From the Department of Pathology and Programs in
§ Immunology and ¶ Genetics,
Department of
Biochemistry, Tufts University School of Medicine, Boston,
Massachusetts 02111
Received for publication, September 13, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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The transcription factor TFII-I can bind
specifically to several DNA sequence elements and is implicated in both
basal and activated transcription. There are four alternatively spliced isoforms of TFII-I, all characterized by the presence of six I-repeats, R1-R6, each containing a potential helix-loop-helix motif implicated in protein-protein interactions. These isoforms exhibit both homomeric and heteromeric interactions that lead to nuclear localization. In this
study we mapped two distinct regions in TFII-I that affect its DNA
binding. Deletion of either of these regions led to abrogation of DNA
binding and transcriptional activation from both the V TFII-I is a unique transcription factor, because it can function
both as a basal factor and as an activator (1-11). Consistent with
these functional activities, it has been shown to bind a core promoter
element, Inr, and various upstream elements apparently through distinct
DNA binding domains (1, 4, 6-11). The primary structure of TFII-I is
compatible with its multifunctional properties, consisting of six
direct reiterated I-repeats, R1-R6, each containing a putative
helix-loop-helix motif, HLH,1
but only one basic region preceding R2 (4, 9). The latter, by analogy
with more conventional HLH proteins, was postulated to constitute a DNA
binding domain (12, 13). The I-repeats, by virtue of having the
potential HLH motifs, are conjectured to present protein-protein
interaction surfaces (4, 9, 12). More recently, it has been shown that
there are altogether four alternatively spliced isoforms of TFII-I (8).
All isoforms contain the I-repeats and the basic region and exhibit
both homomeric and heteromeric interactions with themselves that lead
to their preferential nuclear localization (8). Although each isoform individually bound to DNA and activated transcription both from the
V Here we searched for potential DNA binding domains and regions
responsible for homomeric interactions. The deletion of either the
N-terminal 90 amino acids, including a putative leucine zipper but
containing an intact basic region, or a basic region (containing an
intact N terminus) led to a loss of binding to the V Cell Culture
COS7 cells were grown in Dulbecco's modified Eagle's medium
(Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 50 units of penicillin/ml, and 50 µg of streptomycin (Life Technologies, Inc.). The cells were maintained at 37 °C in a 5% CO2 environment.
Plasmids
TFII-I has four alternatively spliced isoforms: p46 (Amino Acids 1-428)--
The region between amino
acids (1) was PCR-amplified by using the following primers:
primer 1, 5'-GTGGATCCACCATGGGCAG-3'; primer 2, 5'-AGGCGGCCGCCTTTCTTAATTT-3'. The final product was digested with
BamHI and NotI and ligated to pEBG vector.
R1 (Amino Acids 104-176)--
primer 1, 5'-GTGGATCCATTGAAACACTCAGAAAA-3'; primer 2, 5'-GGGCGGCCGCTAAAAAAGGTCTCTT-3'. The PCR-amplified product was
gel-extracted, digested with BamHI and NotI, and
ligated to pEBG.
R2 (Amino Acids 309-397)--
primer 1, 5'-GTGGATCCTTCGAGAAATGGAATGCT-3'; primer 2, 5'-GGGCGGCCGCAAGCTCATCTTTC-3'. The PCR-amplified product was
gel-isolated, digested with BamHI and NotI, and
ligated with pEBG vector.
All the mutant constructs were confirmed by DNA sequencing.
Eukaryotic Expression and Purification of Wild Type and
Mutant TFII-I
COS7 cells were transfected with 7.5 µg of expression plasmids
of either wild type TFII-I Western Blot Analysis
The protein samples were separated by 10% SDS-PAGE, transferred
onto nitrocellulose membrane by semi-dry blotting method, and
processed as described previously (8). The primary antibodies were
used as follows: anti-TFII-I, 1:2500; anti-GST (Sigma), 1:3000; and
anti-GFP (CLONTECH, Palo Alto, CA) 1:300. Either
the secondary anti-rabbit (Zymed Laboratories Inc.,
South San Francisco, CA; 1:8000 dilution) or anti-mouse (Roche
Molecular Biochemicals, Indianapolis, IN; 1:4000 dilution) horseradish
peroxidase-linked antibodies were incubated in TBS containing 0.05%
Tween-20. All Western blots were developed by enhanced
chemiluminescence (Renaissance, PerkinElmer Life Sciences).
Electrophoretic Mobility Shift Analysis
The EMSA reactions in Fig. 1 was performed with either an Inr
probe derived from the V GST Pull-down Assay
Whole cell extracts (200 µg) from COS7 cells, cotransfected
with GFP-tagged wild type TFII-I (either Transient Transfection and Indirect Immunofluorescence
COS7 cells were transfected with either the GST construct alone
or the GST-tagged wild type TFII-I or its mutants as described (8).
After 30-h post-transfection, cells were fixed with 4% paraformaldehyde and prepared for indirect immunofluorescence (8). For
immunodetection, monoclonal anti-GST antibody (Sigma) was used as a
primary antibody at a dilution of 1:4000, and Alexa 594 goat anti-mouse
IgG (H+L) (Molecular Probes) as a secondary antibody at a dilution of
1:30,000. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI)
dye (Sigma). Immunofluorescence was detected using a fluorescent microscope (Nikon, E400) with a 100× objective.
Reporter Assays
Transient transfection and luciferase reporter assays were done
as described (6, 8). Each experiment was done in triplicate and
repeated twice.
Multiple Interdependent DNA Binding Regions in TFII-I--
Based
on its multiple DNA sequence element, recognition properties, and
indirect competition experiments, it was postulated that TFII-I might
have more than one distinct DNA binding domains/regions (1). To gain
insight into its DNA binding regions/domains, we generated several
mutants of TFII-I and tested their ability to bind to the V Transcriptional Properties of the Mutants--
We next tested the
transcriptional properties of the mutants as compared with the wild
type protein on both the V Nuclear Localization of the Wild Type and Mutant Forms of
TFII-I--
It could be argued that the lack of transcriptional
activity of the TFII-I mutants is due to a lack of their proper nuclear localization. This is particularly relevant for the Regions Necessary for Homomerization--
TFII-I has four
alternatively spliced isoforms that can undergo both homo- and
heteromerization with each other (8). Furthermore, either homo- or
heteromerization of isoforms facilitates nuclear translocation. Given
the fact that
Either the GFP-tagged The Role of the I-repeats 1 and 2--
The lack of homomeric
interaction of Since the initial discovery of TFII-I as an Initiator binding
protein (1), this transcription factor has remained intriguing. Although additional functions ranging from roles in signal transduction to a potential role in genetic disorders have been attributed to this
complex molecule (reviewed in Ref. 8), some of the more fundamental
information regarding its DNA binding ability and protein-protein
interaction potentials have remained largely unknown. Given its broad
potential biological role, we believe that it is important to determine
these fundamental properties of TFII-I. In an attempt to understand the
DNA binding and protein-protein interaction capabilities of TFII-I, we
have begun its structure-function analysis and generated selective
mutants and analyzed them in various functional assays.
One of the most interesting features of the TFII-I sequence is the
presence of six highly conserved 90-residue I-repeats, each of which
contains a potential helix-loop-helix (HLH)-like domain that is
implicated in homo- and heterodimerization of conventional HLH proteins
(reviewed in Ref. 12). However, unlike the conventional HLH proteins,
in which a basic region that directly mediates DNA contact precedes the
HLH regions, most of the repeats in TFII-I lack a basic region
preceding them. The only notable exception is a basic region just
before I-repeat 2 (BR, amino acids 301-306). Given this feature, and
the fact that the anti-TFII-I antibody raised against the BR ablates
the DNA binding ability of TFII-I (5-7), we surmised that the BR could
either directly or indirectly affect the DNA binding of TFII-I.
Consistent with this notion, deletion of the BR results in a DNA
binding-deficient mutant that failed to bind to both a consensus Inr
sequence and an upstream activation site. However, it should be noted
that, although the TFII-I recognition element from the c-fos
promoter lies upstream of the transcription start site, the sequence
matches a consensus Inr element (9). Thus, the BR may recognize only
this sequence albeit in a context-dependent fashion. If so,
then there might be other DNA recognition surfaces present in TFII-I
that could bind diverse DNA elements, such as an E-box (1, 4). Although identification of the BR as a DNA binding domain was important, it was
somewhat expected. More surprising was the lack of significant DNA
binding property of the Given the lack of DNA binding ability of the Although the nuclear translocation of the mutants was normal, the lack
of homomeric interactions of the and
c-fos promoters. The I-repeats, as expected, were capable of mediating homomeric interactions either individually or in combination. Unexpectedly, an additional homomeric interaction domain
was found within the N-terminal end of TFII-I that includes a putative
leucine zipper motif. These data suggest a model in which TFII-I
undergoes regulated homomeric interaction mediated by both the
N-terminal end and the I-repeats.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and c-fos promoters, a particular combination of
isoforms differentially regulated the two promoters (8). We and others (14-16) have also shown that a variety of extracellular signals mediating through cell surface receptors, including growth factor receptors, lead to enhanced tyrosine phosphorylation and increased transcriptional activity of TFII-I. Given these unusual structural and
functional features, it is important to determine the regions/domains in TFII-I that is/are responsible for its DNA binding properties and
the role of the I-repeats in mediating protein-protein interactions.
Inr and the
c-fos upstream sites. Consistent with their lack of DNA
binding activities, these mutants failed to transcriptionally activate either the V
or the c-fos promoters in transient
transfection assays. Interaction studies revealed that deletion of
either the basic region or repeats R6 through R3 from the C-terminal
end had no significant effects on homomeric interactions when compared with the wild type TFII-I. However, the N-terminal 90-amino
acid-deleted mutant, containing all the repeats, failed to exhibit
homomeric interactions with the wild type protein. Interestingly, the
I-repeats R1 and R2, either in combination or individually, could also
mediate homomeric interactions. So, although the repeats can mediate
homomeric interactions, they alone are insufficient and additionally
require the N-terminal 90 amino acids. Based on these results, we
propose a model for regulated homomeric interactions of TFII-I
involving both the N-terminal end and the I-repeats containing the HLH motifs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(977 amino
acids),
(978 amino acids),
(998 amino acids), and
(957 amino acids) (8). The construction of wild type expression plasmids of
TFII-I
- and
-isoforms (used in this study), pEBGII-I, pEBBGFPII-I, and p70 mutant plasmid pEBGp70 were detailed elsewhere (6,
8). TFII-I mutants used here were generated by a PCR-based strategy. In
the first round, two PCR reactions were done. In the first reaction, a
restriction enzyme-specific sequence 5' to the deleted region was used
as a forward primer (primer 1) and a TFII-I-specific sequence, just
downstream of the deleted sequence, was used as a reverse primer
(primer 2). In the second reaction, a TFII-I-specific sequence, just
upstream of the deleted sequence, was used as a forward primer (primer
3) and a restriction enzyme-specific sequence 3' to the deleted
sequence was used as a reverse primer (primer 4). The products of each
reaction (0.25 µg) were mixed, and the second round of PCR was
performed using the primers 1 and 4. The final PCR product was
gel-purified and digested with restriction enzymes, the recognition
sequences of which were used as the primers 1 and 4. The
restriction-digested PCR product was ligated into TFII-I-expressing
plasmid, pEBGII-I or pEBBGFPII-I. The specific sequences of these
primers and the corresponding restriction enzymes used are
detailed as follows:
N20 (Deletion of Amino Acids 1-20)--
Reaction
1: primer 1, 5'-ATTCCCCTCTAGAAATAATTTTG-3'; primer 2, 5'-CATGAGGAATGTCACCATATGGCTGCCGCG-3'. Reaction 2: primer 3, 5'-ATGGTGGTGACATTCCTCATGTC-3'; primer 4, 5'-GGAGAGATGCATAAAAT- GAAATCT-3'. The final product was
gel-isolated, digested with XbaI and NsiI, and
then ligated with pEBGII-I.
N90 (Deletion of Amino Acids 1-90)--
Reaction
1: primer 1, 5'-GTGGATCCACCATGGGCAG-3'; primer 2, 5'-CATGAGGAATGTCACCATATGGCTGCCGCG-3'. Reaction 2: primer 3, 5'-ATAGGATCCATGCATAAAATGAA-3'; primer 4, 5'-GGGTTTACGTAGATCAGTGATG-3'.
The purified PCR product was digested with BamHI and
SnaB1, and then ligated with pEBGII-I.
linker (Deletion of Amino Acids
232-252)--
Reaction 1: primer 1, 5'-AATGAGCTACCGCAGCCACCAGTCCCG-3'; primer 2, 5'-TTGAATGTTATATTGATAATAATC-3'. Reaction 2: primer 3, 5'-CAATATAACATTCAAGATGATGATTATTCT-3'; primer 4, 5'-GGGTTTACGTAGATCAGTGATG-3'. The final product was gel-isolated,
digested with Acc65I and SnaB1, and then ligated with pEBGII-I.
BR (Amino Acids 301-306)--
Reaction 1: primer 1, 5'-AATGAGCTACCGCAGCCACCAGTCCCG-3'; primer 2, 5'-GAGTTCAACTTCGAGAAATGGAAT-3'. Reaction 2: primer 3, 5'-GAAGTTGAACTCAGCATTGGCGGG-3'; primer 4, 5'-TTGAATGTTATATTGATAATAATC-3'. The PCR product was digested with
Acc65I and SnaB1 and ligated with pEBGII-I.
-isoform (pEBGII-I) or its various mutants
(pEBG
N90II-I, pEBG
BR), and the recombinant proteins were isolated
as described previously (6, 8).
promoter (17) or with a probe derived from
upstream sequences of the c-fos promoter (10). The EMSA was
performed as described previously (8).
- or
-isoforms) and either GST-tagged wild type TFII-I or the GST-tagged mutants, were
subjected to GST pull-down assay as described (8). The precipitated
proteins were separated by 10% SDS-PAGE, Western-blotted, and probed
with an anti-GFP antibody (CLONTECH). The blots
were stripped to remove the anti-GFP immune complex (17) and reprobed with anti-GST antibody (Sigma).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-derived
Inr and the c-fos-derived upstream sequence elements. Of
particular interest to us were two mutants. The first mutant (
N90)
was created such that the entire N-terminal end (first 90 amino acids)
before I-repeat 1, including the putative leucine zipper, was
deleted. The second mutant (
BR) was created such that the basic
region (amino acids 301-306) preceding I-repeat 2 was deleted,
because, by analogy with known basic-helix-loop-helix (bHLH) proteins,
this could constitute a bona fide DNA binding domain (11,
12). EMSA was performed with the wild type and mutant TFII-I proteins
on probes containing either V
-derived Inr sequences or
c-fos-derived upstream sequences overlapping the serum
response element (Fig. 1). For this and
all subsequent experiments, unless otherwise stated, the
-isoform
was used (Ref. 8, see also "Experimental Procedures"). Compared
with the wild type protein (WT), neither the
N90 nor the
BR gave
any significant shifted complex on any of the probes (nearly 6-fold
reduction in DNA binding between the wild type and either of the
mutants). The amount and integrity of various proteins used for EMSA
were monitored by Western blot assay (data not shown). Although the amount of
N90 used for EMSA was slightly less than the wild type TFII-I, increasing it even 2-fold did not produce any appreciable DNA
binding (data not shown). That the lack of appreciable DNA binding
capability of these mutants is due to specific and not general mutation
in TFII-I is proven by the fact that the p70 mutant exhibits greater
than wild type levels of DNA binding activity (6). Hence, we conclude
that both the N-terminal 90 amino acids and the basic region are
required for DNA binding and, although they may constitute separate DNA
binding regions/domains, neither one alone is sufficient to mediate DNA
binding.
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Fig. 1.
DNA binding activity of wild type and mutant
TFII-I EMSA with V Inr (lanes
1-4) and c-fos (lanes
5-8). Lanes 1 and 5, vector only
(probe); lanes 2 and 6, wild type TFII-I
(WT, 82 ng); lanes 3 and 7,
BR (84 ng); lanes 4 and 8,
N90 (68 ng).
and c-fos promoters. For this
assay, we chose an additional mutant (p70) in which the C-terminal 222 amino acids, containing an activation domain, are deleted (6). Although
the wild type TFII-I stimulated the V
promoter 3-fold, none of the
mutants gave any significant transcriptional stimulation and the
transcriptional activity of
BR and p70 mutants were similar to or
below the basal levels (Fig.
2A).
N90 failed to give any
significant stimulation. Like the V
promoter, the wild type and the
BR and p70 mutants behave similarly to the c-fos promoter
(Fig. 2B). Although the transcriptional activities of
BR
and p70 mutants were similar to or below the basal levels, the
N90
mutant failed to produce any significant transcriptional activation
(Fig. 2B). From these experiments we conclude that the DNA
binding domains and the C-terminal activation domain of TFII-I are
necessary for its transcriptional activation either as a basal factor
or as an activator. Control Western blot analysis from these
experiments (Fig. 2, lower panels) showed that the
expression levels and integrity of the mutants were comparable to that
of the wild type and thus, the differences in the functional activities
were not due to alteration in protein levels.
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Fig. 2.
Transcription function of TFII-I
mutants. A, the top panel shows the
transactivation potentials of individual and combinations of wild type
and TFII-I mutants on the basal V promoter. COS7 cells were
transiently cotransfected with 600 ng of V
and 400 ng of either pEBG
vector alone (vector) or pEBG plasmids expressing TFII-I
mutants,
N90,
BR, and p70. To normalize the transfection
efficiency, 35 ng of plasmid encoding renilla luciferase was also
included in all transfections. The result is an average of two
independent experiments done in triplicate. The bottom panel
shows control Western blot analysis of lysates from a representative
transfection assay. B, the top panel shows the
transactivation potentials of wild type and mutant TFII-I on the
c-fos promoter. COS7 cells were cotransfected with 600 ng of
the c-fos promoter either with 400 ng of empty vector
plasmid alone (top panel, vector) or with 400 ng
of plasmid encoding wild type TFII-I (WT) or mutant TFII-I
(
N90,
BR, and
p70). The result shown is an average of two independent
experiments done in triplicate. The bottom panel shows
Western blot analyses of lysates from transfection assays either in the
absence or in the presence of hEGF.
BR mutant, because a classical nuclear localization signal (NLS) is rich in basic
amino acids (18) and thus, the basic region might constitute an NLS or
contribute to the proper localization of TFII-I. However, like the wild
type TFII-I, all of the mutants (
N90,
BR, and p70) preferentially
localized to the nucleus when ectopically expressed in COS7 cells (Fig.
3). Therefore, none of the mutations impaired the nuclear localization signal of TFII-I, and the lack of the
transcriptional function is indeed due to a lack of either the DNA
binding or the transcriptional activation domains.
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Fig. 3.
Nuclear localization of wild type and mutant
TFII-I. Localization of the TFII-I mutants was compared
with the wild type by expressing them in COS7 cells as GST fusion
proteins. COS7 cells were transfected with 600 ng of expression plasmid
encoding either GST alone (A, B, and
C), or wild type TFII-I (D, E, and
F), or
N90 (G, H, and
I), or
BR (J, K, and L),
or p70 (M, N, and O). 30 h
post-transfection, cells were fixed with 4% paraformaldehyde and the
ectopically expressed proteins were visualized by indirect
immunofluorescence with monoclonal anti-GST antibody and Alexa 594 goat
anti-mouse IgG secondary antibody (A, D,
G, J, and M). Nuclei were stained with
DAPI (B, E, H, K, and
N). Superimposition (Merge) of Alexa 594 and DAPI images is
shown in the bottom panels (C, F,
I, L, and O). Images were obtained by
using a Nikon E400 fluorescence microscope with a 100×
objective.
N90,
BR, and p70 mutants readily translocate to the
nucleus, we anticipated that all of them would exhibit homo- and
perhaps heteromerization capabilities. Furthermore, because of the
presence of a putative leucine zipper (LZ) toward the N-terminal end
(amino acids 23-44) of TFII-I that is conserved in all human isoforms
and in mouse TFII-I (19, 20), we reasoned that it might be involved in
either homo- and/or heteromerization. To test this idea and to further
identify other potential interaction domains, we constructed a series
of deletion mutants (as GST fusion proteins) and analyzed their homo-
and heteromerization potentials. All the mutants are schematically shown in Fig. 4. In
N20, the first 20 amino acids from the N-terminal end, including the first acidic
cluster, are removed, but the putative LZ remains intact (4).
N90 is
described above. In
linker, part of the linker region between R1 and
R2 (amino acids 232-252) is removed, but the LZ remains intact. This
mutant is particularly interesting, because this deletion creates a
shorter version of TFII-I that is naturally absent in humans but
present in mice (20). Therefore, we wanted to test whether such a
mutant/isoform can have homomeric and heteromeric interactions. The p46
is a deletion mutant that contains only the first 428 amino acids from the N-terminal end (containing only repeats 1 and 2), and when expressed, migrates as a 46-kDa protein. The p70 mutant, containing the
first four repeats, has been described before (Ref. 6, see also Fig.
4).
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Fig. 4.
Schematic diagram of TFII-I mutants. The
schematics of wild type and various deletion constructs of the
TFII-I -isoform. The open boxes represent the I-repeats
(denoted as R1-R6), and the closed box denoted
LZ represents the putative leucine zipper motif between
amino acids 23 and 44. The functional nuclear localization sequence
(amino acids 278-284) and the basic region (amino acids 301-306) are
marked as NLS and BR, respectively.
- (Fig.
5A) or the
-isoforms (Fig.
5B) were used as baits to determine homo- and heteromeric
interactions in GST pull-down assays. These proteins were ectopically
co-expressed in COS cells with either the GST-tagged wild type
- or
-isoforms or with various mutants derived from the
-isoform. GST
protein (GST) was used as a negative control (shown only
with the
-isoform, Fig. 5A, lane 1). The GST
pull-down precipitates were analyzed by Western blot first by an
anti-GFP antibody (top panels) and, after stripping, by an
anti-GST antibody (middle panels). The bottom
panel shows the quantity of GFP-tagged TFII-I proteins expressed
in whole extracts. Except for
N90 (lane 3), all other mutants interact with the
-isoform (Fig. 5A, top
panel). The lack of interaction of the
-isoform with
N90 is
not due to lack of protein expression, because the GFP-tagged
-isoform is expressed comparably in all lanes (bottom
panel). This is contrary to our expectations, because
N90
readily translocates to the nucleus (Fig. 3). The slightly diminished
amounts of GFP-TFII-I in the last two lanes directly reflect the lower
amount of GST-tagged p70 and
BR (lanes 7 and
8) pulled down under our assay conditions and do not
constitute a true quantitative difference with the other
mutants. Also, the amount of GFP-TFII-I expression in these two lanes
was less than the other lanes (see bottom panel). The slightly higher mobility of the
N20 mutant (lane 2) is
due to its hyperphosphorylation (data not shown). In contrast to the
-isoform, the
-isoform clearly showed interactions with
N90 and all other mutants (Fig. 5B, top panel). Once
again, the expression of the GFP-
-isoform was comparable in all
lanes (bottom panel). Thus, the first 90 amino acids appear
to be important for homomerization of the
-isoform but not for its
heteromerization with the
-isoform. Moreover, the first 428 amino
acids (as in p46) contain information that is sufficient to mediate
both homo- and heteromerization. The
linker mutation had no
significant effects in interaction with either isoforms suggesting that
this form of TFII-I, although only naturally present in mice, can have
both homo- and heteromeric interactions.
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Fig. 5.
The N-terminal 90 amino acids of TFII-I are
required for homomeric interactions. A, either
GST alone (lane 1) or GST-tagged wild type TFII-I
(lane 4) or its mutants: N20 (lane 2),
N90
(lane 3),
linker (lane 5), p46 (lane
6), p70 (lane 7), and
BR (lane 8) were
used as baits for GST pull-down assays. COS7 cells were cotransfected
with 7.5 µg each of expression plasmids encoding the GST baits and
GFP-tagged wild type TFII-I
(top panel, lanes 1-8).
36 h post-transfection, whole cell lysates were prepared and 200 µg of lysate was subjected to GST pull-down assay. Blots were first
probed with anti-GFP antibody (upper panel) and then
stripped and reprobed with anti-GST antibody (middle panel).
Western blot analysis shows comparable expression of GFP-tagged TFII-I
in various lanes. B, the N-terminal 90 amino acids are
dispensable for heteromeric interactions with
-isoform of TFII-I. As
in A, COS7 cells were cotransfected with 7.5 µg each of
expression plasmids encoding either GST-tagged wild type TFII-I
(lane 3) or its various mutants (lanes 1,
2, and 4-6) with the GFP-tagged wild type
TFII-I
-isoform (lanes 1-6) and processed as in
A. Western blot analysis shows comparable expression of
GFP-tagged TFII-I
in various lanes.
N90, despite its nuclear localization, can be
reconciled if
N90 interacts with itself but not with the wild type
-isoform. We further hypothesized that such potential interactions
between
N90 could be mediated by the I-repeats. To test whether the
two molecules of
N90 interact with each other and whether the first
two I-repeats (as in p46) were necessary and sufficient to mediate such
interactions, we employed GFP-tagged
N90 as bait. GFP-
N90 was
ectopically coexpressed with GST-tagged
N90, GST-tagged R1, or
GST-tagged R2, and GST-pull-down assay was performed. Western blot
analysis with an anti-GFP antibody showed that GFP-
N90 was brought
down by GST-
N90, suggesting that
N90 interacts with itself (Fig.
6, top panel, lane
1). In addition, both R1 and R2 were also capable of interacting
with
N90, although to a much lesser extent than the
N90 with
itself (Fig. 6, top panel, lanes 2 and
3). The blot was stripped and reprobed with an anti-GST
antibody to show comparable amounts of GST-
N90 (lane 1,
second panel), GST-R1 and GST-R2 (lanes 2 and
3, third panel) were precipitated in the
pull-down assay. The bottom panel is a Western blot showing
comparable amounts of GFP-
N90 expression in each extract. We
emphasize that the interactions of R1 and R2 with
N90 are weak and
it is likely that both R1 and R2 are necessary for a robust and
physiological interaction. It is also possible that these repeats fail
to reach the nucleus (note that they lack the NLS). Together, these
results indicate that, although the I-repeats can mediate homomeric
interactions, in the context of the full-length TFII-I, the N-terminal
90 amino acids are also required for such interactions.
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Fig. 6.
I-repeats can mediate protein-protein
interactions. The expression plasmids coding for GST- N90,
I-repeat 1 (GST-R1) or I-repeat 2 (GST-R2) were
cotransfected with GFP-
N90 in COS7 cells. 36 h
post-transfection, lysates were prepared and 300 µg of lysate was
subjected to GST pull-down assay. The precipitates were separated by
10% SDS-PAGE and Western-blotted. The blot was cut into two halves and
processed simultaneously. The top part (70-230 kDa) of the
blot was first probed with anti-GFP antibody to visualize GFP-
N90
(top panel), stripped, and reprobed with anti-GST antibody
to visualize GST-
N90 (second panel). The bottom
half (70-20 kDa) of the blot was only probed with anti-GST
antibody to visualize either GST-R1 or GST-R2 (third panel).
The expression of
N90-GFP was checked by Western blot analysis of
each lysates (50 µg) with anti-GFP antibody (bottom
panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N90 mutant. The region that appears to be
responsible for such a phenotype includes a putative leucine zipper,
and it is very likely that this leucine zipper mediates a homo- or
heteromeric interaction required for DNA binding (12, 13). However,
homo- or heteromerization per se may not be sufficient for
DNA binding, because the
N90 mutant homomerizes with itself (see
below). Thus, the N-terminal region may directly contact DNA or
indirectly contribute to DNA binding by altering the native structure,
and removal of this region will expose a negative inhibitory domain
that covers the DNA binding domain.
BR and
N90 mutants,
it is not surprising that they fail to activate the
TFII-I-dependent reporters. Together with the p70 mutant,
which exhibits DNA binding but lacks an activation domain (6), it is
also not surprising that the
BR mutant that lacks DNA binding
activity did not exhibit any significant transcriptional activity.
Hence, proper transcriptional function of TFII-I requires both its DNA
binding capabilities and its transcriptional activation domain. In this
regard, it behaves like a classical transcription factor with an
N-terminal DNA binding domain and a separable C-terminal activation domain.
N90 mutant with the wild type
TFII-I
-isoform was puzzling at first glance. This is largely because
we have previously shown that homomeric or heteromeric interactions
lead to preferential nuclear occupancy (8). We argued that, although
N90 failed to interact with the wild type
-isoform, it could
interact with itself leading to nuclear localization. Interaction
studies revealed that indeed
N90 interacts with itself. These
observations raise a very interesting question: What is the role of the
N-terminal end? The N-terminal end of the protein contains the leucine
zipper motif (4) that is also present in all the isoforms of human and
mouse TFII-I (8, 19, 20). Moreover, several TFII-I-related proteins
contain this motif approximately in the same position
(21-26).2 Based on our
observations, we propose that the
-isoform remains in a
"closed" conformation until the LZ motif becomes available to
interact with other partners: either another molecule of
or another
molecule of
(Fig. 7). In contrast to
the
-isoform, the
-isoform can assume a constitutively open
conformation. Although secondary interactions can happen through the
I-repeats in the absence of the LZ motif,
N90, without the LZ cannot
induce conformational changes in the wild type
(closed
conformation). However,
N90 can interact readily with
(open
conformation). Thus, we conjecture that the regulated availability of
the leucine zipper, perhaps by phosphorylation or other signal-induced
events, might control the extent of complex formation and consequently,
nuclear function of TFII-I. Such a mechanism of regulated zipper
interaction via phosphorylation has been proposed for the I
B
kinase complex formation (reviewed in Ref. 27).
View larger version (17K):
[in a new window]
Fig. 7.
Model for regulated homomeric
interaction. a, the -isoform remains in a
"closed" conformation. The N-terminal LZ is denoted by four
parallel lines, and the repeats are depicted as black
boxes. Only the first two repeats are shown. b,
signal-induced conformational changes may lead to availability of the
leucine zipper and subsequent coiled-coiled homomeric interactions.
Subsequent secondary interactions can occur via the direct repeats.
c, homomeric interactions involving the
-isoform may not
primarily require the LZ but may require the direct repeats. The
additional 20 amino acids between the repeats may introduce a
"kink" and make the conformation constitutively "open."
d,
N90, lacking the LZ, does not interact with the
-isoform because the LZ is required to induce conformation changes
in the closed
-isoform. e, interactions of
N90 with
the constitutively open
-isoform do not require LZ. f,
lack of LZ also makes the direct repeats constitutively available for
interactions in
N90. For the sake of simplicity, we have depicted
the interactions as dimeric.
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ACKNOWLEDGEMENTS |
---|
We are grateful to rotating student Mireia Guerau for her contribution. We thank Roy laboratory members for their input, valuable suggestions, and reading of the manuscript. We also thank Mike Byrne for DNA sequencing that was done at the Tufts University School of Medicine.
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FOOTNOTES |
---|
* This work was supported by Grant RPG-98-104-01-TBE from the American Cancer Society and Grant AI45150 from the National Institutes of Health (to A. L. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6715; Fax: 617-636-2990; E-mail: ananda.roy@tufts.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008411200
2 I. Tusie-Luna and A. L. Roy, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: HLH, helix-loop-helix motif; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; GFP, green fluorescence protein; EMSA, electrophoretic mobility shift assay; DAPI, 4,6-diamidino-2-phenylindole; WT, wild type; NLS, nuclear localization signal.
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