIID complexes, termed
TFIID
(42). B-TFIID is not thought to contribute to polymerase II
transcription (see 41 , and TFIID
has only been reported to
have a stimulatory effect on the activator TEF-1 (43). The 1
M KCl fraction contains additional TFIID complexes
including TFIIDß (42). TFIID complexes from the 1 M
fraction have been described to be involved in transactivation by a
number of activation domains including VP16, the estrogen receptor
activation domains, the AH synthetic activator, and activation domains
from Zta, E1A, and Sp1 (37, 38, 43). The fractions were incubated with
GST-
1, and the supernatants and pellets were probed for TBP using
Western blotting and an anti-TBP antibody. While the interactions
detected in these experiments were weak, we observed preferential
binding of GST-
1 to TBP-containing complexes from the 1
M P11 fraction, and no interactions were detected with
TFIIIB complexes in the 0.3 M P11 fraction under the same
conditions (Fig. 3D
). Taken together, these results suggest that the
1 domain of GR can interact specifically with TFIID complexes
involved in transcription by RNA polymerase II.
The
1 Domain of GR Can Interact with Recombinant Human TBP
Transactivation domains from a number of activator proteins have
been reported to interact with TFIID, either via interactions with the
TBP subunit or with the TAF subunits (see Introduction).
Although we have not so far seen reproducible interactions between
1
and TAF subunits (see Discussion), we have observed
interactions between
1 and TBP. Similar to the interactions with
TFIID shown in Fig. 3B
, TBP interacts with the GST-
1 and
GST-
1core fusion proteins but not with GST alone (Fig. 4A
). Interaction of both proteins with
TBP is severely reduced by the H1A mutation that substantially reduces
the activity of the
1 and
1core domains in vivo (11).
As a further control, the binding of GST-hTBP to a series of LexA DBD
fusion proteins was tested since it is known that the Lex-
1c fusion
protein is transcriptionally active in yeast both in vivo
(9) and in vitro (I. J. McEwan, unpublished results).
Purified GST-hTBP or GST alone (Fig. 4B
) was immobilized on
glutathione-agarose beads, and equivalent amounts of beads were
incubated with recombinant purified Lex proteins (Fig. 4C
). Binding of
the Lex fusion proteins was then detected by Western blot using an
antibody to LexA DBD. The results showed that there was a strong
interaction between Lex-
1core and GST-TBP that was strongly reduced
by the H1A mutant (Fig. 4D
, upper panel). There was no
interaction between LexA DBD alone and GST-TBP (Fig. 4D
, upper
panel) or between GST and any of the LexA fusion proteins tested
(Fig. 4D
, lower panel).

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Figure 4. 1 Interacts with TBP
GST proteins were immobilized on glutathione agarose and tested for
binding to recombinant purified hTBP (A) or recombinant purified LexA
fusion proteins (D). The protein remaining in the supernatants (S)
after incubation and the protein pelleted with the beads (P) after
extensive washing
were analyzed by SDS-PAGE and Western blotting. In each case,
the pellet fractions were 8-fold concentrated relative to the load and
supernatant fractions. A, Western blot using an antibody against hTBP
and showing the interactions of hTBP with the GST activation domain
proteins shown in Fig. 3A . The diluted hTBP loaded onto the beads is
marked L. B, Coomassie-stained gel of GST and GST-TBP proteins used in
panel D. C, Western blot using an antibody against LexA DBD showing the
Lex fusion proteins used in panel D. D, Western blots using an antibody
against LexA DBD and showing the results of incubation of the Lex
fusion proteins with GST-TBP-bound beads (upper panel)
or GST-bound beads (lower panel).
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DISCUSSION
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In this study we show that the TFIID complex is necessary for the
transactivation activity of the GR
1 domain in vitro. The
1 domain can interact with TFIID directly, and at least part of the
interaction appears to involve direct contact between
1 and TBP. The
physical interactions observed are specific for the TFIID complex since
no interaction was observed with the analogous complex (TFIIIB)
involved in transcription of RNA polymerase III genes. Furthermore, a
reduced activity mutant form of
1 was severely reduced in its
ability to interact with both the intact TFIID complex and the TBP
subunit. This mutant also reduces hormone-dependent gene activation by
the intact GR in transient transfection experiments in mammalian cells
(11).
These findings are significant because no target factors have
previously been identified that might account for the activity of the
GR N-terminal transactivation domain. Early studies showed that the N
terminus of the GR (3, 4, 5, 6), and the
1 domain in particular (4), is
critical for glucocorticoid signaling such that a receptor derivative
lacking
1 was reported to have only 510% of wild type activity
(4). The TFIID complex has been shown to be important for the activity
of a number of activator proteins in vitro (see
Introduction). Evidence from these studies suggests that
interaction with the activator protein helps to recruit TFIID to the
promoter and thus facilitates binding of TBP to DNA and subsequent
assembly of the transcription complex. The importance of recruiting TBP
is also suggested by in vivo experiments in yeast, which
show that artificial recruitment of TBP alleviates the requirement for
activator proteins (45, 46). The TFIID recruitment mechanism could help
to explain previous reports in which both the intact GR and the
isolated N-terminal transactivation domain have been shown to
facilitate transcription complex formation in vitro
(28, 29, 30).
Recent evidence from studies in yeast suggests that TFIID may not be
important for activation of all genes in vivo (47, 48). The
data show that while TBP was essential for activation of all the genes
tested, mutations that drastically reduced the levels of the TAF
subunits had no effect on the activation of most of the genes studied.
However, the TAFs are essential proteins, and it appears that they are
absolutely required for in vivo activation of some genes in
both yeast and Drosophila (47, 48, 49). Thus recruitment of
TFIID by the
1 domain is likely to play a role in the activation of
at least some GR-responsive genes in vivo. The yeast results
suggest that there may be other strategies to ensure the recruitment of
TBP that do not involve the TAF subunits. One possibility is that
activator proteins interact directly with TBP (as we have shown for
1) and thus recruit it to transcription complexes independently of
the TAF subunits. While the simplicity of this model is attractive, the
interaction between activators (including
1) and TBP is relatively
weak, and so far there is no evidence to suggest that it would be
sufficient to recruit TBP independently in vivo. The
relative weakness of interactions between activation domains and TBP
may explain why the
1-TBP interaction was not detected previously
using less sensitive methods (8). Interestingly, it has been reported
that interaction of the VP16 activation domain with endogenous TBP in
yeast nuclear extracts requires an accessory protein called Ada2 (50),
which has also been identified in human cells (51). Ada2 is part of a
transcriptional adaptor complex, one function of which appears to be to
facilitate the recruitment of TBP by activator proteins. Thus the Ada
adaptor could represent an alternative to TFIID and facilitate
recruitment of TBP to TFIID-independent promoters. Interestingly, we
have shown in a parallel study that the Ada adaptor also plays a role
in the function of the GR
1 domain (52).
As stated in the Introduction, many activators that function
via TFIID have been shown to make contacts with the TAF subunits of the
TFIID complex. Within the family of nuclear receptors, the estrogen
receptor (ER) has been shown to interact with human
TAFII30
(42) while the progesterone (PR), retinoid X
(RXR), and thyroid hormone (TR) receptors interact with
Drosophila TAFII110 (53, 54, 55). The interaction of
TAFII110 with the PR is with the DBD and thus although it
may be important it does not explain the function of the activation
domains. TAFII110 was recently shown to interact with the
ligand-binding domain of the TR and although the interaction does not
require the AF-2 activation function, TAFII110 did enhance
transactivation activity substantially. In the case of RXR, TBP
interacted more strongly than TAFII110, and the TBP
interaction required the AF-2 transactivation domain while the
TAFII110 did not. In similar studies to those above, we
have tried to identify interactions with a range of
Drosophila and human TAFs, but no reproducible interactions
have been observed. Thus, while we cannot rule out that
1 interacts
with TFIID via TAFs, our present results indicate a role for
interactions between
1 and the TBP subunit of TFIID, as has been
described for the RXR AF-2 activation domain.
It could be suggested that our observation of direct interactions
between
1 and TBP is contradictory to our demonstration that the
whole TFIID complex is required for
1-mediated transcriptional
activation because
1 should be able to recruit TBP in the absence of
the TAFs. However, there are two complementary explanations that can
account for our observations: 1) The interaction with TBP could be only
one of several interactions required to recruit TFIID to promoters such
that as yet unidentified interactions with the TAF subunits could also
be important; 2) The TAF subunits of TFIID could play an essential role
in
1-mediated activation other than recruitment of the complex to
the promoter. Consistent with the latter suggestion, protein kinase and
histone acetylase enzyme activities have recently been associated with
TAFII250 (56, 57).
In conclusion, we suggest that recruitment of TFIID can account for
part of the mechanism by which the GR activates at least some target
genes. Previous studies, using cell-free transcription systems, have
suggested that the GR
1 domain may also function at one or more
steps in transcriptional activation that follow transcription complex
formation (30). That activator proteins can facilitate transcriptional
activation by several mechanisms is supported by the variety of
interactions that they make with other proteins. Thus, for VP16
interactions with TBP (58), TFIIB (59), TFIIA (60), TFIIH (61),
Drosophila TAFII40 (62), and its human homolog
hTAFII32 (63), Drosophila TAFII60
(64), the yeast coactivator Ada2 (50, 65), and the human coactivator
PC4 (66) have been reported. It remains to be determined to what extent
interactions with different targets represent independent alternative
activation strategies as opposed to constituent parts of a single
concerted mechanism.
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MATERIALS AND METHODS
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HeLa Nuclear Extract Preparation and Chromatography
Cell line LTR
3 (38), which expresses HA-tagged TBP, was
obtained as a kind gift from A. J. Berk (University of California,
Los Angeles, CA). Nuclear extract was prepared from 6l suspension
cultures as described by Dignam et al. (40), except that
extraction of nuclear proteins was performed using KCl at a final
concentration of 300 mM. Nuclear extract was dialyzed
against buffer D [20 mM HEPES-KOH, pH 7.9, 100
mM KCl, 0.2 mM EDTA, 20% (vol/vol) glycerol, 1
mM dithiothreitol (DTT), 0.2 mM
phenylmethylsulfonylfluoride (PMSF)] for direct use in in
vitro transcription assays or for fractionation. LTR
3 nuclear
extracts were fractionated by chromatography on phosphocellulose
(Whatman, Clifton, NJ) as described (40), and the fractions were
dialyzed against buffer D, except for the 1 M fractions
that were to be used for subsequent TFIID affinity purification.
Affinity purification was performed as described (38) using protein-A
Sepharose 4FF (Pharmacia, Piscataway, NJ) that had been covalently
coupled to monoclonal antibody 12CA5 (Boehringer Mannheim,
Indianapolis, IN) at a concentration of 1 mg monoclonal antibody /ml
beads. Elution of affinity- purified TFIID was performed in 2 x
50 µl using peptide YPYDVPDYA (synthesized by Neosystem Laboratoire,
Strasbourg, France) at a concentration of 1 mg/ml in buffer D
containing 200 mM KCl, 50 µg/ml BSA, 0.1% NP-40.
Yeast Whole Cell Extract and Chromatography
Yeast strains, derived from the diploid strain SEY6210.5 and
expressing HA-tagged yTAFII130 or HA-tagged Brf1 (39) were
obtained as a kind gift from P. A. Weil (Vanderbilt University
School of Medicine, Memphis, TN). Whole cell extracts were prepared
from 4l cultures according to Woontner et al. (67) and were
fractionated by Bio-Rex 70 (Bio-Rad, Richmond, CA) chromatography as
described (39, 68) to obtain 600 mM KAc fractions
containing high-salt, TBP-containing complexes.
Overexpression and Purification of Recombinant Proteins
Plasmids expressing GST-TBP (pGEX-KG-TBP) and GST-VP16
(pGEX-CS-VP16AD; 67) were kindly provided by A. Kouzarides (Cambridge,
UK) and S. A. Johnston (University of Texas, Dallas, TX)
respectively. Plasmid GST-
1 was constructed by subcloning an
EcoRI-XbaI (blunted) fragment (containing hGR
residues 77262 and additional N-terminal residues NSSSVPG) from
plasmid pEhGR770 (8) in-frame into pGEX-4T-3 (Pharmacia).
GST-
1(H1A), containing mutations F191A, I193A, L194A, L197A, F199A,
was constructed by swapping the wild type
HindIII-StyI fragment (from GST-
1) encoding
hGR amino acids 120218 for a mutated fragment from plasmid
pRS315
1(H1Ala) (gift from T. Almlöf, Stockholm, Sweden).
pRS315
1(H1Ala) contains the full-length
1 sequence mutated at the
required positions (11). GST-
1core (
1c) was constructed by
subcloning of hGR residues 187244 from plasmid
YEp
2-
1c as an in-frame fragment into the
EcoRI site of pGEX-5X-2 (Pharmacia). GST-
1c(H1A) was
constructed identically to the wild type except that cDNA containing
the five alanine mutations was subcloned from
YEp
2-
1c(H1Ala) (cDNA obtained as a gift from T.
Almlöf). The YEp
2-
1c plasmids contain the
1core sequence flanked by EcoRI (5') and BamHI
(3') sites and will be described elsewhere. Both GST-
1c and
GST-
1c(H1A) were sequenced from upstream of the fusion junctions
with GST until downstream of the stop codons because it was observed on
SDS PAGE that they ran with apparently different molecular masses. The
only differences between the sequences were due to the described
mutations. Proteins were expressed and purified according to the
manufacturers instructions (Pharmacia). Production of the LexA DBD
(Lex), Lex-
1core (
1c), and Lex-
1c(H1A) will be described
elsewhere. The plasmid expressing Gal(1100) was created by
introducing Gal4(94100) and a linker containing 3' EcoRI
and BamHI sites into pRJR1 (70) by PCR (pRJRI was a kind
gift from R. J. Reece, University of Manchester, Manchester, UK).
The sequence after Gal(100) was CTTAAGTTCCTAGG. The sequence of the PCR
fragment was confirmed by sequencing. Gal(1100)-
1 was created by
subcloning of hGR residues 77262 into the in-frame BamHI
site of Gal(1100). The histidine-tagged Gal4 proteins were expressed
and purified as previously described (70). Plasmid pET-6HishTBP, for
the expression of hTBP, was obtained as a gift from M. Meyer (EMBL,
Heidelberg, Germany). Human TBP was prepared from the vector in
accordance with the manufacturers recommendations for the pET series
(Novagen, Madison, WI).
Cell-Free Transcription Assay
In vitro transcription reactions were performed in 25
µl reaction volumes that contained 100 ng (0.04 pmol) template
pG5HMC2AT (37) (a gift from R. G. Roeder,
Rockefeller University, New York, NY) along with approximately 60 µg
HeLa nuclear extract, 050 pmol activator proteins, 80 µg/ml BSA, 12
mM HEPES-KOH, pH 7.9, 6 mM MgCl2,
12% (vol/vol) glycerol, 60 mM KCl, 0.1 mM
EDTA, 0.4 U Prime RNase Inhibitor (5 Prime to 3 Prime, Inc), 5
mM DTT, 0.4 mM each of ATP and CTP, 4
µM UTP, 5 µCi
-32P-UTP (>400 Ci/mmol,
Amersham, Arlington Heights, IL), 0.2 mM
3'-O-methyl-GTP (Pharmacia), 4 mM phosphoenyl
pyruvate (Boehringer Mannheim). Reactions were allowed to proceed for
55 min at 30 C, after which they were stopped by incubation at room
temperature for 15 min with 200 µl stop buffer [10 mM
Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA, 0.5%
glycogen, 0.4 U RNase T1 (Boehringer Mannheim)], extracted with
phenol-chloroform, and precipitated with ethanol. Transcription
products were resolved on 7% (29:1) polyacrylamide-7 M
urea gels and quantified by PhosphorImager analysis using a BAS2000
bioimaging analyzer (Fuji Film, Tokyo, Japan). Where indicated, HeLa
nuclear extract was heat treated in a water bath at 46 C or 47.5 C for
15 min and then recooled before its addition to the transcription
reaction (36). Reactions containing heat-treated extracts were
supplemented with approximately 2.5 ng TBP in the form of purified
rhTBP, TFIID from 1 M phosphocellulose fractionated HeLa
nuclear extract, or affinity-purified TFIID. rhTBP was also added at
10- fold higher amounts (
25 ng). TBP concentrations in TFIID
fractions were estimated by semiquantitative Western blotting using the
ECL system (Amersham) employing rhTBP as a standard and detecting TBP
with a primary monoclonal anti-TBP antibody (Promega, Madison, WI).
Protein-Protein Interaction Assays
GST fusion proteins were bound to glutathione agarose beads
(Sigma, St. Louis, MO) at an approximate concentration of 1 mg
protein/ml beads. The packed volume of beads was 25 µl per
experiment. Purified proteins [LexA DBD, Lex-
1c, Lex-
1c(H1A)
each at approximately 20 µg/ml; rhTBP at approximately 2 µg/ml],
were added to the beads in eight-bead volumes of IP buffer [20
mM HEPES-KOH, pH 7.9, 10% (vol/vol) glycerol, 100
mM KCl, 0.2 mM EDTA, 5 mM
MgCl2, 1 mM DTT, 0.2 mM PMSF]
supplemented with 0.01% NP-40 and 1 mg/ml BSA. Incubation with
rotation was carried out overnight at 4 C, and the supernatants were
recovered. Alternatively, 600 mM KAc fractions from Bio-Rex
70 chromatography of yeast whole cell extracts were added at
approximate protein concentrations of 1.5 mg/ml in six-bead volumes of
YB [20 mM HEPES-KOH pH 7.6, 150 mM KAc, 10%
(vol/vol) glycerol, 5 mM Mg(Ac)2, 1
mM EDTA, 0.02% NP-40, 1 mg/ml BSA, 1 mM DTT,
0.2 mM PMSF]. Incubation with rotation was carried out for
2 h at 4 C, and the supernatants were recovered. Alternatively
HeLa nuclear extracts fractionated on phosphocellulose were added to
the beads at approximate concentrations of 0.5 mg/ml (1 M
P11) or 1.3 mg/ml (0.3 M P11) in six-bead volumes of IP
buffer. Incubation with rotation was carried out overnight at 4 C, and
the supernatants were recovered. In all cases, the beads were then
washed four times with 20 vol of the corresponding incubation buffer,
and the beads or initial supernatants were boiled with SDS sample
buffer. The proteins were separated by SDS-PAGE, transferred to
nitrocellulose, and probed with the appropriate antibody. Blots were
developed using the enhanced chemiluminescence system (Amersham).
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge A. J. Berk (UCLA, Los Angeles,
CA) for cell line LTR
3, P. A. Weil (Vanderbilt University
School of Medicine, Memphis, TN) for yeast strains, T. Almlöf
(Karolinska Institute, Stockholm, Sweden), S. A. Johnston
(University of Texas, Dallas, TX), A. Kouzarides (Cambridge University,
Cambridge, UK), M. Meyer (EMBL, Heidelberg, Germany), R. J. Reece
(University of Manchester, Manchester, UK), R. G. Roeder
(Rockefeller University, New York, NY), and R. Tjian (University of
California, Berkeley, CA) for plasmids. We are very grateful to T.
Almlöf and A. Henriksson for communicating results before
publication. We thank A. J. Berk, Q. Zhou (MIT, Boston, MA) and
R. J. Reece for helpful advice during this project and E. Treuter
for interesting discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Anthony P. H. Wright, Department of Biosciences at Novum, S-141 57 Huddinge, Sweden.
This work was supported by Swedish Medical Research Council Grant
13x-2819 and Swedish Natural Science Research Council Grant
K-KV9756301. J. F. was supported by a Travelling Research
Fellowship from The Wellcome Trust, UK (Grant reference number:
040181/Z/93/Z/LEC/cg).
Received for publication March 13, 1997.
Revision received June 5, 1997.
Accepted for publication June 18, 1997.
 |
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