(Received for publication, June 13, 1995)
From the
The transcription factor USF1 belongs to the family of basic helix-loop-helix proteins that are involved in the regulation of various important cellular processes. Here we characterized the factors involved in the activation of transcription by upstream stimulatory factor 1 (USF1) in a reconstituted class II gene transcription system. Activation of transcription by both wild type USF1 and a GAL-USF (amino acids 1-94 of the yeast activator protein GAL4 fused to amino acids 17-196 of USF) fusion protein required the presence of at least one positive cofactor. A novel positive cofactor (PC5) that functions specifically through the activation domain of USF1 was partially purified and biochemicaly distinguished from previously described positive cofactors. The mechanism by which PC5 mediates activation of transcription through USF1 was investigated in order-of-addition experiments. PC5 had to be present during binding of transcription factor (TF) IID to the TATA box to observe transcriptional activation. However, this event alone did not result in transcriptional activation, which also required the presence of the activator and of PC5 after binding of TFIID. Hence, PC5 may enter transcription during binding of TFIID to function in concert with the activator during subsequent steps in transcription.
Upstream stimulatory factor (USF) ()was originally
described as an activity derived from HeLa nuclear extract that binds
to an element (E-box) upstream of the adenovirus major late
promoter(1, 2, 3) . Purification of USF (4) led to identification of two polypeptides of 43 (USF1) and
44 kDa (USF2). The corresponding cDNAs were
cloned(5, 6) , and the proteins encoded by the cDNAs
were shown to form homo- and heterodimers. All three different USF
dimers bind to the E-box element in vitro and in
vivo(6) .
Sequence analysis showed USF1 and USF2 to be members of the basic helix-loop-helix (bHLH)/leucin zipper family of transcription factors. This family of transcription factors also includes the mammalian proteins MYC, MAD, MAX, MXI1, TFEB, TFE3, and AP-4, which represents a subfamily of the large family of bHLH transcription factors (for review see (7) and (8) ). Homo- and heterodimers of bHLH proteins via binding to E-boxes regulate a variety of genes that are involved in a number of important cellular processes ranging from differentiation to cell cycle regulation and apoptosis. In addition to its important role in regulation of the adenoviral major late promoter, USF is involved in the regulation of cellular genes, including the murine metallothionein I gene(9) , the rat gamma-fibrinogen gene(10) , the human growth hormone gene(11) , the p53 gene(12) , and the cardiac ventricular myosin light chain 2 gene(13) .
USF has been widely used as a model activator to study mechanisms of transcriptional activation by bHLH transcription factors. In nuclear extracts recombinant USF1, indistinguishable from purified USF, activates transcription of the major late promoter(14) . The bHLH/leucin zipper region at the carboxyl terminus of USF1 is sufficient for DNA binding (5) but does not have an intrinsic activation potential(15) . Transcriptional activation is dependent on the amino-terminal half of the polypeptide, and a fusion of this region to the GAL4 DNA-binding domain gives rise to an transcriptionally active protein(15) .
Activation of
transcription appears to require interactions between the regulatory
sequence-specific DNA-binding proteins and the general transcription
machinery. There is evidence for functional interaction of USF with
TFIID (3, 16, 17, 18, 19) with
one of the TBP-associated factors (TAF55)(20) .
USF also binds cooperatively with TFII-I to both USF recognition sites
and sequences close to the initiation sites of
transcription(21, 22) . Although recombinant USF1 is
able to activate transcription in nuclear extracts indistinguishable
from natural USF(14, 15) , activation potential in
reconstituted transcription systems is substantially reduced during
purification of natural USF(16) .
The activity of partially purified USF can be restored in vitro by including an upstream factor stimulatory activity(23, 24) . Four different positive cofactors (PC1, PC2, PC3/Dr2/TopoI, and p15/PC4) ((25, 26, 27, 28, 29) ; reviewed in (30) and (31) ) have been isolated from this fraction that are essential for stimulation of transcription by activators in highly purified class II gene transcription systems (32) . At least in one example (p15/PC4), these coactivators interact with activators (29) and the general transcription factor TFIIA(29) . More recently, functional analysis revealed that PC4, in conjunction with an acidic activator, stimulates transcription predominantly during TFIID-TFIIA promoter complex formation(33) .
Here, we have analyzed transcriptional activation by recombinant USF1 and the USF1 activation domain fused to the minimal DNA-binding domain of GAL4 in a reconstituted class II gene transcription system. We have isolated and partially purified a novel cofactor termed PC5. PC5 displayed specificity for the USF1 activation domain and, in contrast to previously identified PCs, had no effect on transcription in the presence of a GAL4 DNA-binding domain. Mechanistic studies suggest that PC5 enters transcription during and function after binding of TFIID to the promoter.
HeLa nuclear extracts
derived from epitope-tagged TBP-expressing HeLa cells (37) were
fractionated on phosphocellulose (P11, Whatman) essentially as
described(38) . PC2 and PC3 were isolated from the P11 0.85 M KCl fraction, which was dialyzed to buffer HP (10 mM Hepes-KOH, pH 8.2, 100 mM NaPO, pH 7.6, 200
mM KCl, 1 mM phenylmethylsulfonyl fluoride, 0.01%
Nonidet P-40, 20% glycerol) containing 5 mM imidazole and
applied to a nickel-agarose column (QIAGEN Inc.). PC3, which is
identical to topoisomerase I and contains a large number of clustered
histidine residues, and also PC2 could be purified from this crude
fraction on nickel columns. The column was washed with buffer HP
containing 15 mM imidazole, and the activity was eluted using
buffer HP containing 100 mM imidazole. PC2 and PC3 were
separated according to their different native sizes of 500 and 300 kDa,
respectively(25, 26) , using Superose 12 (SMART)
chromatography. The column (load 50 µl, 70 µg of protein) was
developed in buffer P (200 mM NaPO
, pH 7.6, 0.2
mM EDTA, 10% glycerol). 1.5 and 3.0 µl of the peak
fractions (fraction size, 50 µl) of PC3 and PC2, respectively, were
used for transcription reactions.
PC4 (p15) was expressed and purified as described(27) . 50 ng of purified protein was used in transcription reactions.
Figure 4: Analysis of the mechanism of activation by PC5 and GAL-USF. A, digestion of templates in between the GAL4 recognition sites and the TATA box after the DA complex formation abolishes activation by GAL-USF. As indicated, GAL-USF and PC5 (heat-treated P11 0.5 M KCl fraction) were incubated together with the templates TFIID (DA) and TFIIA for 50 min. Afterwards, templates were digested with SphI for 20 min, followed by a 60-min incubation period with the remaining GTFs and nucleotides (NTPs). B, PC5 acts independent of TFIID concentrations and does not function if added after DA complex formation. As indicated, GAL-USF and PC5 were included in the preincubation together with TFIID and TFIIA (lanes 1-4) or added after the preincubation (lanes 5-6). Standard (lanes 1, 2, 5, and 6) or 4-fold higher concentrations of TFIID (lanes 3 and 4) were used. After a 50-min preincubation period, the remaining GTFs and NTPs were added, and transcription reactions were allowed to proceed for 60 min.
Figure 1:
Fractionation of PC5. A, purification scheme of PC5. B, specificity of
highly purified PC5 (Superose 12 fraction). PC5 was tested in
transcription reactions in combination with GAL94 (94),
GAL-USF (USF) and GAL4-AH (AH). Templates contained
the HIV promoter carrying five GAL4 recognition sites (GAL)
and the major late core promoter (ML). C,
specificity of heat-treated PC5 purified on MonoQ columns. A fraction
eluting at 350 mM KCl from the MonoQ column was tested in a
purified transcription system for the ability to mediate effects of
GAL94 (94) and GAL-USF (USF). Fold activation was
calculated by comparing transcription level of the HIV promoter
normalized to levels of the internal control (ML core promoter). D, determination of PC5 on Superose 12 columns. Eluting
fractions (3 µl of 50-µl fractions) and the corresponding load (L, 0.2 µl of 10-fold concentrated heat-treated P11 0.5 M KCl fraction) were tested for their capacity to mediate
activation by GAL-USF, which was present in all reactions. Molecular
sizes as determined by standard proteins are
indicated.
Figure 3: Activation by recombinant USF1 is mediated by different PCs. A, effects of a crude PC5 fraction (heat-treated P11 0.5 M KCl fraction) on activation of the major late wild type promoter (MLWT) by E. coli-expressed USF1 in a purified transcription system. B, effects of PC2 and PC5 (Superose 12 fraction of PC5, Fig. 1B) on basal and USF1-dependent transcription of the major late wild type promoter (MLWT).
PC5 appears to be distinct from PC1 to PC4 by several criteria. On
Superose 12 columns, PC5 eluted according to a native size of 300 kDa (Fig. 1A). This distinguishes PC5 from most of the PCs,
specifically PC2 (500 kDa)(26) , PC4 (50-100
kDa)(27) , and PC1 (400 kDa). PC5, if purified as
shown in Fig. 1C, did not contain topoisomerase I
activity (PC3), which eluted according to a similar native size on gel
filtration columns(25) . PC5 could be further distinguished
from PC1 and PC4 by its chromatographic behavior on strong anion
exchanger columns. In contrast to PC4 and PC1, which pass through MonoQ
ion exchanger columns at low salt (40 mM KCl)(23, 27) , PC5 binds tightly to
MonoQ-Sepharose, from which it elutes at 250-360 mM KCl (Fig. 1C). Furthermore, in contrast to most of the PCs,
PC5 activity was found to be resistant to heat treatment for 15 min at
55 °C. When the cofactors were heat-treated at identical salt (100
mM KCl) and protein concentrations (0.5 mg/ml) only PC5 and
PC4 retained cofactor activity (data not shown). Thus, PC5 fractions
appear to contain a novel coactivator distinct from previously defined
PCs. We have subsequently used a heat-treated P11 0.5 M KCl
fraction (see Fig. 1A) to study the mechanism of PC5
function. This PC5 fraction had similar specific activity as highly
purified PC5 (Fig. 1B) and apparently was not
contaminated by other PCs, because it eluted as a single peak from
Superose 12 according to a native size of 300 kDa (Fig. 1D) and displayed functional properties
indistinguishable from purified PC5.
Figure 2:
Specificity of different PCs for
activation domain of USF1. A, GAL94 (94), GAL-USF (USF)m and GAL4-AH (AH) were tested in a purified
transcription system containing PC5 (heat-treated P11 0.5 M KCl fraction) or recombinant purified PC4. Transcripts below the
position of the internal control transcripts do not originate from the
major late core promoter (ML) but represent terminated
HIV promoter transcripts (lane 9). B, comparison of
activation levels of GAL94 (white bars), GAL-USF (shaded
bars), and GAL4-AH (black bars) mediated by different
PCs. Transcription levels in each lane were normalized to levels of
major late core promoter. Fold activation was calculated by comparing
normalized transcription levels in the presence and the absence of
activators. Experiments with the different PCs were reproduced at least
two times.
PCs that were active in combination with GAL-USF (PC1, PC2, and PC5) were also tested for their capacity to facilitate activation of transcription by the recombinant wild type USF1 protein, which was expressed in and purified from Escherichia coli. In these experiments the adenovirus major late promoter was used as a template that carries a single USF recognition site located between positions -54 and -60(39) . These experiments could not be performed with crude PC5 fractions because these fractions activated the major late promoter in the absence of exogeneously added recombinant USF1 (Fig. 3A). Consistent with this observation, we detected substantial amounts of native USF in crude PC5 fractions by gel shift experiments (data not shown). However, more purified PC5, as well as PC2 and PC1, were depleted of USF and facilitated activation of transcription by USF1 (Fig. 3B, lanes 6 and 3, 3.5- and 3.2-fold activation, respectively, and data not shown). In the absence of PCs, USF1 had no effect on transcription in a highly purified transcription system. Thus, PCs that are active in conjunction with GAL-USF also facilitate activation of transcription by the wild type USF1 protein.
In this study we have characterized a novel cofactor (PC5) that stimulates transcriptional activation by USF1 in vitro. In order-of-addition experiments we show that PC5 initiates stimulation of transcription during binding of TFIID to the promoter but completes its effects in conjunction with an activator after DA complex formation.
Previous investigations suggested that USF1 requires at
least one additional factor to activate transcription, which is
apparently removed during purification of the
protein(3, 4, 15, 16) . This
analysis suggests that this missing factor may be a member of the
family of PCs. Although none of the PCs directly coelute with native
USF, we could indeed detect PC5 activity in fractions that contain
native USF. Furthermore, PC5 is heat stable and binds to DE52 columns,
suggesting that it may have contributed to USF activity during the
first purification
steps(1, 3, 4, 16) . It seems
reasonable to assume that crude transcription systems contain PCs and
that these factors contribute to transcriptional activation. Upon
purification of general transcription factors, PCs are at least in part
removed, leading to reduction of effects of activators on
transcription(23) . In agreement with this interpretation, PCs
do not strongly affect activation of transcription by USF in nuclear
extracts. ()
For the first time, we have systematically
compared effects of different PCs in conjunction with an activator, in
this example the bHLH/leucin zipper protein USF1. Evidence is provided
that PC5 displays specificity for the USF1 activation domain. Very
little is known about specificities of PCs for domains of regulatory
proteins that are essential for transcriptional activation in
vivo. Weak preferences of the coactivator PC2 for the AH domain in
GAL4-AH (26, 42) and of PC1 fractions for the acidic
carboxyl-terminal activation domain of NFB subunit p65 (TA-1
domain), and not vice versa, have been demonstrated
previously(26, 43) . Preferences of certain PCs for
defined activators are most easily explained if we assume that these
two classes of transcription factors interact with each other. Indeed,
at least in the case of PC4, direct interactions with VP16 and other
activation domains have been reported(29, 44) .
However, in most examples functional interactions of activators with
PCs remain to be demonstrated.
Other putative targets of activators
include TFIIB (45) and the TAFs (Refs. 32, 39, 46, and 47;
reviewed in (48) ). In all cases investigated TAFs were
essential for or strongly increased the activator-dependent
transcription in vitro (Refs. 32, 37, and 41; reviewed in (48) ). Analyses included USF, which was recently shown to
interact with TAF55, one of the integral components of the
TFIID complex(20, 36) . In previous investigations we
and others provided evidence that PCs are distinct of
TAFs(26, 32) . This was subsequently confirmed by
cloning of some members of the
PCs(25, 27, 28, 29) . Also in the
case of PC5 we have no evidence for a relationship of this factor to
members of the TBP-associated factors. PC5 did not coelute with TFIID,
and our transcription system contained the complete native TFIID
complex.
We have begun to study the mechanisms by which PC5 mediates activation of transcription by USF1. This analysis revealed clear differences to the mode of action of PC4. Effects of PC4 were found to be strictly dependent on TFIID concentrations leading to moderate (down from 10-fold to 3-fold) stimulation at saturating TFIID concentrations(33) . In contrast, activation by GAL-USF through PC5 did not depend on TFIID concentrations. This observation is in agreement with the assumption that PC5 does not stimulate binding of TFIID, as does PC4, but acts on subsequent steps of preinitiation complex formation.
We had previously analyzed the activation mechanism of PC4 by introducing a combined order-of-addition/restriction digest protocol. In this experiment the activator could be removed in active transcription complexes through restriction digest from general factors after formation of stable DA complexes(33) . These experiments confirmed that PC4 acted mostly on binding of TFIID. However, they also demonstrated moderate effects of PC4 on subsequent steps of preinitiation complex formation. Utilizing this protocol we found that PC5, independent of the TFIID concentration, stimulates transcription after DA complex formation. Basal levels of transcription were observed in reactions in which templates were preincubated with activator, PC5, TFIIA, and limiting concentrations of TFIID if templates were digested after DA complex formation. However, like PC4, PC5 failed to activate transcription when added after binding of TFIID and TFIIA to the promoter. As discussed previously, the activator itself binds quantitatively to its recognition sites after formation of DA complexes (33) . Thus, one likely explanation for these observations might be that PC5 fails to enter into the transcription complex, if added after binding of TFIID, whereas it does stimulate an event during assembly of the other general transcription factors (such as TFIIB or RNA polymerase II). It could equally well enhance transcription after initiation of transcription (e.g. during promoter clearance or elongation by RNA polymerase II). Both topological promoter mutants and mutants of general transcription factors will be required to further elucidate the mechanism by which positive cofactors stimulate transcription.