From the Biotechnology Centre of Oslo, University of Oslo, Oslo 0316, Norway
Received for publication, August 30, 2000, and in revised form, February 26, 2001
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ABSTRACT |
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TCF11 is a bZIP transcription factor of
the CNC subfamily. It has been implicated in the regulation of the
antioxidant response and is vital during embryonic development, but its
precise biological functions have not yet been fully worked out.
Structural characterization of the gene and several of its products
indicates that complex regulatory mechanisms are employed. To
investigate how altering the structure of the gene products might
influence their activity we have mapped functional domains within the
protein. We show that two separate domains are required for
transactivation by full-length TCF11: an N-terminal acidic domain and a
serine-rich stretch adjacent to the CNC-bZIP domains. A naturally
occurring shorter isoform (identical to LCR-F1) produced by internal
initiation of translation is unable to transactivate in our assay.
However, the shorter form could interfere with the transactivating
ability of the longer form, which indicates a control mechanism for
keeping the activity of TCF11 at a desired level. We show that TCF11
and the closely related CNC-bZIP factor p45 NF-E2 show different cell type-specific activation patterns with full-length TCF11 being active
in COS-1 cells but silent in erythroid cells (K562), whereas p45 NF-E2
is active in K562 cells and silent in COS-1 cells. Domain swapping
experiments show that cell type-specific activity is not fully
determined by dimerization/DNA binding domains or transactivation domains alone. The resulting profile of activity is most likely achieved by interaction of the domains and their cell-specific environment.
TCF11 (and the isoform Nrf1) is a widely expressed human
transcription factor of the CNC-bZIP family (1, 2), most closely related to the family members p45 NF-E2, Nrf2, and Nrf3 (3-5). The importance of TCF11 was demonstrated by two independent
inactivations of the gene in mouse, showing that TCF11 is vital during
development (6, 7), but the precise biological role of TCF11 has not yet been defined. It has been shown that fibroblasts derived from TCF11/Nrf1-null embryos have enhanced sensitivity to the toxic effects
of oxidant compounds (8). This is likely to be because of reduced
glutathione level in these cells. TCF11 binding and transactivation of
the promoter of the catalytic subunit of TCF11 is widely expressed (1) and has the potential of complex
regulation. Among the different cDNA clones that were originally isolated, both alternative transcription start sites and alternative poly(A) sites were observed. Several different isoforms caused by
alternative splicing were isolated (1), one of them identical to the
cDNA sequence of Nrf1 (2). In a later study another isoform was
identified without the serine-rich domain, which specifically interacts
with the tumor necrosis factor- The activity of TCF11 may also be regulated at a number of additional
levels. First, tcf11 transcripts are translated as two major
isoforms with the possibility for translational initiation from an
internal methionine cluster to yield a short isoform of 447 amino acids
(originally described as LCR-F1 (11)). Full-length TCF11 ranges from
728 to 772 amino acids depending on alternative splicing within an
acidic N-terminal domain (1). Second, dimerization with different
partners may alter the transactivating activity of TCF11. The small Maf
proteins MafF, MafG, and MafK have been identified as putative partners
of TCF11 (12, 13). More recent results show that these factors,
originally thought to be widely expressed, are differentially expressed
during development and might therefore regulate TCF11 in a
tissue-specific manner (14, 15). Both MafG and MafK repress
transactivation when coexpressed with TCF11 compared with the level of
reporter gene transcription induced by TCF11 alone in transient
transfections (13, 16). However, these factors are also partners of
several other bZIP proteins (for an overview, see Ref. 17), such as the
erythroid-specific transcription factor p45 NF-E2 (13, 18) and more
widely expressed family members. Competition between partners might
thus regulate TCF11 activity. Third, post-translational modifications
such as phosphorylation may play a role in regulating of TCF11
activity. TCF11 has several potential phosphorylation sites, and casein kinase II has been shown to phosphorylate an isoform of TCF11 specifically and thereby render it active in binding to the tumor necrosis factor- Given the potential for complex regulation and interaction, we wished
to map functional domains within TCF11, using a transactivation assay
in cultured cells. The activities of three naturally occurring differential translation products have been compared, and we used deletion and fusion constructs to examine subdomains more extensively. Two different regions in the TCF11 protein are required to gain full
transcription factor activity in transient transfections in COS-1 and
HeLa cells. However, even full-length TCF11 is inactive in K562 cells,
whereas the closely related protein p45 NF-E2 is active under the same
conditions. We demonstrate that even though TCF11 and p45 NF-E2 have a
high degree of similarity in their DNA binding domains, one does not
interfere with the transactivation abilities of the other through the
NF-E2 site when coexpressed in transient transfections. Chimeric
fusions between TCF11 and p45 NF-E2 show that the DNA binding and
dimerization domains from p45 NF-E2 can replace the corresponding
domains from TCF11 and give a protein with the functional
characteristics of TCF11. However, this occurs only when the fusion
includes the serine-rich stretch, a domain that is not present in p45
NF-E2.
Expression Constructs
The expression construct pcA20 producing TCF11 longer form was
made by cloning the full-length sequence of tcf11, clone
pZcEA20 (5' to the EcoRV site at bp 3550, accession no. X77366) (1) into the expression vector pcDNA3
(Invitrogen). In the same way, clone pZcEA56 was used to make an
expression construct pcA56 producing a variant of the TCF11 longer
form, and pZcEA52 was used for the construct pcA52 producing only the
shorter form. An expression construct pcA20IntMut producing only the
full-length TCF11 was made by mutating the four internal methionine
residues (Met-318, Met-321, Met-323, and Met-326) to leucine residues
(QuickChange site-directed mutagenesis, Stratagene). All deletion
constructs were made from the full-length sequence of tcf11
(pcA20). In the TCF11 Fusion Constructs
TCF11 and p45 NF-E2 fusion constructs were made by cloning
PCR-generated p45 NF-E2 fragments (verified by sequencing)
into the appropriate sites in the TCF11 expression construct pcA20 as follows.
The Transactivating Domain (TAD) from p45 NF-E2 Fused to the DNA
Binding Domain (DBD) of TCF11 without the Serine-rich Domain--
The
sequence coding for the TAD fragment was PCR amplified with primers 1 and 2 and ligated to the HindIII (polylinker of pcDNA3)/BamHI (bp 2263) fragment of pcA20. The construct
was named NATDB (p45 NF-E2 activating domain
fused to TCF11 DNA binding domain).
The TAD from p45 NF-E2 Fused to the DBD of TCF11 Including the
Serine-rich Domain--
The sequence coding for the TAD fragment was
PCR amplified with primers 1 and 3 and ligated to the
HindIII/BsgI (bp 2003) fragment of pcA20 (named NASerTDB).
TAD from TCF11 without the Serine-rich Domain Fused to DBD from
p45 NF-E2--
The sequence coding for the DBD fragment was PCR
amplified with primers 4 and 5 and ligated to the BsrGI
(1599)/XbaI (polylinker of pcDNA3) fragment of pcA20
(named TANDB).
TAD from TCF11 Including the Serine-rich Domain Fused to DBD from
p45 NF-E2--
The sequence coding for the DBD fragment was PCR
amplified with primers 6 and 5 and ligated to the PmlI (bp
2302)/XbaI fragment of pcA20 (named TASerNDB).
Primer 1: 5'-GCG CAA GCT TGG ACA CTA CCC GCA
GCC TCA TCT C-3', covering bp 215-238 in the p45 NF-E2 sequence
(AC L09600 (3)) and containing an additional HindIII site.
Primer 2: 5'-CGC AGC TGG ATC CCC CTG ATG CAG GTC ATA
AGA TGG TGG GGG AAG GGA GAA GC-3', covering bp 586-559 and
containing an additional BamHI site.
Primer 3: 5'-AAA TTC CTC CTC CAC CTG GGA GGC CTG CAC
CTC ATA AGA TGG TGG GGG AAG GGA GAA GCC TGC-3', covering bp
586-555 and containing an additional BsgI site.
Primer 4: 5'-GGA ACC TTG TAC ACA CCC TTG GCC TTA GAG
TCA TCC TCC GGT CC-3', covering bp 942-967 and containing an
additional BsrGI site.
Primer 5: 5'-GCA CCA GTG TCC ACT CTC TAG ACC-3',
covering bp 1484-1461 with the XbaI site.
Primer 6: 5'-TTA ATT CAC GTG GCC TTA
GAG TCA TCC TCC-3', covering bp 943-962 and containing an
additional PmlI site.
The part of the p45 NF-E2 sequence in the different primers
is shown in bold, and the enzyme sites are underlined.
Cell Culture
COS-1, HeLa, and HepG2 cells were cultured in Dulbecco's
modified Eagle's medium (1 g/liter glucose, Life Technologies, Inc.) supplemented with 10% fetal calf serum, 2 mM
L-glutamine, and 50 units/ml penicillin-streptomycin at
37 °C in 5% CO2. K562 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 50 units/ml penicillin-streptomycin, sodium pyruvate, and 0.25% glucose at 37 °C in 5% CO2.
Transient Transfections
COS-1, HeLa, or HepG2 cells were transfected at 50-80%
confluence either using a standard calcium phosphate procedure (16) or
FuGENE 6, following the supplier's instructions (Roche Molecular Biochemicals). For calcium phosphate transfection, 5 µg of TCF11 expression construct, 2 µg of PBGD3.2Luc reporter (16), and 1 µg of internal control (pRSVCAT)/10-cm dish were used. The luciferase gene in the PBGD3.2Luc reporter is under the control of a part of the porphobilinogen deaminase (PBGD) gene promoter. It has been
shown previously that TCF11 activates the reporter through the single
NF-E2 site in the promoter region (16). A total of 1.0-2.2 µg of
DNA/3.5-cm dish was used during FuGENE 6 transfections together with
2-6 µl of transfection reagent. The DNA mixture consisted typically
of 0.2 µg of PBGD3.2Luc reporter construct, various amounts of
different TCF11 and/or p45 NF-E2 expression constructs, and empty
pcDNA3 vector to the required total amount of DNA.
K562 cells were transfected with DEMRIE-C transfection reagent (Life
Technologies, Inc.) following the supplier's instructions. A mixture
of 4 µg of DNA together with 4 µl of DEMRIE-C transfection reagent
was used to transfect 2.0 × 106 cells. The DNA
mixture consisted typically of 0.8 µg of PBGD3.2Luc reporter
construct, 0.4 µg of internal control (pRSVCAT), various amounts of
TCF11 and/or p45 NF-E2 expression constructs, and empty vector to
achieve the required total amount of DNA.
Luciferase, CAT, and Protein Assay
The cells were harvested at two days post-transfection in a
lysis buffer (50 mM Tris-MES, pH 7.8, 1 mM
dithiothreitol, 0.1% Triton X-100). The luciferase activity was
measured on a MicroLumat Plus luminometer (EG&G Berthold). 5-50 µl
of cell extract was diluted in luciferase mixture buffer (10 mM Mg(OAc)2, 50 mM Tris-MES, pH
7.8, 2 mM ATP) to a total volume of 200 µl. 100 µl of 1 mM luciferin (Roche Molecular Biochemicals 1626353) was
added to each sample. CAT activity was measured using a standard
protocol (16). Luciferase activity was normalized either by comparison with CAT activity or with the total protein content in the sample, determined by the Bio-Rad protein assay with 3-5 µl of the cell extract.
The levels of luciferase induction after transfection with TCF11 or
other expressing constructs are given as relative activity compared
with the activity obtained by the empty vector alone.
Western Blot Analysis
COS-1, HeLa, or K562 cells were transfected as described
previously. After 2 days of incubation the cells were harvested using standard methods for cells growing either in monolayer or in suspension (21). Protein extracts were separated using 10% SDS-polyacrylamide gel
electrophoresis (running buffer: 25 mM Tris, 200 mM glycine, 0.1% SDS) then electroblotted onto
nitrocellulose membranes (blotting buffer: 20% (v/v) methanol, 200 mM glycine, and 25 mM Tris). After transfer,
the membranes were blocked with 5% bovine serum albumin in TBST (165 mM NaCl, 10 mM Tris, pH 8, 0.1% Tween 20) for
1 h at room temperature. Blots were incubated with primary
antibody (anti-TCF11 diluted 1:16,000 in TBST containing 0.5% bovine
serum albumin (12)) at 4 °C overnight, followed by incubation with secondary antibody (anti-rabbit horseradish peroxidase diluted 1:5,000
in TBST containing 0.5% bovine serum albumin (Amersham Pharmacia
Biotech)) for 1 h at room temperature. The immune complexes were
visualized using enhanced chemiluminescence (ECL, Amersham Pharmacia
Biotech) and detected with x-ray film.
MBP Pulldown Assay
In vitro dimerization between TCF11 or MafG and
different TCF11 variants, p45 NF-E2, or chimeric proteins was performed
as described previously (12). Briefly, MBP, MBP-TCF11-A, and MBP-MafG were bacterially expressed and purified on amylose resin (New England
Biolabs). The different variants of TCF11, p45 NF-E2, and the chimeric
proteins were translated in vitro using the TNT-coupled in vitro transcription/translation system (Promega) in the
presence of [35S]methionine and incubated with the
desired MBP fusion by gently mixing overnight at 4 °C in
dimerization buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40) containing protease inhibitors. Unbound proteins were removed by washing three times with the dimerization buffer. Bound proteins were eluted by denaturation in
SDS-PAGE sample buffer (2% SDS, 0.1 M dithiothreitol, 60 mM Tris-HCl, pH 6.8, 10% glycerol, and 0.001% bromphenol
blue), separated by SDS-PAGE, and analyzed by autoradiography.
Two Alternative Translation Products of TCF11 Show Different
Transactivating Ability--
In vitro assays have
previously revealed two major translation isoforms of TCF11 (2), the
second, smaller isoform presumably being produced by translation
initiation from an internal cluster of ATGs (Fig.
1). We confirm this observation both in
cell culture and with in vitro transcription/translation
assays. This is not unexpected because there is a non-optimal Kozak
sequence at the initial methionine (22). TCF11 produced from a
full-length cDNA, where both isoforms can potentially be produced,
can specifically bind to and transactivate through the NF-E2 site in
the PBGD promoter (16). We wished to investigate if either or both of
the major translation products were involved and if they showed any
differences in their ability to induce expression of a reporter. To
test this, we have used a transient transfection assay expressing two
differentially spliced cDNAs that can produce both full-length and
internally initiated protein and a truncated cDNA that can only
produce the short isoform. The reporter induction occurs through a
single NF-E2 site in the assay.
Fig. 1 shows a schematic representation of the full-length TCF11
protein with functional domains indicated, a variant of the protein
caused by alternative splicing (long variant), and the shorter protein
resulting from internal translation initiation. Initiation from the
internal methionine cluster yields a short isoform of 447 amino acids,
whereas full-length TCF11 is 772 amino acids (1). The long variant
transcript yields a product of 731 amino acids (Fig. 1) because of the
omission of exons 3a and 4 in the N-terminal acid-rich domain
(23).
In a transient transfection assay the different protein products show
variation in their ability to induce luciferase expression. COS-1 cells
transiently transfected with clones that produced either of the longer
translation products (long and long variant) showed a significant
luciferase induction (Fig. 2a,
second and third bars). In contrast, in
cells where only the shorter translation product can be produced, no
luciferase induction above background level was observed (Fig.
2a, fourth bar). The same results were obtained
with transiently transfected HeLa or HepG2 cells (data not shown). The
omission of the two small exonic sequences (exons 3a and 4; 41 amino
acids) in the acid-rich domain of the long variant of TCF11 did not
show a detectable difference in the ability to activate the reporter
under the conditions used (Fig. 2a, compare second and third bars).
To examine the expression of the different translation products the
proteins were analyzed by Western blot (Fig. 2b). The full-length clone gave rise to three different equally expressed protein forms: two large proteins of ~160 and 140 kDa, and a shorter protein of 65 kDa, which is the expected size for the internally initiated translation product (Fig. 2b, second
lane) (2). The alternatively spliced clone gave rise to two
different translation products: a longer form of 140 kDa and the
internally initiated product (Fig. 2b, third
lane). The incomplete clone gave only the shorter protein form
(Fig. 2b, fourth lane). The total amount of TCF11
protein detected was much higher when only the shorter protein form was produced.
To examine if the observed inability of TCF11 short form to activate
the reporter in the transient transfection assay was caused by a
difference in dimerization ability, an MBP pulldown assay was
performed. The dimerization ability of different in vitro
translated variants of TCF11 was tested (Fig. 2c). The long, long variant, and the short form all showed specific dimerization with
both the MBP-TCF11-A fusion protein (Fig. 2c,
sixth, eighth, and ninth lanes) and
MBP-MafG (data not shown) (12). No dimerization was observed in the
control reaction between long TCF11 and MBP (Fig. 2c,
fifth lane). This implies that dimerization ability is not
altered in the various forms.
To characterize further the activity of the short form, a transient
transfection assay with coexpression of TCF11 short form and MafG was
performed (Fig. 3). This showed that
TCF11 short form in the presence of MafG was still not able to
transactivate expression under these conditions (Fig. 3,
sixth and seventh bars), although an MBP pulldown
assay showed that the two proteins could dimerize (data not shown). On
the other hand MafG expressed alone repressed reporter expression (Fig.
3, fifth bar) whereas coexpression with TCF11 short form
reduced the repression caused by MafG (Fig. 3, seventh bar).
However, MafG coexpressed with p45 NF-E2 activated reporter expression
(Fig. 3. eighth bar).
Because transactivation in our transient transfection assay in COS-1,
HeLa, and HepG2 cells was achieved only under conditions where either
of the longer proteins was produced, we wished to examine if the
observed activation was dependent on the expression of a longer form
alone or a combination of a longer and the shorter form. To address
this we produced a mutated construct that produces only the longer
protein form (Fig. 1, long mutated). In the mutated form four
methionine residues (Met-318, Met-321, Met-323, and Met-326), which are
possible internal translation initiation start sites, were changed into
leucine residues, and thereby internal initiation was prevented. This
was confirmed by in vitro expression (Fig. 2c,
second lane) and Western blotting (Fig.
4a, third lane), and the protein product was shown to dimerize with both MBP-TCF11-A (Fig. 2c, seventh lane) and MBP-MafG (data not
shown). Expression of the mutated form in the transient transfection
assay showed an enhanced transactivation ability compared with TCF11
long form (Fig. 4b, compare second and
third bars). This enhanced activity was observed when
several different amounts of DNA were used in the transfections (Fig.
4c, compare sixth, seventh, and
eighth bars with third, fourth, and
fifth bars). When either the longer form or the longer
mutated form was coexpressed with TCF11 short form the transactivation
abilities were reduced (Fig. 4c, compare ninth
through twelfth bars with third through
fifth bars, and thirteenth through
sixteenth bars with sixth through
eighth). This implies that the presence of short form TCF11
can actually interfere with the activity of TCF11 long form.
To examine if the introduced leucine residues had any effect on the
protein expression, extracts from transiently transfected cells were
analyzed by Western blot. The mutated long form was expressed at the
same level as TCF11 long form (Fig. 4a, compare third with second lane). Coexpression of the
short form with either long or long mutated form had no effect on the
level of protein expression (Fig. 4a, fifth and
sixth lanes).
From these results we conclude that the region N-terminal to the
internal methionine cluster is necessary for transactivation under
these conditions. Within this stretch there is an acid-rich region that
may function as a transactivation domain. However, omission of two
small stretches within this region did not influence the
transactivation. Interestingly, there is an additional acidic domain
just N-terminal of the serine stretch (amino acids 428-474, Fig. 1)
which is also present in the shorter protein produced by internal
initiation. This is clearly not sufficient to induce reporter
expression in our assay in COS-1, HeLa, or HepG2 cells although it may
have an additional effect.
Domains of TCF11 Important for Transactivating
Potential--
Because the long variant of TCF11, lacking 41 amino
acids in the N-terminal acidic domain, showed the same activity as the full-length protein in transient transfections, we constructed plasmids
that express mutant proteins to determine more closely which regions of
TCF11 are important for the transactivating ability. In three of these
constructs, areas around the N-terminal acidic region were deleted
(Fig. 1:
The same results were obtained when these four deletions were used in
transient transfections of HeLa cells (data not shown).
To confirm that the reduced or absent activity was not caused by a
lower level of TCF11 protein, extracts of cells transfected with the
wild type or mutant constructs were analyzed by Western blot. All of
the mutant proteins that were less active than the wild type in the
transfection assay were present in approximately equal (
In addition, an MBP pulldown assay was used to analyze the ability of
the mutant proteins to dimerize with MBP-TCF11-A or MBP-MafG (Fig. 5c).
All four mutant proteins showed the same dimerization abilities as
TCF11 long form (Fig. 5c, compare seventh through tenth lanes with sixth lane in both the upper and
the lower figure).
Taking these results together we conclude that the presence of both the
serine-rich domain and the N-terminal acid-rich domain is necessary for
TCF11 to maintain full transactivating capacity in COS-1, HeLa, and
HepG2 cells.
Opposite Transactivation Ability of TCF11 and p45 NF-E2 in COS-1
and K562 Cells--
Structural comparisons between TCF11 and p45 NF-E2
(Fig. 6) show a high degree of similarity
in the C-terminal parts of the proteins. The DNA binding domain shows
85% (22 amino acids identical in a stretch of 26 amino acids (2))
identity in amino acid residues. Immediately N-terminal to this region,
the CNC domain shows 67% (29 amino acids of 43 amino acids (11))
identity. The dimerization domain is less conserved: 39% (14 amino
acids out of 36 amino acids (2)). It is therefore not surprising that
both factors can bind to and transactivate through the same DNA
sequence if they bind as homodimers or heterodimerize with the same
partners (16, 18). In contrast to the widely expressed TCF11 (1, 2),
p45 NF-E2 is specifically expressed in hematopoietic cells (3, 24). It
was postulated that p45 NF-E2 could regulate the expression of globin
genes during development together with MafK (25). However, more
recently it was shown that p45 NF-E2-deficient mice only show a mild
erythroid defect (26, 27), and it has therefore been speculated that
other members of the CNC-bZIP family might compensate for the p45 NF-E2
deficiency (6). Two different reports show that a compound deficiency
of Nrf2 and p45 NF-E2 in mice did not introduce any additional
defects in globin gene expression compared with p45 NF-E2 deficiency
alone (28, 29). We therefore wanted to compare directly the activity of
TCF11 and p45 NF-E2 proteins through the NF-E2 site in COS-1 cells and in an erythroid cell line (K562).
TCF11 longer form induced luciferase activity in COS-1 cells (Fig.
2a, second bar, and Fig.
7a, second bar) as
well as in several other cell lines (16). In contrast, p45 NF-E2 did
not induce the reporter through the NF-E2 site when transiently
transfected into COS-1 cells (Fig. 7a, fourth
bar), although analyses of cell extracts by Western blot showed
the presence of p45 NF-E2 protein (see Fig. 9a, fourth
lane). The lack of reporter induction in COS-1 cells was
consistent with earlier reports that in non-hematopoietic cells, p45
NF-E2 has to be coexpressed with one of the small Maf proteins to
achieve detectable transactivation in a transient transfection assay
(Fig. 3) (13, 18), indicating that p45 NF-E2 lacks an activation
partner in COS-1 cells. When both TCF11 and p45 NF-E2 were coexpressed
in COS-1 cells, the level of luciferase induction did not change
compared with that of TCF11 alone (Fig. 7a, compare
fifth and sixth bars with second and
third bars, respectively). Analysis of cell extracts showed
that both TCF11 and p45 NF-E2 were expressed (see Fig. 9a,
fifth and sixth lanes). This implies that p45
NF-E2 in this context does not compete directly with TCF11 for binding
to the NF-E2 site in the reporter construct in COS-1 cells, and p45
NF-E2 does not appear to interfere with the transactivation activity of
TCF11.
In the erythroid K562 cell line the ability of TCF11 and p45 NF-E2 to
induce a reporter through a single NF-E2 site was reversed compared
with the results from COS-1 cells. K562 cells transiently transfected
with p45 NF-E2 expression vector showed significant luciferase
induction above background level (Fig. 7b, fourth
bar). In these cells the dimerization partner MafK is known to be
present (13). In contrast, neither TCF11 long nor short form showed any
ability to influence the production of luciferase reporter through the
single NF-E2 site (Fig. 7b, second and
third bars) despite previous findings that MafK may dimerize
with TCF11 (13). This is in contrast to the results reported by
Caterina et al. (11), where a fusion of the short form of
TCF11 with the Gal4 DNA binding domain had transactivating ability in
K562 cells, using a reporter construct with two Gal4 binding sites.
Surprisingly, when both TCF11 and p45 NF-E2 are coexpressed, the level
of luciferase induction did not change compared with expression of p45
NF-E2 alone (Fig. 7b, compare fifth and
sixth bars with fourth bar). This implies that
the two transcription factors do not compete for binding to the NF-E2
site in the reporter construct in K562 cells and that TCF11 does not
appear to interfere with the activity of p45 NF-E2.
Transactivation Abilities of Fusion Products from TCF11 and p45
NF-E2 in COS-1 and K562 Cells--
To investigate further the
differences in activities of TCF11 and p45 NF-E2 in the two different
cell types, fusion constructs between the transactivation domain of
TCF11 and the DNA binding domain of p45 NF-E2 and vice versa were
prepared and tested in COS-1 and K562 cells. Because there is no region
in p45 NF-E2 similar to the serine-rich domain of TCF11, fusions were
made both with and without this domain to investigate its possible role
in the activity differences (Fig. 6).
In COS-1 cells only the fusion between the transactivating domain of
TCF11, including the serine-rich stretch, and the DNA binding domain of
p45 NF-E2 (TASerNDB) was able to induce the reporter to the same level
as TCF11 long form (Fig. 8a,
third bar). These results showed that the DNA binding and
zipper domain of TCF11 could be replaced by the corresponding part of
p45 NF-E2. However, a similar construct without the serine-rich stretch
(TANDB) did not induce the reporter to the same extent (Fig.
8a, fourth bar). To test if this difference in
transactivation ability was caused by changes in their dimerization
potential we compared their ability to associate with TCF11. We showed
that TANDB, which lacks the serine-rich domain, had a reduced ability
to dimerize with MBP-TCF11-A compared with TASerNDB (Fig.
9c, compare eighth lane with seventh lane). However, even the latter
chimeric protein showed reduced dimerization compared with TCF11 long
form (Fig. 9c, compare seventh lane with
sixth lane). On the other hand, both of these chimeric
proteins showed the same dimerization ability with MBP-MafG as TCF11
long form (Fig. 9d, compare seventh and eighth lanes with sixth lane).
The reciprocal constructs, with the transactivating domain of p45
NF-E2 and the DNA binding and dimerization domains of TCF11, showed
that the putative transactivating domain of p45 NF-E2 (30, 31) was
unable to induce the reporter. No change was seen when the serine-rich
domain from TCF11 was included (Fig. 8a, fifth and sixth bars). These two fusion proteins showed no change
in the dimerization ability compared with TCF11 long form (Fig. 9, c and d, compare ninth and tenth
lanes with sixth lane). Analysis of cell extracts
showed that all of the chimeric proteins were expressed (Fig.
9b, fourth through seventh lanes). In
K562 cells only p45 NF-E2 was active, and none of the fusion constructs
was able to reproduce this transactivation pattern (Fig. 8b,
compare third through sixth bars with
second bar).
Thus the opposite activities of TCF11 and p45 NF-E2 in different cell
types cannot be entirely determined by the transactivation domain or
the DNA binding and dimerization domains alone but require the correct
combination of the bZIP and the transactivation domains.
TCF11 is a bZIP transcription factor of the CNC subfamily, as is
p45 NF-E2. Whereas p45 NF-E2 has an expression pattern restricted to
hematopoietic cells (3, 24), TCF11 is widely expressed (1). Both
proteins can bind to and transactivate through the same DNA sequence
and form heterodimers with the small Maf proteins like MafK and MafG
(12, 13, 16, 18). We show here that despite their similarity, these
proteins have different cell type-specific activities that are not
determined by specific transactivation domains alone but by the
combination of transactivation domain and bZIP domain. This indicates
cooperation or interplay between the domains and interaction with
cell-specific environmental factors.
Tcf11 transcripts are translated as two major products, one
from the initial ATG and another from an internal ATG cluster. The
existence of a poor Kozak sequence around the initial methionine may be
important to allow initiation in vivo to occur also from the
internal methionine residues, leading to production of both the long
and short protein forms. We have demonstrated that both protein forms
are present in extracts from transiently transfected cells (Figs.
2b and 4a), and we confirm that mutating all four potential internal start codons abolishes production of the smaller protein. Transfection with the 5'-incomplete clone that can only produce the shorter form showed a clearly higher protein expression. This might be caused by differences in the stability of the transcripts or the proteins and/or less efficient translation initiation from the
initial ATG. Interestingly, the protein amount produced from the
initial ATG did not change appreciably when the internal ATGs were
mutated, indicating that there is not a competitive relationship between initial and internal initiation.
No transactivation activity was observed when only the shorter version
of TCF11 was produced, whereas protein products from the originally
isolated cDNA clones (1) (including the longer forms) caused
transactivation in COS-1, HeLa, and HepG2 cell lines. An interesting
question arising from this observation is why are two isoforms with the
same dimerization and DNA binding domains but with different activities
produced? We suggest that the non-optimal translation initiation
sequence around the first methionine leading to the possibility of
internal initiation is likely to be an important regulatory mechanism
for keeping the positive transactivating form of TCF11 at a desired,
limited level. In addition, the shorter protein may influence, directly
or indirectly, the functioning of the more active longer protein. This
hypothesis was supported with the observation that a mutated construct
incapable of internal translation initiation showed enhanced reporter
induction in transient transfections. In addition, this observed
elevated induction was reduced to approximately the same level as wild
type when the mutated construct was cotransfected with an incomplete
clone that only produces the short form (Fig. 4c). The use
of alternative translation initiation to generate protein isoforms with
distinct transcriptional properties has been reported previously for
other genes, like the Wilms' tumor (32) gene and Egr3 (33).
Only expression of the long TCF11 protein provides the combination of
the two domains which we demonstrate to be essential for full
transactivation: the N-terminal, acidic domain, and the serine-rich
stretch (Figs. 2a and 5a). The ability of an
acid-rich region to act as a transactivation signal has been reported
for several transcription factors (for review, see Ref. 34). The serine-rich region has several potential phosphorylation sites, which
may be important for TCF11 to retain full activity in the cell systems
analyzed. This region is present both in the long and short forms and
is therefore necessary but not sufficient to effect transactivation in
COS-1, HeLa, and HepG2 cells. In K562 cells however, even the
full-length protein with both domains does not transactivate, whereas
p45 NF-E2 shows an opposite pattern, being active in K562 cells and not
in COS-1 and HeLa cells. Coexpression of TCF11 and p45 NF-E2 did not
essentially change the transactivation pattern, indicating that TCF11
and p45 NF-E2 factors did not compete directly with each other for
partners and/or DNA in our assay (Fig. 7, a and
b).
The domain swapping experiments involving the transactivation and bZIP
domains of p45 NF-E2 and TCF11 shed some more light on the cell
specific activities of the two proteins. The fusion joining the
transactivation domains of TCF11 and the bZIP domain of p45 NF-E2
(TASerNDB) was active in COS-1 cells. However, the similar chimeric
protein without the serine-rich domain (TANDB) was not active. Our
deletion analysis similarly showed that the serine-rich region is
necessary for full transactivation by TCF11, but the domain swapping
experiment also shows that the TANDB chimera dimerizes less efficiently
with MBP-TCF11. The results indicate that the serine stretch, in
addition to being an activation domain, is important for dimerization.
It might be that the serine-rich domain alters the dimerization
characteristics of the chimeric protein, and it might promote
homodimerization, which was shown previously not to be favored for p45
NF-E2 in its natural context (13). This experiment demonstrates that in
this fusion context, the bZIP domain of p45 NF-E2 can dimerize and bind
DNA in COS-1 cells. On the other hand, the N-terminal part of p45 NF-E2
was inactive when fused to the bZIP of TCF11, with or without the serine-rich domain, although both proteins showed dimerization with
MBP-TCF11-A. The results of these experiments indicate that the
inactivity of NASerTDB and NATDB in COS-1 cells is related to an
inability to transactivate rather than an inability to dimerize and
bind to DNA. Surprisingly, the N-terminal 83 amino acids of p45 NF-E2
in this context cannot reiterate the activity of the heterodimer
p45NF-E2/MafG in cotransfections (Fig. 3) (13).
In K562 cells none of the fusion proteins was able to transactivate the
reporter (Fig. 8b). The N-terminal part of TCF11 most likely
either lacked interacting cofactor(s) or was not modified in a way
necessary for transactivation, for example by phosphorylation. TCF11
may be phosphorylated both by casein kinase II and mitogen-activated protein kinase Erk2 (10). We have found that mutating a casein kinase
II site in TCF11 increases the protein's ability to induce the
luciferase reporter (data not shown), confirming the complex regulation
of this transcription factor. However, more surprising was the lack of
transactivation when the N-terminal 83 amino acids of p45 NF-E2 were
fused to TCF11 CNC-bZIP because the p45 NF-E2 domain has been shown
previously to transactivate in K562 cells when coupled to the GAL4 DNA
binding domain (30). However, it has been shown that removal of amino
acids 176-209 in p45 NF-E2 reduced the protein's activity in the MEL
cell line CB3 (19). It is possible that this domain is also required to
obtain a transcriptionally active chimeric protein together with the
DNA binding and dimerization domains of TCF11. Alternatively, the
inactivity of this fusion might indicate that p45 NF-E2 is dependent on
strong DNA binding to activate transcription and that sufficiently
strong binding is not achieved. The strength of binding may become
critical in the presence of more strongly binding complexes, for
example native NF-E2, which is present in K562 cells and is known to
bind DNA very efficiently (35). Extending this idea, it is possible
that the TCF11 CNC-bZIP domain is unable to bind DNA in K562 cells. This could be caused by the lack of a specific dimerization partner or
by dimerization with a partner which results in a complex with low
binding affinity for the NF-E2 site within the reporter plasmid or by
incorrect modification of TCF11 which might prevent binding to DNA.
From this we conclude that TCF11 and p45 NF-E2 need interplay between
their respective transactivation and DNA binding/dimerization domains
in addition to interaction with cell-specific partners and/or cofactors
to gain full activity as a transcription factor.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamylcysteine synthetase have recently been
observed.1 In addition, TCF11
has been shown to transactivate through the hARE response element in
the NAD(P)H:quinone oxidoreductase-1 promoter (9), a site that is
important for the induction of NAD(P)H:quinone oxidoreductase-1 in
response to xenobiotics and antioxidants. These observations indicate a
role for TCF11 in the regulation of the antioxidant response. In
addition, TCF11 has also been suggested to play a role in tumor
necrosis factor-
regulation, because specific interaction
with the tumor necrosis factor-
promoter has been demonstrated in
the cell line DC18 (10).
promoter to stimulate transcription
(10). These observations indicate that differential processing of TCF11
transcripts is a prominent feature and may be important in regulating
TCF11 activity.
promoter (10). Fourth, TCF11 might interact with
other tissue-specific, yet unidentified cofactors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
173-315 construct (numbers referring to the
amino acid residues deleted) the sequence between the two
BglII sites was deleted (restriction sites at bp 1108 and
1537 in the tcf11 sequence). In the TCF11
12-125
construct the sequence between the XmnI site (bp 624) and
the first XhoI site (bp 962) was deleted. In the TCF11
12-315 construct the sequence between the XmnI (bp
624) site and the second BglII site (bp 1537) was deleted,
and in the TCF11
469-558 construct the sequence between a
PvuII site (bp 1996) and the NarI (bp 2569) site
was deleted. The p45 NF-E2 expression construct was kindly provided by
Dr. Paul Ney. In this construct the murine p45 NF-E2 is
under the control of the polypeptide chain elongation factor 1
(19,
20). For in vitro translation, p45 NF-E2 was
subcloned into the pcDNA3 vector using the EcoRI and the
NotI sites.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the different
isoforms and mutated or deleted versions of TCF11 indicating structural
motifs within the protein. The putative N-terminal transactivating
domain (acid-rich), an internal acid-rich domain (acid-rich), the
serine-rich domain, the CNC domain (CNC, highly conserved among the
family of the CNC-bZIP factors), and the basic (DNA binding domain)
leucine zipper (dimerization domain) (bZIP) are shown. The two
different translation initiation start sites, which are the initial
methionine (M) and the internal methionine cluster (MMMM), are
also marked. Full-length TCF11 (long), a natural variant of the long
form (long variant), the internal initiated shorter protein form
(short), a mutated version of the long form where the four internal
methionine residues are changed to leucine residues (LLLL, long
mutated), and four deletion constructs are represented. The long
variant lacks a total of 41 amino acids in the N-terminal acid-rich
domain because of alternative splicing. In 173-315, amino acid
residues 173-315 have been removed in the N-terminal acid-rich domain.
In
12-125 most of the protein N-terminal to the acid-rich domain is
deleted, and
12-315 has a deletion covering the whole acid-rich
domain in addition to most of the N terminus. The serine-rich domain
has been deleted in
469-558.
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Fig. 2.
Panel a, luciferase
activity induced in transient transfected COS-1 cells expressing TCF11
long form, the TCF11 long variant, and the TCF11 short form. The
PBGD3.2Luc reporter and internal control (pRSVCAT) were
cotransfected with either empty pcDNA3 vector (first bar
from left) or one of the TCF11 expression constructs
(second through fourth bars). The cells were
transfected by a standard calcium phosphate method using a total of 10 µg of DNA/10-cm dish. Luciferase activity has been normalized to CAT
activity and is shown relative to the activity given with
cotransfection of the empty vector. The induction shown is the
average of eight experiments, and the error bars reflect the
standard deviation of each mean value. Panel b,
detection of the different translation products using Western blot
analysis. Whole cell extracts were prepared from COS-1 cells
transiently transfected with the different TCF11 constructs expressing
wild type isoforms as shown in Fig. 1. After SDS-PAGE and transfer to a
nitrocellulose membrane, immunodetection with a polyclonal antibody
against the C terminus of TCF11 (12) revealed the different translation
products as indicated ( ). The arrowheads on the
left (
) indicate the position of size standards with the
size of each shown in kDa. Extracts of cells transfected with
pcDNA3 show background staining with this antibody.
Panel c, in vitro dimerization between
MBP-TCF11-A (12) and different in vitro translated variants
of TCF11 was tested in an MBP pulldown assay (sixth through
ninth lanes). TCF11 long form incubated with MBP alone is
used as a control for binding specificity (fifth lane). 10%
of the input of 35S-labeled proteins is shown in the
first through fourth lanes.
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Fig. 3.
Luciferase activity induced in transient
transfected COS-1 cells expressing TCF11 short form, p45 NF-E2, and
MafG either alone or in combinations. The PBGD3.2Luc was
cotransfected with either empty pcDNA3 vector (first
bar), the incomplete TCF11 expression construct (second
bar), the p45 NF-E2 expression construct (third bar),
the MafG expression construct (fourth and fifth
bars), a combination of TCF11 with different amounts of MafG
(sixth and seventh bars), or a combination of
MafG and p45 NF-E2 (eighth bar). Luciferase activity was
normalized to the total protein content and is relative to the activity
given with the empty vector. The induction shown is the average of
three experiments, and the error bars reflect the standard
deviation of each mean value. The cells were transfected by using
FuGENE 6 transfection reagent and a total of 1.3 µg of DNA/3.5-cm
dish.
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Fig. 4.
Panel a, detection of the different
translation products using Western blot analysis. Whole cell extracts
were prepared from COS-1 cells transiently transfected with different
TCF11 constructs expressing either TCF11 long form, long mutated, or
short form as shown in Fig. 1. After SDS-PAGE and transfer to a
nitrocellulose membrane, immunodetection with a polyclonal antibody
against the C terminus of TCF11 (12) revealed the different translation
products as indicated ( ). The arrowheads on the
left (
) indicate the positions of size standards with the
size of each shown in kDa. Extracts of cells transfected with
pcDNA3 show background staining with this antibody. Panel
b, luciferase activity induced in transient transfected COS-1
cells expressing TCF11 long form, long mutated, and short form. The
PBGD3.2Luc was cotransfected with either empty pcDNA3 vector
(first bar) or one of the TCF11 expression constructs
(second through fourth bars). Luciferase activity
was normalized to the total protein content and is relative to the
activity given with the empty vector. The induction shown is the
average of three experiments, and the error bars reflect the
standard deviation of each mean value. The cells were transfected by
using FuGENE 6 transfection reagent and a total of 2.2 µg of
DNA/3.5-cm dish. Panel c, luciferase activity induced in
transient transfected COS-1 cells expressing TCF11 long form, long
mutated, and short form either alone or in combinations. The
PBGD3.2Luc was cotransfected with either empty pcDNA3 vector
(first bar), one of the TCF11 expression constructs
(second through eighth bars), or a combination of
the incomplete clone together with either the full-length
(ninth through twelfth bars) or the mutated
construct (thirteenth through sixteenth bars).
Luciferase activity was normalized to the total protein content and is
relative to the activity given with the empty vector. The induction
shown is the average of three experiments, and the error
bars reflect the standard deviation of each mean value. The cells
were transfected by using FuGENE 6 transfection reagent and a total of
2.2 µg of DNA/3.5-cm dish. The different amounts of DNA used in each
transfection are shown.
173-315,
12-125,
12-315), and one construct lacks
a region spanning the serine-rich domain (Fig. 1:
469-558). When
these constructs were used in transient transfections in COS-1 cells,
significant differences in luciferase induction were observed. Although
no reduction in the reporter activity was seen when amino acids 12-125
were deleted (Fig. 5a,
fourth bar), the activity was abolished when part of (amino
acids 173-315) or the complete (amino acids 12-315) N-terminal acidic
domain was deleted (Fig. 5a, third and
fifth bars). These results indicate that the acid-rich
domain is necessary for the transactivation ability of TCF11 and that
deletion of the last 143 amino acids in the region (amino acids
173-315) was enough to abolish most of the transactivating ability. On
the other hand omission of exons 3a and 4 at the N terminus had no
effect. In addition, the serine-rich domain, a stretch of 56 amino
acids with more than 60% serine residues (35 serine residues out of 56 amino acids), was also shown to be essential for the full
transactivating capacity of TCF11. Construct
469-558 showed a
significant reduction in the induced luciferase level compared with
wild type (Fig. 5a, sixth bar).
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Fig. 5.
Panel a, luciferase induction by
full-length TCF11 compared with the four deletion constructs
( 173-315,
12-125,
12-315, and
469-558) represented in
Fig. 1. The 3.2PBGDLuc reporter was cotransfected with either
empty pcDNA3 vector (first bar) or one of the TCF11
constructs (second through sixth bars) in COS-1
cells. Luciferase activity was normalized to the total protein content
and is relative to the activity given with the empty vector. The
induction shown is the average over a number of experiments
(n), and the error bars reflect the standard
deviation of each mean value. The cells were transfected by using
FuGENE 6 transfection reagent and a total of 1 µg of DNA/3.5-cm dish.
Panel b, detection of the different translation products
using Western blot analysis. Whole cell extracts were prepared from
COS-1 cells transiently transfected with TCF11 long form and the
different TCF11 deletion constructs shown in Fig. 1. After SDS-PAGE and
transfer to a nitrocellulose membrane, immunodetection with a
polyclonal antibody against the C terminus of TCF11 (12) revealed the
different translation products as indicated (
). The
arrowheads on the left (
) indicate the
position of size standards with the size of each shown in kDa. Extracts
of cells transfected with pcDNA3 show background staining with this
antibody. Expression from the transfected constructs is seen in
addition to this background pattern. Panel c, in
vitro dimerization between MBP-TCF11-A (upper figure)
or MBP-MafG (lower figure) (12) and different in
vitro translated mutant proteins of TCF11 were tested in an MBP
pulldown assay (seventh through tenth lanes).
TCF11 long form incubated with MBP alone is used as a control for
binding specificity (upper figure, fifth lane).
10% of the input of 35S-labeled proteins is shown in the
first through the fourth lanes in the upper
figure and the first through the fifth lanes
in the lower figure.
173-315) or
higher amounts (
12-315 and
469-558) compared with the wild type
(Fig. 5b, compare third, fifth, and
sixth lanes with second lane). Interestingly, the only
deletion construct that showed a reduced protein level was the
N-terminal deletion
12-125 (Fig. 5b, compare
fourth lane with the second lane) where the
transactivating ability was the same as for the wild type (Fig.
4a, compare fourth bar with second
bar).
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Fig. 6.
Schematic representation of the complete
TCF11 long form, p45 NF-E2, and the different chimeric TCF11/NF-E2
proteins. The different shaded boxes indicate the same
domains as given in Fig. 1. In the chimeric constructs the
numbers indicate the amino acid residues in the protein of
origin. Numbers above the drawing indicate domains from
TCF11, whereas numbers below the drawing indicate a domain
from p45 NF-E2 (also dot-filled). The names of the different
constructs are given as p45 NF-E2 or TCF11 (N or
T) Activating domain fused with p45 NF-E2 or
TCF11 (N or T) DNA Binding
domain. When the serine-rich region is incorporated, Ser is added to
the name of the chimeric protein.
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Fig. 7.
Panel a, transient transfection of COS-1
cells showing the effect on luciferase induction when TCF11 is
coexpressed with p45 NF-E2. The PBGD3.2Luc reporter was
cotransfected with either empty pcDNA3 vector (first
bar), TCF11 longer or shorter form alone (second and
third bars, respectively), p45 NF-E2 alone (fourth
bar), or TCF11 together with p45 NF-E2 (fifth and
sixth bars). Luciferase activity was normalized to the total
protein content and is relative to the activity given with the empty
vector. The induction shown is the average over a number of experiments
(n), and the error bars reflect the standard
deviation of each mean value. The cells were transfected by using
FuGENE 6 transfection reagent and a total of 1.3 µg of DNA/3.5-cm
dish. Panel b, transient transfection of K562 cells
measuring the effect on luciferase induction when TCF11 is coexpressed
with p45 NF-E2. The PBGD3.2Luc reporter and internal control
(pRSVCAT) were cotransfected with either empty pcDNA3 vector
(first bar), TCF11 long or short form alone
(second and third bars, respectively), p45 NF-E2
alone (fourth bar), or TCF11 together with p45 NF-E2
(fifth and sixth bars). Luciferase activity was
normalized to the CAT activity and is relative to the activity obtained
with cotransfection of the empty vector. The induction shown is the
average over a number of experiments (n), and the
error bars reflect standard deviation of each mean value.
The cells were transfected by using DEMRIE-C (Life Technologies, Inc.)
transfection reagent and a total of 1.3 µg of
DNA/2.0×106 cells.
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Fig. 8.
Panel a, transient transfection of COS-1
cells measuring the effect on luciferase induction of the different
TCF11/p45 NF-E2 chimeric proteins (Fig. 6). The PBGD3.2Luc
reporter was cotransfected with either empty pcDNA3 vector
(first bar), TCF11 long form (second bar), or one
of the chimeric constructs (third through sixth
bars). Luciferase activity was normalized to the total protein
content and is relative to the activity given with the empty vector.
The cells were transfected by using FuGENE 6 transfection reagent and a
total of 1 µg of DNA/3.5-cm dish. The induction shown is the average
of six experiments, and the error bars reflect the standard
deviation of each mean value. Panel b, transient
transfection of K562 cells measuring the effect on luciferase induction
with the different TCF11/p45 NF-E2 chimeric proteins (Fig. 6). The
PBGD3.2Luc reporter and internal control (pRSVCAT) were
cotransfected with either empty pcDNA3 (first bar), p45
NF-E2 (second bar), or one of the chimeric constructs
(third through sixth bars). Luciferase activity
was normalized to the CAT activity and is relative to the activity
obtained with cotransfection of the empty vector. The cells were
transfected by using DEMRIE-C transfection reagent and a total of 1 µg of DNA/2.0 × 106 cells. The induction shown is
the average of six experiments, and the error bars reflect
the standard deviation of each mean value.
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Fig. 9.
Panel a, detection of the different
translation products following single and cotransfection with TCF11 and
p45 NF-E2 expression vectors using Western blot analysis. Whole cell
extracts were prepared from COS-1 cells transiently transfected with
TCF11 long and short form and p45 NF-E2, each alone or together. After
SDS-PAGE and transfer to a nitrocellulose membrane, immunodetection
with a polyclonal antibody against the C terminus of TCF11 (12) was
performed. This antibody recognizes both TCF11 and p45 NF-E2 proteins, which
have similar C termini. The different translation products are
indicated by an arrow ( ). The arrowheads on
the left (
) indicate the position of size standards with
the size of each shown in kDa. Panel b, detection of the
different chimeric translation products after transfection using
Western blot analysis. Whole cell extracts were prepared from COS-1
cells transiently transfected with the different constructs. After
SDS-PAGE and transfer to a nitrocellulose membrane, immunodetection
with a polyclonal antibody against the C terminus of TCF11 (12)
revealed the different translation products as indicated (
). The
arrowheads on the left (
) indicate the
positions of size standards with the size of each shown in kDa.
Panel c, in vitro dimerization between
MBP-TCF11-A (12) and the different in vitro translated
chimeric (TCF11/p45 NF-E2) proteins were tested in the MBP pulldown
assay (seventh through tenth lanes). Dimerization
between MBP-TCF11-A and TCF11 long form is used as a positive control
of the assay (sixth lane). 10% of the input of
35S-labeled proteins is shown in the first
through fifth lanes. Panel d, in
vitro dimerization between MBP-MafG (12) and the different
in vitro translated chimeric (TCF11/p45 NF-E2) proteins was
tested in the MBP pulldown assay (seventh through
tenth lanes). Dimerization between MBP-MafG and TCF11 long
form is used as a positive control of the assay (sixth
lane). 10% of the input of 35S-labeled proteins are
shown in the first five lanes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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: Biotechnology Centre
of Oslo, University of Oslo, 1125 Blindern, 0316 Oslo, Norway. Tel.: 47-22840510; Fax: 47-22840501; E-mail:
annebko@biotek.uio.no.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M007951200
1 Myhrstad, M. C. W., Husberg, C., Murphy, P., Nordström, O., Blomhott, R., Moskaug, J. Ø., and Kolstø, A. B. (2001) Biochim. Biophys. Acta 1517, 212-219.
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction; TAD, transactivating domain; DBD, DNA binding domain; Luc, luciferase; PBDG, porphobilinogen deaminase; RSV, Rous sarcoma virus; CAT, chloramphenicol acetyltransferase; MES, 4-morpholineethanesulfonic acid; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis.
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