(Received for publication, December 1, 1995; and in revised form, March 5, 1996)
From the
The proximal or core promoter of a typical eukaryotic protein
coding gene comprises distinct elements, TATA and/or initiator (Inr).
The existence of TATA or Inr at the core promoter suggests that the
mechanism of transcription initiation mediated by these two genetic
elements may be different. Accordingly, it has been demonstrated that
the transcriptional requirements for the TATA-containing, Inr-less
(TATAInr
) promoters are different
from the transcriptional requirements for the TATA-less, Inr-containing
(TATA
Inr
) promoters. Although both
types of promoters require the transcription initiation factor (TFIID)
in addition to other common initiation factors, a
TATA
Inr
promoter requires accessory
component(s). Here we have employed in vitro analyses to
address the transcription factor requirements for a
TATA
Inr
promoter. We demonstrate
that in addition to TFIID, a naturally occurring
TATA
Inr
promoter requires TFII-I, an
Inr element-dependent transcription factor. Consistent with its Inr
element-dependent activities, TFII-I is dispensable for a
TATA
Inr
promoter. Furthermore, we
demonstrate that both TFII-I and TFIID activities in nuclear extracts
are temperature-sensitive. However, TFII-I is heat-inactivated at
temperatures lower than that required to inactivate TFIID. Therefore,
differential heat treatment of nuclear extracts provides an assay to
discriminate between transcriptional requirements at
TATA
Inr
and
TATA
Inr
promoters.
Transcription initiation of protein coding genes is brought
about by RNA polymerase II and a set of general transcription
initiation factors(1, 2, 3) . These factors,
in a combinatorial fashion, can direct transcription initiation of a
variety of eukaryotic promoters in an in vitro assay(1, 2, 3) . The core promoter
region of a typical eukaryotic gene consists of a TATA box and/or an
Inr ()element(4, 5) . The presence of
distinct core promoter elements in different genes suggests distinct
transcriptional strategies. However, the differences in mechanism of
transcriptional initiation mediated by TATA or Inr elements have yet to
be elucidated. Biochemical complementation assays employing
heat-treated nuclear extracts have demonstrated that the transcription
factor requirements for a TATA
Inr
promoter are different from a
TATA
Inr
promoter(6) . The
TATA-binding transcription factor complex TFIID is required for both
promoters(7) , and heat treatment of nuclear extracts (at 49
°C for 15 min) renders the TFIID inactive(6, 8) .
Hence, a heat-treated nuclear extract was incapable of transcribing
either a TATA-containing or a TATA-less promoter unless supplemented
with exogenous TFIID(6) . Exogenously added TFIID could restore
only a TATA
Inr
promoter activity but
not a TATA
Inr
promoter activity,
suggesting that in addition to TFIID, another heat-sensitive
component(s) was required for the TATA
Inr
promoter(6) . The mechanism of action of this component
is unclear. Because TFIID is required for a TATA-less promoter, it is
possible that such a factor may serve to anchor TFIID to a
TATA
Inr
promoter in the absence of a
TATA box(9) .
Transcription factor requirements for
TATAInr
promoters are controversial.
Several factors have been reported as Inr element-binding proteins
including TFII-I(10, 11, 12) ,
USF(10, 13) , YY1(14, 15) , RNA
polymerase II(16) , and a member of the TBP-associated factors
(TAF; 6, 17-20), although other studies have suggested that a TAF
may not bind directly to an Inr element(21) . However, these
observations may not be mutually exclusive. The differences possibly
indicate redundancy in Inr element-mediated interactions, which may be
condition- and/or promoter context-dependent. In addition, it is
possibile that structurally (and perhaps functionally) different
classes of Inr elements exist.
We wish to elucidate the molecular
mechanisms of transcription initiation mediated via an Inr element in
TATAInr
promoters. Here, we report
that TFII-I (10, 11, 12) is necessary for
transcription of a naturally occurring
TATA
Inr
but not for a
TATA
Inr
promoter. For our analyses,
we have used the T cell receptor variable region-derived (V
)
promoter (22) as a model TATA
Inr
promoter and the B cell immunoglobulin heavy chain-derived (IgH)
promoter (23, 24) as a model
TATA
Inr
promoter. We provide three
lines of evidence in support of a requirement of TFII-I for the
TATA
Inr
V
promoter. In each
case we have selectively blocked the transcription of the V
promoter and subsequently restored its transcriptional activity by
exogenous addition of purified TFII-I. We demonstrate that: 1)
Immunodepletion of nuclear extracts with an anti-TFII-I antibody
completely abrogates transcription of the
TATA
Inr
V
promoter, which is
restored by addition of purified TFII-I. Importantly, these antibodies
have no effect on the TATA
Inr
IgH
promoter. 2) TFII-I binds specifically to the V
Inr element. Thus,
an oligonucleotide containing the wild type V
Inr element
sequences efficiently inhibits V
transcription; exogenously added
TFII-I restores V
transcription. A control oligonucleotide
containing the mutant V
Inr sequence does not inhibit V
transcription. 3) In addition to TFIID, TFII-I is
temperature-sensitive. Thus, heat treatment of nuclear extracts impairs
both TFII-I and TFIID activities. However, the two activities are
affected at different temperatures. Heat treatment of nuclear extracts
at 42 °C ablates TFII-I but not TFIID activity, whereas heat
treatment at 49 °C destroys TFIID activity as well. Transcriptional
complementation assays using heat-treated nuclear extracts demonstrate
that although TFIID is both necessary and sufficient for a
TATA
Inr
promoter, TFII-I is
additionally required for the TATA
Inr
V
promoter.
Figure 4:
Differential heat treatment of nuclear
extracts discriminates between a TATAInr
and a TATA
Inr
promoters. a,
TFII-I activity is temperature-sensitive because heat treatment of
nuclear extracts interferes with TFII-I binding. A predominant mobility
shift was observed in HeLa nuclear extract (lane 1). The
binding was due to TFII-I because the mobility shift was inhibited by
an anti-TFII-I antibody (lane 2). Heat treatment of the
nuclear extract either at 42 °C for 6 min (lane 3) or 42
°C for 15 min (lane 4) blocked TFII-I binding. b,
heat treatment of nuclear extract at 42 °C for 6 min abolished
V
transcription (compare lanes 1 and 2). Neither
TFII-I (lane 3) nor TFIID (lane 4) completely rescued
the heat induced block of V
(TATA
Inr
) transcription. The
marginal effect observed with the TFIID fraction (lane 4) was
due to contamination with TFII-I (not shown). However, simultaneous
addition of TFII-I and TFIID completely rescued the heat-induced block
of the V
transcription (lane 5). c, contrary to
the TATA
Inr
V
transcription,
heat treatment of nuclear extract either at 42 °C for 6 min (lane 2) or 42 °C for 15 min (lane 4) did not
block TATA
Inr
IgH transcription
(compare with lane 1). In fact, the mild heat treatment
produced a stimulatory effect. Furthermore, the addition of a TFIID
fraction to the mildly heat-treated nuclear extract had no significant
effects (lanes 3 and 5).
For peptide block experiments, the anti-TFII-I antibody was preincubated for 30 min at 0 °C prior to probing with 2.5, 0.25, or 0.025 mg/ml of the synthetic peptide derived from the putative DNA binding region of TFII-I.
Figure 1:
Immunodepletion of TFII-I affects a
TATAInr
(V
) but not a
TATA
Inr
(IgH) promoter. a, EMSA
of HeLa and Jurkat nuclear extracts (lanes 1 and 2)
demonstrates a major complex that is blocked by an anti-TFII-I antibody
(
I; lanes 3 and 5) but not by an anti-TBP
antibody (
T; lanes 4 and 6). A preimmune serum
had no effect on TFII-I mobility shift (not shown). For this EMSA we
used an AdML promoter-derived Inr element. Identical results were
obtained with all Inr elements tested under our conditions. b,
sequence comparison of different Inr elements. The initiating
nucleotide is indicated by the arrow. c, in vitro transcription using a linearized V
template. The run-off
transcript from the V
template was not affected by mock depletion
of Jurkat nuclear extract with a preimmune (pI) serum (compare lanes 1 and 2). The transcription was abolished
completely upon immune depletion with the anti-TFII-I antibody (I, lane 3). d, the anti-TFII-I antibody (I) severely
decreased the V
transcription (compare lanes 1 and 2). However, the transcription was restored completely upon
exogenous addition of a purified preparation of TFII-I (lane
3). As before, a preimmune (pI) serum had negligible
effects on the V
transcription (lane 4). e,
immunodepletion of TFII-I from Jurkat nuclear extract had no
significant effect on IgH transcription (compare lanes 1 and 3). Similarly, the control antibody (preimmune serum, pI) had no effects on IgH transcription (lane
2).
Figure 2:
Specificity of the anti-TFII-I antibody. a, the anti-TFII-I antibody specifically recognized
p120/TFII-I in Jurkat nuclear extract (lane 1) and in purified
TFII-I (lane 2). The cross-reactive 125-kDa band in Jurkat
nuclear extract is a modified form of TFII-I (not shown). Importantly,
the antibody reactivity can be blocked by pretreatment with the
antigenic peptide, either 2.5 (lanes 3 and 4) or 0.25
mg/ml (lanes 5 and 6) but not with 0.025 mg/ml (lanes 7 and 8). b, antibody depletion (TFII-I dep.) leads to removal of TFII-I from a Jurkat nuclear
extract but mock depletion (mock dep.) has no appreciable
effect compared with undepleted extract (undep.). c,
the immune precipitate (I ppt) obtained from antibody
depletion shows the presence of substantial amount of TFII-I, whereas
the precipitate from mock depletion (pI ppt) has no
significant amount of TFII-I.
Figure 3:
Binding of TFII-I to the Inr element is
essential for TATAInr
transcription. a, a purified preparation of TFII-I (HeLa-derived) binds to
the V
Inr element (lane 2). The binding was specific
because it was competitively inhibited by a wt V
Inr element
containing oligonucleotide (lane 3) but not by a mutant
oligonucleotide (mut, lane 4). b, V
transcription by Jurkat nuclear extract (lanes 1 and 7) was competitively inhibited by a wt Inr oligonucleotide (lanes 3 and 8). Neither an E-box containing
oligonucleotide (lane 2) nor a mut Inr oligonucleotide (lane 4) inhibited V
transcription. The wt Inr
oligonucleotide-mediated block of transcription (lane 8) was
restored upon the addition of the purified preparation of TFII-I (lane 9). An oligonucleotide containing the wild type TATA box
sequences from the AdML promoter blocked the V
transcription (lane 5); a mutant TATA oligonucleotide did not block
transcription (lane 6).
Sequence comparison
between different promoters reveals that the V promoter contains a
consensus Inr element (Fig. 1b). The V
promoter
(kind gift from Dr. D. Loh) is typically expressed in T
cells(22) . Thus, we employed a T cell-derived nuclear extract
(Jurkat) for all of the in vitro transcriptional assays. We
used the anti-TFII-I antibody to deplete TFII-I from a
transcriptionally competent Jurkat nuclear extract (Fig. 1c). Employing an undepleted nuclear extract (lane 1), the run-off assay from the linearized V
promoter (containing wild type sequences from -480 to +260)
produced an accurately initiated 260-nucleotide major transcript. Mock
depletion of Jurkat nuclear extract with a control antibody (preimmune
serum) had no appreciable effects on transcription (lane 2).
However, immunodepletion with an anti-TFII-I antibody severely impaired
the V
transcription (lane 3).
To demonstrate that the
antibody treatment causes depletion of only TFII-I, we added back
TFII-I, exogenously, to an immunodepleted Jurkat nuclear extract (Fig. 1d). Antibody-mediated inhibition of
transcription (lane 2) was completely restored by exogenous
addition of TFII-I (lane 3). Mock depletion (with preimmune
serum) had very little effect (lane 4). Therefore, an
anti-TFII-I antibody inhibits transcription from a
TATAInr
promoter, which can be
restored by exogenous addition of TFII-I.
As a control for promoter
specificity, we employed the TATAInr
IgH (23, 24) promoter (Fig. 1e).
The minimal (-47 to +1) IgH promoter does not exhibit any
tissue type specificity in vitro and therefore can be
transcribed by a T cell (Jurkat) nuclear extract. Most importantly,
TFII-I depletion of a Jurkat nuclear extract did not affect the
TATA
Inr
IgH promoter. Thus, the
level of the 400-nucleotide transcript produced from the IgH promoter
remains unaltered in undepleted (lane 1), mock-depleted (lane 2), and immunodepleted (lane 3) Jurkat nuclear
extracts. These data clearly demonstrate that TFII-I is required for a
TATA
Inr
promoter but not for a
TATA
Inr
promoter.
Consistent with the
DNA binding analyses, the wild type Inr oligonucleotide competitively
inhibited transcription from the V promoter in nuclear extracts (Fig. 3b, lanes 3 and 8), whereas the
mutant oligonucleotide failed to inhibit transcription (lane
4). Most importantly, the Inr oligonucleotide-mediated inhibition
of transcription was restored upon the addition of TFII-I (lane
9). As an additional control of DNA binding specificity, an E-box
containing oligonucleotide was used (lane 2). Although TFII-I
can bind to both an Inr element and an E-box (dual specificity), an
E-box containing oligonucleotide cannot block TFII-I binding to the Inr
element and vice versa(10) . Accordingly, the E-box containing
oligonucleotide failed to abrogate Inr-dependent transcription. Taken
together, these results demonstrate that TFII-I binds specifically to
the V
Inr element and that this binding is necessary for the
V
transcription initiation.
TFIID has been shown to be required
for transcriptional activity of TATA-containing as well as TATA-less
promoters(7, 9) . Consistent with this notion, an
oligonucleotide containing only a wild type (lane 5) but not a
mutant TATA box (lane 6) competitively inhibited the V
transcriptional activity. It is important to note that TFIID is
necessary but not sufficient to direct Inr-dependent V
transcription (see below).
Next, we employed
the heat-treated nuclear extracts in in vitro transcriptional
assays with the V (Fig. 4b) and IgH (Fig. 4c) promoters. Heat treatment of a Jurkat nuclear
extract at 42 °C for 6 min led to abrogation of the V
transcription (Fig. 3a, lane 2). Surprisingly,
the addition of TFII-I did not alleviate the transcriptional block (lane 3), suggesting that this heat treatment interfered with
additional component(s). The Addition of a partially purified TFIID
fraction in the absence of exogenous TFII-I did not rescue the
transcriptional block either (lane 4). However, the addition
of both TFII-I and TFIID simultaneously rescued completely the
heat-induced block of V
transcription (lane 5),
suggesting that the additional component was present in the TFIID
fraction. Therefore, in addition to TFII-I and TFIID, a third
component, which is present in the TFIID fraction, is required for a
TATA
Inr
promoter. Although the exact
identity of this component is presently unknown, the existence of such
a component (activity) has been described before(6) . Similar
results were obtained when nuclear extracts were heat treated at 42
°C for 15 min (not shown). Furthermore, only TFIID (and not TBP)
was effective in these complementation assays (not shown).
TFII-I is
an Inr element-dependent factor and therefore is not required for an
Inr-less promoter. Accordingly, mild heat treatment (42 °C for 6
min) of nuclear extracts did not abolish the
TATAInr
IgH promoter activity (Fig. 4c). In fact, we observed a reproducible increase
in the IgH promoter activity upon mild heat treatment (compare lanes 1 and 2). Because TFIID activity was not
affected at 42 °C, the addition of TFIID had no effect on IgH
transcription at this temperature (lane 3). Similarly, heat
treatment of nuclear extracts at 42 °C for 15 min had no negative
effect on IgH transcription (the background was reduced under these
conditions), and subsequently added TFIID had no appreciable effect on
transcription (lanes 4 and 5). Under similar
conditions, nuclear extracts heat treated at 42 °C for 15 min
failed to transcribe the V
promoter (data not shown). Taken
together, our data demonstrate that transcription factor requirements
between the TATA
Inr
and
TATA
Inr
promoters are different.
TATA
Inr
promoters require TFII-I,
whereas the TATA
Inr
promoters do
not.
The control region of typical eukaryotic messenger RNA coding
genes is comprised of proximal (core) and distal (enhancer) promoter
regions(1) . The core promoter region consists predominantly of
two elements: the TATA box and/or the Inr element, which can be present
either alternately (TATAInr
or
TATA
Inr
) or in limited cases
simultaneously
(TATA
Inr
)(26) . To understand
the various transcriptional strategies that exist in nature, it is
important to elucidate why different genes have adopted different core
promoter elements and how these elements mediate transcription.
Transcription initiation in eukaryotes is mediated by a set of
general transcription factors that assemble at the core promoter to
form the preinitiation complex(27, 28, 29) .
The core promoter structures are different for different genes.
Consequently, the preinitiation complexes (containing general
transcription initiation factors), which assemble at different core
promoter elements (TATA or Inr) are
different(10, 11) . These experiments, however,
employed a composite TATAInr
core
promoter and were reconstituted with purified and/or recombinant
proteins(10, 11) . To distinguish between the
mechanisms of transcription initiation mediated by TATA and Inr, we
employed nuclear extracts to transcribe the naturally occurring
TATA
Inr
(V
) and
TATA
Inr
(IgH) promoters. Our
analyses demonstrate that the mechanisms of promoter utilization and
the requirement of transcription factors are distinct for the two
classes of promoters.
We present multiple approaches that were
undertaken to demonstrate differences in promoter utilization. First,
we depleted various nuclear extracts for the transcription factor
TFII-I, which is important for Inr element-containing
promoters(10, 11, 12) . Depletion of TFII-I
by an anti-TFII-I antibody led to complete inhibition of transcription
of a TATAInr
promoter, whereas
TFII-I depletion did not have a negative effect on
TATA
Inr
transcription. Furthermore,
the addition of TFII-I relieved the antibody-mediated inhibition of the
V
promoter, suggesting that the active component was indeed
TFII-I. This conclusion is supported by the fact that the antibody
predominantly recognizes TFII-I in nuclear extracts (as evidenced by
Western blot analysis) and can be effectively blocked by the antigenic
peptide derived from TFII-I (Fig. 2a). However, because
the preparation of TFII-I used to reconstitute the transcriptional
activity is partially pure, involvement of additional components cannot
be ruled out completely.
Second, we demonstrate that an Inr
element-containing oligonucleotide, which was competent in TFII-I
binding, competitively inhibited transcription of a
TATAInr
promoter. This observation
suggests that interactions of TFII-I to the Inr element is necessary
for TATA
Inr
transcription.
Consistent with this suggestion, the Inr oligonucleotide-mediated
inhibition of transcription was relieved by exogenous addition of
TFII-I. Other factors(13, 14, 15, 16, 17, 18, 19, 20) have
been implicated in Inr element binding. However, under the conditions
tested, TFII-I is the predominant factor present in various nuclear
extracts that is responsible for Inr-dependent binding and
transcriptional activities via the V
promoter. This is consistent
with our preliminary data, which indicate that TFII-I is also required
for the V
promoter function in vivo.
TFIID is required
for both TATAInr
and
TATA
Inr
promoters(6, 7) . However, it is questionable
whether or not TATA binding activity of TFIID is required for the
TATA
Inr
promoters(6) .
Accordingly, it has been shown that TATA binding activity is required
for some TATA
Inr
promoters but not
for others (``true'' TATA-less promoters)(6) . The
definition of a true TATA-less promoter is confusing and thus a
TATA-less promoter should be defined by the lack of a consensus TATA
sequence and not on the basis of mechanisms of TFIID binding. The
promoter employed here (V
) is a naturally occurring TATA-less
promoter (lacking a consensus TATA box), a notion further supported by
model building studies. (
)However, V
transcription
requires the TATA binding activity of TFIID. Although we do not know
the exact mechanism of TFIID recruitment to the V
promoter, it is
possible that the binding of TFIID to the promoter may be mediated by
TFII-I interactions because TFII-I interacts with the TATA binding
subunit (TBP) of TFIID(11) .
Finally, we demonstrate that
heat treatment of nuclear extracts affects
TATAInr
and
TATA
Inr
promoters differently. It
has been shown that heat treatment of a nuclear extract, normally
competent for transcription, rendered the extract inactive for
transcription of both types of promoters(6) . The
transcriptional activity of the extract for a
TATA
Inr
promoter could be restored
upon exogenous addition of TFIID(6) . However, for a
TATA
Inr
promoter, the addition of
TFIID was insufficient, suggesting that an additional heat-labile
component(s) was necessary for TATA
Inr
promoters(6) . Here we demonstrate that TFII-I is
heat-labile and is required in addition to TFIID for
TATA
Inr
promoter function. Our
analyses also suggest the existence of a third component that is
required for TATA
Inr
promoters. This
component is present in a partially purified TFIID fraction and is
heat-labile (Fig. 5). It is unclear at present whether this
component is directly associated or merely copurifies with TFIID.
Figure 5:
Factor requirements for
TATAInr
and
TATA
Inr
promoters. A
TATA
Inr
promoter requires binding of
TFII-I to the Inr element. In the absence of a cognate TATA box, this
initial interaction of TFII-I with the Inr element may be necessary to
recruit TFIID and an additional component (?) to the promoter.
The exact order of entry of different factors into the preinitiation
complex is unclear at present. TFII-I is dispensable for a
TATA
Inr
promoter, which, however,
requires TFIID.
Our data reveal that different transcription factor activities can
be targeted by heat treating nuclear extracts at different
temperatures. Thus, although heat treatment of nuclear extracts at 42
°C for 6 min completely ablates
TATAInr
transcription, similar
treatment does not ablate TATA
Inr
transcription. This mild heat treatment does not affect TFIID
activity but destroys other activities (including TFII-I) that are
required for TATA
Inr
transcription.
Therefore, differential heat treatment of nuclear extracts at different
temperatures can be used as an assay to distinguish between
TATA
Inr
and
TATA
Inr
-dependent transcriptional
activities.
In conclusion, we clearly demonstrate the Inr-dependent
function of TFII-I via the TATAInr
V
promoter. It is possible that TFII-I may be necessary for
transcription of other TATA
Inr
promoters as well(30) . Finally, although it appears at
present that different Inr-dependent factors may function through
different promoters or under different conditions, it is likely that
multiple Inr-dependent factors may work in concert for some
TATA
Inr
promoters.