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Department of Pathology (C.S.V., N.R., C.Y., L.R.B., R.A.J.,
F.F.P.) Vanderbilt University Nashville, Tennessee
37232
Laboratory of Molecular Carcinogenesis (M.B.)
National Cancer Institute Bethesda, Maryland 20892
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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There is increasing evidence that the ER-ERE interaction is influenced by other proteins. For example, recombinant human ER purified from either HeLa or yeast cells fails to bind ERE (14, 15). Addition of yeast extract to purified ER restored formation of the ER-ERE complex. Mukherjee and Chambon (14) identified the yeast factor and characterized it as a 45-kDa single-stranded DNA-binding protein, termed ER DNA-binding stimulatory factor. Since then, various other proteins of sizes ranging from 30 to 160 kDa have been reported to associate with ER (16, 17, 18, 19, 20, 21, 22, 23). Some of these interacting proteins were identified by expressing the hormone-binding domain of ER fused to glutathione-S-transferase (GST-AF2). In the presence of 10-8 M 17ß-estradiol, several proteins from ZR-75 human breast cancer cells with molecular masses of approximately 160, 100, and 50 kDa were retained by GST-AF2 preloaded on glutathione-coupled beads (16). Using similar techniques, another group identified 160- and 140-kDa proteins (17). More recently, the cloned human TAFII30, which complexes with TATA-binding protein (TBP), has also been shown to interact directly with ER (18). Neither estradiol nor estrogen antagonists influenced this binding.
Although the number of ER-associated proteins is growing, their interaction and precise role in DNA binding or transcriptional activation remains to be defined. Recently, it was reported that binding of purified progesterone receptor to its response element is enhanced by HMG-1 (24, 25). HMG-1 is a 28-kDa-member of the high-mobility group (HMG) family of nonhistone chromosomal proteins that is involved in diverse aspects of eukaryotic gene expression, including determination of nucleosome structure and stability, as well as transcription and/or replication (26, 27). Higher eukaryotes contain three families of HMG proteins, the HMG-1/-2 family, the HMG-14/-17 family, and the HMG-I/-Y family, each of which contains distinct sequence motifs (28). HMG-1 is an abundant, highly conserved protein present in all vertebrate nuclei that has been shown to nonspecifically bind and bend different DNA structures as well as facilitate the binding of transcription factors to template DNA (28, 29). Two groups conclusively proved the ability of HMG-1 to induce curvature in double-stranded DNA by testing its effect on ligase-dependent cyclization of short linear DNA fragments. Covalently closed circles were formed only in the presence of HMG-1, indicating that HMG-1 is capable of introducing bends into the linear duplex (30, 31). Because the two groups used different DNA fragments, their results also indicate that the effect of HMG-1 is independent of DNA sequence.
In addition, HMG-1 and the related HMG-2 have been shown to facilitate the DNA-binding of general and specific transcription factors, such as TFIID-TFIIA, MLTF, HOX, and octamer transcription factor 2 (Oct2) (32, 33, 34, 35). Thus, HMG-1 should be considered as an "architectural element" (29, 32), which bends DNA and facilitates binding of DNA-binding proteins to their target. Based on this interaction, it has been proposed that HMG-1 may function as a general class II transcription factor by stimulating the formation of transcription initiation complexes of RNA polymerase II and III (36, 37, 38). Conversely, HMG-1 has been proposed to inhibit formation of the preinitiation complex by interacting with TBP, leading to an inhibition of RNA polymerase II transcription (39).
In light of these findings, we decided to determine whether HMG-1 and TAFII30 could facilitate ER to ERE binding and whether that promotion of binding is associated with any changes in the level of estrogen-dependent transcription. We find that recombinant, purified human ER binds to its cognate response element only in the presence of HMG-1. HMG-1 and TAFII30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Moreover, the ER overexpression increased
with time, reaching a peak 48 h post infection. When tested for
ERE binding by electrophoretic mobility shift assay (EMSA), the
overexpressed ER failed to show binding with ERE (Fig. 1B), even though
hormone-binding assays revealed high estradiol binding (Fig. 1C). ER
overexpressed in bacteria (Fig. 1, D and E) similarly failed to bind
with ERE (results not shown). This failure of recombinant ER to bind
ERE, therefore, suggested that additional proteins may be necessary to
promote formation of the complex.
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). In
competition experiments, addition of increasing concentrations of cold
ERE led to a progressive decrease in the intensity of the ER-ERE
complex (Fig. 2B). To determine whether the complex contained ER, we
carried out an antibody supershift analysis. Incubation with the
monoclonal anti-ER antibody, H222, further retarded band migration,
indicating the presence of ER in the complex (Fig. 2C). On the other
hand, using a rabbit antiserum to HMG-1, we were not able to
demonstrate the presence of HMG-1 in the ER-ERE complex. EMSAs carried
out with histone H1 failed to show a promoting effect on ER-ERE
binding, ruling out the possibility of a nonspecific effect of nuclear
proteins on ER-ERE complex formation.
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). This coprecipitation was dependent on the presence
of both the HMG-1 protein and the antibody against HMG-1, confirming
the specificity of the precipitation reaction. When recombinant
TAFII30 or BSA as a control were substituted for ER, no
coimmunoprecipitation occurred. These results suggest that the HMG1-ER
interaction is a property of the native proteins.
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, to promote ER-ERE
binding, HMG-1 must be incubated with the DNA probe before addition of
ER. When the order is reversed, HMG-1 no longer facilitates ER-ERE
complex formation. In addition, the promotion of ER-ERE binding brought
about by HMG-1 is concentration-dependent (Fig. 4). Moreover, if DNA
bending by HMG-1 is a critical factor for ER-ERE complex formation, the
length of the oligonucleotide as well as the position of the
palindromic ERE within the synthetic oligo deserve consideration. Two
25-mers containing the ERE consensus sequence, either at the end or in
the middle of the oligo, were employed instead of the usual 35-mer. The
binding of ER to these two 25-mers differed significantly when tested
in the presence of HMG-1. Figure 5A, lane 1, shows
binding obtained with the usual 35-mer used in previous EMSAs. With the
25-mers, when the ERE consensus is placed at the end of the oligo, a
significant reduction in ER-ERE interaction was observed. In contrast,
when the ERE is centrally placed within the oligo, ER-ERE binding is
restored to levels higher than that observed with the asymmetric 25-mer
but still lower than that obtained with the 35-mer. The effect of HMG-1
was also dependent on the sequence flanking the ERE consensus. When the
ERE consensus was flanked symmetrically by GCs to create another
symmetric 25-mer, ER-ERE binding mediated by HMG-1 was reduced as
compared with the symmetric 25-mer flanked by mostly ATs. Yet, the
symmetry of this GC-rich 25-mer ERE allowed it to bind ER at a much
higher level than did the asymmetric 25-mer (Fig. 5A, compare lane 2
vs. 4). Competition experiments showed that increasing
amounts of cold 25-mer suppressed ER-ERE complex formation in a
dose-dependent manner, whereas equimolar concentrations of cold 15-mer
containing the ERE without flanking sequences reduced the ER-ERE
formation much less (Fig. 5B). Thus, the effectiveness of HMG-1 to
stimulate ER-ERE binding in the EMSA depends on oligonucleotide length
and position of the ERE within the nucleotide.
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). By itself, TAFII30 neither
promotes ER-ERE binding nor does it further induce the HMG-1-mediated
ER-ERE binding. In light of these findings, we assessed the functional
role, if any, that HMG-1 and TAFII30 might have on
transcription initiation. The results of the in vitro
transcription are presented in Fig. 7. When the plasmid
pLovTATA, which is devoid of any ERE, is used, only basal level or no
transcription can be obtained with ER alone or in the presence of HMG-1
or TAFII30 or both (lanes 1–3). Similarly, when the
ERE-containing plasmid, pERE2LovTATA, was used,
transcription remained basal in the presence of ER alone or in
combination with HMG-1 or TAFII30 (lanes 4, 5, and 7).
However, transcription was enhanced 20-fold by addition of
TAFII30 to ER and HMG-1 (lane 6). Addition of estradiol
further increased transcription (lanes 9–11). The hormone induction
was approximately 5-fold for ER alone, ER plus HMG-1, and ER plus HMG-1
and TAFII30.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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HMG-1 has previously been shown to enhance the sequence-specific DNA
binding of the progesterone receptor (24, 25). However, the binding of
the retinoic acid receptor 
to its cognate responsive element is not
enhanced by HMG-1 (34), indicating that the effect of HMG-1 on DNA
binding is not generalized for all nuclear receptors. HMG-1 also
enhances the sequence-specific DNA binding of HOX proteins,
developmentally active transcription factors (34). Addition of HMG-1 to
the HOX-DNA binding reaction did not result in the formation of slower
migrating complexes in EMSA, indicating that a DNA-HMG1-HOX ternary
complex was not formed or dissociated very rapidly, similar to the lack
of a ternary ERE-HMG1-ER complex in the present study. In any case,
coimmunoprecipitation experiments demonstrated that HMG-1 formed
protein-protein contacts with HOX proteins in the absence of DNA (34),
again similar to the HMG1-ER interaction in this study. HMG-1 and the
closely related HMG-2 were also shown to increase the sequence-specific
DNA binding of Oct proteins (35). Interestingly, HMG-2 protein,
although not present in the Oct-DNA complex detected by EMSA, forms
protein-protein contacts with Oct, as demonstrated by
coimmunoprecipitation. Thus, HMG1/2 is capable of enhancing the
sequence-specific DNA binding of several unrelated transcriptional
activators and of establishing protein-protein contacts with these same
activators in the absence of DNA.
It has been suggested that HMG1-like proteins may exert a ‘DNA chaperone’ action by binding transiently to DNA, bending it into a thermodynamically unfavorable conformation and then exchanging with the protein that must eventually form a stable complex with its DNA target (42). This scenario is indeed attractive also in the context of ER-mediated transcription. HMG-1 enhanced binding of ER to ERE in a time-dependent fashion, i.e. the promotion of binding was apparent only when HMG-1 was incubated with the probe before the addition of ER. However, the ‘DNA chaperone’ model does not necessarily predict any form of direct protein-protein interaction as demonstrated for HOX, Oct, and ER. To account for the protein-protein interaction, Zappavigna et al. (34) favor an alternative interpretation, although not mutually exclusive with the one described above. They propose that the physical contact between HMG-1 and its protein partner directs both to adjacent or overlapping DNA segments, generating a complex that is endowed with both geometric and sequence specificity.
Using purified ER, we sought to determine how the length as well as the position of the ERE within the oligonucleotide might affect the HMG-1-mediated ER-ERE binding. We found that when the ERE was positioned at the very end of the oligonucleotide, a significant reduction in binding was noted. Flanking sequences around the ERE were also important factors contributing to the ER-ERE complex formation induced by HMG-1. ERE flanked by ATs bound ER at a higher yield than did the ERE flanked by GCs. While HMG-1 has no consensus DNA-binding site, it shows a preference for binding AT-rich sequences (38). In terms of length, we have also noted a significant reduction in binding when a 25-mer ERE was used in place of the usual 35-mer. We believe that this reduction in binding is a result of decreasing the DNA site HMG-1 needs for efficient interaction with sequences flanking the ERE. The footprint size of HMG-1 was determined to be 14 [plusm] 3 bp (28). Therefore, it is conceivable that as the number of bases flanking the ERE is reduced, HMG-1-mediated ER-ERE binding becomes affected as well. In the case of PR, the effect of HMG-1 on DNA binding was assessed in relation to the position of the progesterone response element within a 142-bp oligonucleotide (25). In EMSA, Prendergast et al. (25) found a subtle difference in band migration that was dependent upon the position of the progesterone response element and reflected the degree of DNA flexure induced by HMG-1. In this study, no difference in migration was noted between the 35-mer and 25-mer, because both are much smaller that the 142-bp PRE.
Although HMG-1 plays a clearly defined role in DNA binding, its function in transcriptional activation remains uncertain (36, 37, 38, 39). For this reason, we decided to determine its interaction with a bona fide ER-specific transcriptional activator, TAFII30 (18). By EMSA, we find that TAFII30 alone does not promote ER-ERE binding and does not have an effect on HMG-1-mediated ER-ERE binding. However, for transcription to be initiated from an ERE-containing template, TAFII30 must be present. Thus, although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. This is consistent with the notion that although DNA bending may be involved in transcriptional regulatory mechanisms, it is not sufficient, by itself, for transcription (43, 44). Therefore, HMG-1 might fall into the growing class of transcription factors that act by bending DNA to facilitate assembly of higher order nucleoprotein complexes (45, 46, 47, 48, 49). In this context, HMG-1 may be providing the structural framework necessary for other transcription factors to interact and function. In fact, as seen by in vitro transcription assay, TAFII30, while not affecting ER-ERE binding, stimulates transcription initiation when in the presence of HMG-1. We observed a 20-fold induction of transcription initiation, even in the absence of hormone, when ER, TAFII30, and HMG-1 are incubated with the test template, pERE2LovTATA. In the presence of hormone, an additional effect on transcription initiation was apparent. We believe that the induction of transcription initiation in the absence of hormone is due to the lack of repressors in our in vitro system. A repressor protein, SSN6, that specifically interacts with the N-terminal AF1 of the ER was identified in yeast (50). Estradiol promotes dissociation of SSN6 allowing interaction of AF1 and AF2 with the transcription apparatus. However, a mammalian counterpart of yeast SSN6 has not been identified. Other repressors have been identified for the thyroid hormone and retinoic acid receptors as SMRT (silencing mediator for retinoid and thyroid receptors) and N-Cor (nuclear receptor corepressor) that have the ability to silence thyroid hormone receptor- and retinoic acid receptor-dependent gene expression (51, 52, 53). Upon addition of ligand, SMRT and N-Cor dissociate from the receptor, thereby permitting ligand-induced transcriptional activation.
In summary, HMG-1 enhances the binding of ER to ERE. The ER thus joins the progesterone receptor and the HOX and Oct proteins as a group of transcription factors whose sequence-specific DNA binding is promoted by HMG-1. These findings are in agreement with the proposed role of HMG-1 as an "architectural element" (29, 32) that bends DNA and facilitates the stable binding of DNA binding proteins to their target. Once formed, the stable ER-ERE complex enhances the recruitment of specific transcription factors, such as TAFII30, that can assume the role of a bridging protein between ER, TBP, and other components of the transcriptional machinery that are essential for ER-induced transcription initiation. In this process, HMG-1 and TAFII30 appear to act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation. Extensive work will be required to define the role of additional proteins in this process and to gain a complete understanding of ER-mediated gene transcription.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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10 plaque-forming units/cell
for protein expression studies and 0.1–1.0 plaque forming units/cell
for virus stock production. The baculovirus transfer vector pVL1392 was
purchased from Invitrogen. The plasmid pSG5 HEGO containing the human
ER cDNA was a generous gift from Dr. Pierre Chambon (Illkirch, France).
Insertion of the ER cDNA into pVL1392 was accomplished by digesting
pSG5 HEGO with EcoRI. This fragment was then cloned into
pVL1392, which had been similarly digested and purified by isolation on
NA45 membranes (Schleicher & Schuell, Keene, NH). This fragment was
oriented by digestion with SmaI and designated pVL1392-hER.
Recombinant baculovirus was produced by cotransfecting 2 x
106 Sf9 cells with AcNPV DNA (1 µg) and pVL1392-hER (2
µg) using the calcium phosphate transfection. The resulting culture
supernatants were harvested after 4 days and screened for homologous
recombination by visual inspection of plaques, which were confirmed by
dot-blot hybridization using the respective 32P-labeled,
nick-translated cDNA probes. Purified recombinant baculovirus was
obtained after three rounds of plaque purification and designated
Ac-hER. Sf9 cells (9 x 106) infected with Ac-hER were
harvested at 24, 48, and 72 h post infection by centrifugation,
and lysed in 500 µl of buffer A (20 mM HEPES, pH 7.5, 0.1
mM EDTA, 40 mM KCl, 20% glycerol) containing 5
mM dithiothreitol (DTT), 1 mM
phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, 2.5 µg/ml
pepstatin, and 2.5 µg/ml leupeptin. The lysates were clarified by
centrifugation at 14,000 x g for 10 min and stored
frozen at -70 C.
Purification of ER from Baculovirus Extracts
Approximately 35 x 106 cells were homogenized
in 3.2 ml of ice-cold extraction buffer (0.4 M KCl, 10
mM Tris, pH 7.9, 1 mM EDTA, 5 mM
DTT, 10% glycerol) containing protease inhibitors as listed above. The
resulting homogenate was centrifuged for 3 min at 4 C in an Eppendorf
microfuge at 14,000 rpm. The salt concentration of the supernatant (3
ml) was reduced to 40 mM KCl with extraction buffer
containing no salt. The sample was loaded onto a Heparin-Sepharose
column that had been preequilibrated with extraction buffer containing
40 mM KCl. The column was washed with the same buffer used
for equilibration and step-eluted with extraction buffer containing
200, 400, and 800 mM KCl, respectively. A 10-µl aliquot
of the 400 and 800 mM fractions containing ER was used for
protein determination by standard methods to adjust the protein
concentration to 1.0 mg/ml using incubation buffer (10 mM
Tris, pH 7.9, 1 mM EDTA, 10 mM
monothioglycerol, 10% glycerol) containing protease inhibitors before
carrying out EMSAs.
Bacterial Expression and Purification of His-tagged ER and
TAFII30
The ER cDNA was amplified using the sense primer,
5'-CGGGATCCATGACCATGACCCTCCACACCAAAGC-3' and the antisense primer,
5'-GGGGTACCCGTGTGGGAGCCAGGGAGCT-3'. TAFII30 cDNA was
amplified from a cDNA stock of the normal mammary cell line, HBL-100.
Primers for amplification of TAFII30 were the sense primer,
5'-CGGGATCCAGCTGCAGCGGCT CC-3' and the antisense primer,
5'-GGGGTACCTACATTTAGGTTGGGTGGCTCAG GTG-3'. Both sets of primers were
designed to contain BamHI and KpnI sites,
respectively, at the 5'-ends. The amplification reaction was carried
out in 100 µl volume containing 10 mM Tris-HCl, pH 8.3,
50 mM KCl, 1.5 mM MgCl2, 200
mM each of the four deoxyribonucleotides, native
Pfu DNA Polymerase (2.5 U; Stratagene, La Jolla, CA), and
each oligonucleotide at either 50 ng/ml or 150 ng/ml. Amplification
conditions for ER cDNA consisted of a denaturing step at 97 C,
annealing at 67 C, and extension at 72 C for a total of 20 cycles. For
TAFII30, conditions consisted of a denaturing step at 95 C,
annealing at 64 C, and extension at 72 C for a total of 30 cycles. The
amplified cDNAs were purified using the QIAEX gel purification kit
(QIAGEN, Chatsworth, CA), digested with BamHI and
KpnI, and repurified by the same method. Ligation of each
cDNA into the similarly digested vector pQE-30 (QIAGEN), which encodes
an N-terminal hexahistidine tag, followed. Each ligated vector/insert
was used to transform M15 Escherichia coli strain (QIAGEN)
as described by the manufacturer. A picked colony harboring the correct
size insert (as judged by restriction digest and DNA sequencing) was
used to express the ER or TAFII30 protein. Expression
conditions consisted of growing the cells in Luria-Broth (supplemented
with kanamycin at 25 mg/ml and Ampicillin at 100 mg/ml) at 30 C until
an A600 reading of 0.6 was reached, induction with 0.5
mM isopropyl-ß-D-thiogalactopyranoside, and
collection of cells 1.5 h after induction. The cells were lysed by
sonication and freeze/thaw cycles. Tagged ER or TAFII30 was
purified using the Ni-NTA resin as specified by the manufacturer
(QIAGEN).
HMG-1
Human HMG-1 was purified as previously reported (54, 55).
Histone H1
Histone H1 was purified by 5% perchloric acid extraction
followed by ion exchange chromatography on Amberlite IRC-50 columns
(ICN Pharmaceuticals, Costa Mesa, CA) (56).
ER Immunoblotting
Whole-cell lysates from either Sf9 or E. coli were
separated by SDS-PAGE and transferred to Immobilon-P membranes
(Millipore; Bedford, MA) in 25 mM Tris, 192 mM
glycine, 0.025% SDS, and 15% methanol for 2 h at 200 mA.
Nonspecific binding was blocked with nonfat dry milk, and the blots
were incubated with rat anti-human ER monoclonal antibodies D547 or
H222 (Abbott Laboratories, North Chicago, IL). The filters were
washed four times with Tris-buffered saline/0.05% Tween-20 and bound
antibody was detected with enhanced chemiluminescence (Amersham;
Arlington Heights, IL).
Hormone-Binding Assay
The ER content of whole-cell extracts from the baculovirus or
bacterial system was determined using dextran-coated charcoal to absorb
free hormone in a six-point binding assay of
17ß-[3H]estradiol. Scatchard analysis was performed to
calculate binding capacity and affinity.
Oligonucleotides
All double-stranded oligonucleotides used in EMSAs were
purchased from Integrated DNA Technologies (Coralville, IA). EREs used
were: 1) 35-mer ERE of the Xenopus vitellogenin A2 gene
5'-GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT-3'; 2)
two 25-mers from the vitellogenin A2 gene differing in the position of
the ERE, 5'-AGGTCACAGTGACCTGATCAAAGTT-3' and
5'-AAGTCAGGTCACAGTGACCTGA TCA-3'; 3) a
consensus ERE flanked by GCs,
5'-GGCCCCGGTCACAGTGACCGG CCCC-3'. The oligos
were dissolved in TE buffer (10 mM Tris, 1 mM
EDTA, pH 8.0), and equimolar amounts of each strand were annealed by
heating to 95 C and cooling to room temperature over a period of
1.5 h. Double-stranded oligos were end-labeled using
[
-32P]ATP and T4 polynucleotide kinase. Free isotope
was removed by passing the labeled, double-stranded oligos through
Chroma spin-10 columns (Clontech; Palo Alto, CA).
EMSA
The assay was performed essentially as previously described
(57). HMG-1 (1 ng) was first incubated with 0.3–0.5 ng of the
32P-labeled double-stranded ERE oligomer for 15 min at 4 C.
Aliquots of partially purified ER (10 µg/ml) and 1.0 µg
poly(deoxyinosinic)·poly(deoxycytidylic)acid were then added to the
reaction mixture for a final volume of 20 µl. After a 15-min
incubation at 4 C, the protein-ERE complexes were separated by
electrophoresis through 4.5% acrylamide (38:2, acrylamide:bis) gels
using 1x TBE buffer (10 mM Tris, 10 mM boric
acid, 0.02 mM EDTA, pH 8.0). Gels were vacuum-dried and
autoradiographed. The ER-containing human breast cancer cell line MCF-7
was used as a positive control for ERE binding. For antibody shift
experiments, ER was preincubated with the monoclonal antibody H222
before the assay.
Coimmunoprecipitation Assays
For coimmunoprecipitation experiments, 25 µl preswollen
protein A insolubilized on Sepharose CL-4B (Sigma, St. Louis, MO) were
washed twice with 500 µl binding buffer (100 mM NaCl, 20
mM Tris, pH 7.0, 10% glycerol, 1% Triton-X). Between
washes, the protein A-Sepharose was recovered by centrifugation for 2
min at 3500 rpm. To the washed beads, affinity-purified anti-HMG-1 (1
µg), purified HMG-1 (100 ng), and 100 ng purified bacterial ER or
TAFII30 were added. The reaction volume was brought up to
400 µl using binding buffer containing DTT and the protease
inhibitors phenylmethylsulfonylfluoride, aprotinin, and leupeptin. The
reactions were incubated overnight at 4 C on a rotating wheel.
Immunoprecipitates were collected by centrifugation, washed three times
with 500 µl binding buffer, and recovered by boiling the precipitate
for 5 min in SDS-sample buffer. The supernatant and pooled washes were
each concentrated using Centricon-10 concentrators (Amicon, Beverly,
MA). Supernatant, wash, and precipitate fractions were run on an
SDS-PAGE gel. Proteins were transferred onto nitrocellulose membranes,
and Western blot analysis was carried out with the ER-antibody, H222
(Abbott Laboratories) or TAFII30 antibody, 2F4 (kindly
provided by Dr. Pierre Chambon). The same membranes were stripped in
100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM
Tris/HCl, pH 7.0, at 50 C and reprobed with the affinity-purified
anti-HMG-1.
In Vitro Transcription Assays
Assays were performed using the test template,
pERE2LovTATA, and the control template, pLovTATA, kindly
provided by Drs. Ming-Jer Tsai and Bert O’Malley (Baylor University,
Houston, TX). Both templates are ultimately derived from
pML(C2AT)19, a plasmid containing a 377-bp
‘G-free cassette’ linked to the TATA box region of the adenovirus-2
major late promoter (58). Accurate initiation 30 bp downstream from the
TATA box is expected to generate a 360-nucleotide transcript devoid of
G residues. As an internal control, the plasmid pMLcas190, which
contains the AdML promoter linked to a G-free cassette of 180 bp (58),
was used. Typical reactions contained the following components in a 30
µl volume: 1) 7.5 mM HEPES, pH 7.6, 60 mM
potassium glutamate, 3.75 mM MgCl2, 0.03
mM EDTA, 1.5 mM DTT, 3% glycerol, and 0.5
mM each ATP, CTP, GTP, 20 µM UTP, and 15
µCi of [-32P]UTP; 2) 500–700 ng ERE-test or control
template and internal control template; 3) ER-containing extract at 100
ng/ml and, when indicated, ER was preincubated with 10-8
M estradiol dissolved in ethanol; 4) 1 ng HMG-1; 5) 10 ng
TAFII30; and 6) 10 U of ribonuclease T1. Reactions were
initiated by adding 5 U of Drosophila embryo nuclear extract
(Promega; Madison, WI). After a 60-min incubation at 30 C, the
transcription reactions were terminated by treatment with 170 µl of
stop solution [20 mM Tris-HCl, pH 7.5, 10 mM
EDTA, 0.5% SDS] containing 200 µg/ml yeast tRNA, and 400 µg/ml
proteinase K. After addition of 200 µl of 7 M urea (in 10
mM Tris-HCl, 1 mM EDTA, pH 8.0), transcripts
were recovered by ethanol precipitation and analyzed by electrophoresis
on 6% acrylamide (38:2, acrylamide-bis), 8 M urea
sequencing gels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by Public Health Service Grant HD-07043 (to C.S.V.) and US Army Breast Cancer Training Grant DAMD-17-94-J4024 (to C.J.Y. and L.R.B.).
Received for publication August 15, 1996. Accepted for publication April 17, 1997.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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D. Das, R. C. Peterson, and W. M. Scovell High Mobility Group B Proteins Facilitate Strong Estrogen Receptor Binding to Classical and Half-Site Estrogen Response Elements and Relax Binding Selectivity Mol. Endocrinol., November 1, 2004; 18(11): 2616 - 2632. [Abstract] [Full Text] [PDF]
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 X. Chen, J. Lechago, A. Ertan, G. Ergun, R. Verm, M. Bridges, C. Johnson, K. Woods, F. Meriano, M. Chirala, et al. Expression of the High Mobility Group Proteins HMGI(Y) Correlates with Malignant Progression in Barrett's Metaplasia Cancer Epidemiol. Biomarkers Prev., January 1, 2004; 13(1): 30 - 33. [Abstract] [Full Text] [PDF]
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 A. L. Amir, M. Barua, N. C. McKnight, S. Cheng, X. Yuan, and S. P. Balk A Direct {beta}-Catenin-independent Interaction between Androgen Receptor and T Cell Factor 4 J. Biol. Chem., August 15, 2003; 278(33): 30828 - 30834. [Abstract] [Full Text] [PDF]
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S. Banerjee and T. K. Kundu The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription Nucleic Acids Res., June 15, 2003; 31(12): 3236 - 3247. [Abstract] [Full Text] [PDF]
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P. Crucitti, A. M. Abril, and M. Salas Bacteriophage Phi 29 Early Protein p17. SELF-ASSOCIATION AND HETERO-ASSOCIATION WITH THE VIRAL HISTONE-LIKE PROTEIN p6 J. Biol. Chem., February 7, 2003; 278(7): 4906 - 4911. [Abstract] [Full Text] [PDF]
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V. S. Melvin, S. C. Roemer, M. E. A. Churchill, and D. P. Edwards The C-terminal Extension (CTE) of the Nuclear Hormone Receptor DNA Binding Domain Determines Interactions and Functional Response to the HMGB-1/-2 Co-regulatory Proteins J. Biol. Chem., July 5, 2002; 277(28): 25115 - 25124. [Abstract] [Full Text] [PDF]
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H. Miyamoto, M. Rahman, H. Takatera, H.-Y. Kang, S. Yeh, H.-C. Chang, K. Nishimura, N. Fujimoto, and C. Chang A Dominant-negative Mutant of Androgen Receptor Coregulator ARA54 Inhibits Androgen Receptor-mediated Prostate Cancer Growth J. Biol. Chem., February 8, 2002; 277(7): 4609 - 4617. [Abstract] [Full Text] [PDF]
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X. Yuan, M. L. Lu, T. Li, and S. P. Balk SRY Interacts with and Negatively Regulates Androgen Receptor Transcriptional Activity J. Biol. Chem., November 30, 2001; 276(49): 46647 - 46654. [Abstract] [Full Text] [PDF]
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 H. WANG, H. YANG, C. J. CZURA, A. E. SAMA, and K. J. TRACEY HMGB1 as a Late Mediator of Lethal Systemic Inflammation Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): 1768 - 1773. [Full Text] [PDF]
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 C. J. Czura, Haichao Wang, and K. J. Tracey Dual roles for HMGB1: DNA binding and cytokine Innate Immunity, August 1, 2001; 7(4): 315 - 321. [Abstract] [PDF]
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 K. B. Ellwood, Y.-M. Yen, R. C. Johnson, and M. Carey Mechanism for Specificity by HMG-1 in Enhanceosome Assembly Mol. Cell. Biol., June 15, 2000; 20(12): 4359 - 4370. [Abstract] [Full Text]
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 C. K. Glass and M. G. Rosenfeld The coregulator exchange in transcriptional functions of nuclear receptors Genes & Dev., January 15, 2000; 14(2): 121 - 141. [Full Text]
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M. Elías-Arnanz and M. Salas Functional interactions between a phage histone-like protein and a transcriptional factor in regulation of phi 29 early-late transcriptional switch Genes & Dev., October 1, 1999; 13(19): 2502 - 2513. [Abstract] [Full Text]
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M. Bustin Regulation of DNA-Dependent Activities by the Functional Motifs of the High-Mobility-Group Chromosomal Proteins Mol. Cell. Biol., August 1, 1999; 19(8): 5237 - 5246. [Full Text] [PDF]
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C. C. Zhang, S. Krieg, and D. J. Shapiro HMG-1 Stimulates Estrogen Response Element Binding by Estrogen Receptor from Stably Transfected HeLa Cells Mol. Endocrinol., April 1, 1999; 13(4): 632 - 643. [Abstract] [Full Text]
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S. Um, M. Harbers, A. Benecke, B. Pierrat, R. Losson, and P. Chambon Retinoic Acid Receptors Interact Physically and Functionally with the T:G Mismatch-specific Thymine-DNA Glycosylase J. Biol. Chem., August 14, 1998; 273(33): 20728 - 20736. [Abstract] [Full Text] [PDF]
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V. Boonyaratanakornkit, V. Melvin, P. Prendergast, M. Altmann, L. Ronfani, M. E. Bianchi, L. Taraseviciene, S. K. Nordeen, E. A. Allegretto, and D. P. Edwards High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells Mol. Cell. Biol., August 1, 1998; 18(8): 4471 - 4487. [Abstract] [Full Text]
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L. E. Romine, J. R. Wood, L. A. Lamia, P. Prendergast, D. P. Edwards, and A. M. Nardulli The High Mobility Group Protein 1 Enhances Binding of the Estrogen Receptor DNA Binding Domain to the Estrogen Response Element Mol. Endocrinol., May 1, 1998; 12(5): 664 - 674. [Abstract] [Full Text]
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L. Jayaraman, N. C. Moorthy, K. G.K. Murthy, J. L. Manley, M. Bustin, and C. Prives High mobility group protein-1 (HMG-1) is a unique activator of p53 Genes & Dev., February 15, 1998; 12(4): 462 - 472. [Abstract] [Full Text]
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T. Imamura, H. Izumi, G. Nagatani, T. Ise, M. Nomoto, Y. Iwamoto, and K. Kohno Interaction with p53 Enhances Binding of Cisplatin-modified DNA by High Mobility Group 1 Protein J. Biol. Chem., March 2, 2001; 276(10): 7534 - 7540. [Abstract] [Full Text] [PDF]
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D. Das and W. M. Scovell The Binding Interaction of HMG-1 with the TATA-binding Protein/TATA Complex J. Biol. Chem., August 24, 2001; 276(35): 32597 - 32605. [Abstract] [Full Text] [PDF]
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