From the University of Washington, Department of Pharmacology, School of Medicine, Health Sciences Center, Box 357280, Seattle, Washington 98195-7280
Received for publication, January 14, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclin D1 is an oncogene that regulates
progression through the G1 phase of the cell
cycle. A temperature-sensitive missense mutation in the transcription
factor TAF1/TAFII250 induces the mutant ts13 cells to
arrest in late G1 by decreasing transcription of cell cycle
regulators, including cyclin D1. Here we provide evidence that TAF1
serves two independent functions, one at the core promoter and one at
the upstream activating Sp1 sites of the cyclin D1 gene. Using in
vivo genomic footprinting, we have identified protein-DNA
interactions within the cyclin D1 core promoter that are disrupted upon
inactivation of TAF1 in ts13 cells. This 33-bp segment, which we termed
the TAF1-dependent element 1 (TDE1), contains an initiation
site that displays homology to the consensus motif and is sufficient to
confer a requirement for TAF1 function. Electrophoretic mobility shift
assays reveal that binding of ts13-TAF1-containing TFIID complexes to
the cyclin D1 TDE1 occurs at 25 °C but not at 37 °C in
vitro and involves the initiator element.
Temperature-dependent DNA binding activity is also observed
for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the
wild-type TAF1 protein. These data suggest that a function of TAF is
required for the interaction of TFIID with the cyclin D1 initiator. Our
finding that recruitment of TFIID, by insertion of a TBP binding site
upstream of the TDE1, restores basal but not activated transcription
supports the model that TAF1 carries out two independent functions at
the cyclin D1 promoter.
Understanding the control of cell proliferation at the molecular
level is a central issue in cancer biology. Normal cells progress
through a well-defined cell cycle consisting of four distinct stages,
G1, S, G2, and M. Progression from one stage to
the next is regulated in part by cyclins that bind and activate cyclin
dependent kinases, an event necessary to drive cells through the
proliferation cycle (1). Cyclin protein levels oscillate throughout the
cell cycle and are thought to be controlled, in part, at the level of
transcription. In support of this model, the deregulated expression of
cyclins A and D1 has been found in several human tumors (2, 3). The
overexpression of cyclin D1 is found in many breast cancers (~50%),
mantle cell lymphomas (~100%), and squamous cell carcinomas
(~64%) and is often associated with a poor prognosis (4-6).
Therefore, a better understanding of the regulatory pathways governing
cyclin D1 promoter activity may lead to the discovery of new
therapeutic targets for the development of anti-cancer drugs.
RNA polymerase II-dependent gene transcription requires the
assembly of more than 20 proteins at the promoter region to form a
functional preinitiation complex. The general transcription factor IID
(TFIID)1 is a large
multisubunit complex consisting of the TATA-binding protein (TBP) and a
set of TBP-associated factors (TAFs) (7). The recruitment of TFIID to
the promoter is the first step in the assembly of the preinitiation
complex. At promoters that contain a TBP binding site, the mode of
recruitment is at least partly via TBP binding to the TATA element.
DNase I protection studies have shown that TFIID, in contrast to TBP,
protects an extended region that includes sequences ~30 bp downstream
of the TATA box (8). These data suggest that one or more of the TAF
subunits most likely contact DNA (9). However, the precise function of
the TAF components of TFIID in the transcription process still remains
to be elucidated.
Many promoters, including cyclin D1, lack a consensus TATA element.
Under these circumstances the TAFs may be more actively involved in the
recruitment of TFIID to promoter sequences. Several TAFs have been
shown to interact directly with promoter DNA (9-11). UV cross-linking
experiments with purified TFIID reveal that TAF1 and TAF2 are in close
contact with the core promoter region (9). In a random site selection
screen, TAF1-TAF2 heterodimers, but not the individual TAFs, were found
to preferentially bind to DNA sequences that fit the initiator
consensus motif (11). These data suggest that TAF1 and TAF2 could play
an important role in TFIID binding under conditions suboptimal for TBP binding.
The ts13 cell line, derived from BHK-21 (baby hamster kidney) cells, is
a temperature-sensitive mutant that arrests in late G1 at
the nonpermissive temperature (39.5 °C) due to a missense mutation
in the TAF1 protein (12-14). The single base pair change identified in
ts13 cells results in a glycine to an aspartic acid substitution at
amino acid 690 of the hamster TAF1 protein (15). Expression of
wild-type human TAF1 is sufficient to complement the ts13 cell cycle
arrest phenotype, suggesting that the point mutation is responsible for
the proliferation defect (16).
Despite having a mutation in a component of the general transcriptional
machinery, ts13 cells do not display a global defect in gene expression
(16-19). Instead, transcription from a subset of genes, including the
cell cycle regulators cyclins A, D1, and E, is dramatically reduced at
39.5 °C. Transfection of wild-type human TAF1 into ts13 cells not
only complements the G1 arrest but also overcomes the
cyclin A and D1 transcriptional defect (20). Conditional mutations in
yeast TAF1 (yTAF1) also induce cells to arrest in G1 and to
exhibit defects in gene transcription (21). DNA microarray analysis of
yTAF1 mutant strains under permissive and restrictive conditions
revealed that ~16% of yeast genes display a requirement for TAF1
function (22). Studies carried out with ts13 mRNA hybridized to a
more limited mouse DNA microarray generated similar results (23). These
data provide additional evidence that inactivation of the TAF1 subunit
of TFIID leads to gene-specific defects in mRNA synthesis. Our
current working hypothesis is that the inability of ts13 cells to
progress through the G1/S phase checkpoint can be
attributed to a disruption in TAF1 function, which compromises the
transcription of a select group of cell cycle control genes. The
availability of the ts13 cell line provides a unique model system to
identify and characterize the specific activity of TAF1 required for
cell cycle gene transcription and ultimately cell proliferation.
Cyclin D1 is a G1/S phase cell cycle regulatory protein
that was isolated from murine bone marrow macrophages as an early responsive gene after stimulation with colony stimulating factor 1. Among the cyclin proteins, expression of the D-type cyclins is one of the earliest events to occur during the G1 phase
of the cell cycle leading to cell division. Cyclin D1 has been shown to
be transcriptionally up-regulated by numerous growth factors, including
nerve growth factor in PC12 cells, estrogen and gestergen in
endometrial cells, and epidermal growth factor in prostate cancer cells
(24-26). The 5'-regulatory region of the cyclin D1 gene has been
isolated from a number of species, including rat, mouse, and human
(27-29). It consists of a TATA-less promoter with a cAMP-responsive
element (CRE) and multiple Sp1 binding sites, through which several
growth factors exert their proliferative effect (30).
For our studies, we have elected to focus on the cyclin D1 promoter,
because decreases in cyclin D1 mRNA levels occur relatively quickly
after ts13 cells have been shifted to 39.5 °C (19). The rapid
response suggests that transcription of cyclin D1 is directly affected
by the ts13 mutation in TAF1. Studies in yeast with conditional alleles
of yTAF1 mapped the TAF1-dependent phenotype to the core
promoter region of CLN2 (31). The analysis of chimeric promoter constructs between the TAF1-dependent cyclin A and
TAF1 independent c-fos genes has revealed that the
core promoter elements of cyclin A are also sufficient for
temperature-sensitive expression in ts13 cells (20). These results
indicate that TAF1 has a unique function at the core promoter of a
subset of genes. The goal of our studies is to elucidate the function
of TAF1 required for efficient promoter activity.
We report here that in vivo protein-DNA interactions within
a 33-bp region of the cyclin D1 core promoter, the TDE1 (for
TAF1-dependent element 1), are altered under conditions
where TAF1 activity is compromised. We demonstrate that TFIID binds to
the TDE1 of cyclin D1 in gel mobility shift assays. The DNA binding
ability of TFIID as well as TAF1-TAF2 heterodimers is compromised at
37 °C in vitro by the ts13 mutation in TAF1. These
results have led us to conclude that the lack of cyclin D1
transcription can be attributed at least in part to the inefficient
recruitment of TFIID to the core promoter. The introduction of a
properly positioned consensus TATA element in the minimal cyclin D1
core promoter is able to fully restore basal transcriptional activity
at 39.5 °C in ts13 cells. However, full recovery of activated
transcription was not observed when similar experiments were carried
out with a longer cyclin D1 promoter fragment containing the upstream
activating Sp1 sites. These findings suggest a dual function for TAF1
in cyclin D1 transcription, one of which is required for efficient TFIID binding to the core promoter to initiate the transcription cycle.
Tissue Culture Cell Lines--
ts13 and ts13R3 cells were grown
in Dulbecco's modified Eagle's media (Invitrogen) supplemented with
10% fetal bovine serum (JRH Biosciences), 2 mM
L-glutamine, and penicillin/streptomycin in a humidified
incubator containing 10% CO2 at 33.5 °C and 39.5 °C,
respectively. To inactivate TAF1 and induce cell cycle arrest, ts13
cells were shifted to 39.5 °C. ts13 and ts13R3 cells that constitutively express HA-epitope-tagged ts13 and wild-type alleles of
human TAF1, respectively, were established as follows. Cells were
transfected with a cytomegalovirus expression vector for the HA-tagged
mutant or wild-type TAF1 protein along with a neomycin expression
plasmid at a ratio of 10 to 1. After 24 h, cells were plated into
media containing 1 mg/ml G418. G418-resistant colonies were isolated
and clonally expanded, and the expression of the HA-tagged TAF1 was
determined by Western blotting with the anti-HA mAb 12CA5. HA-positive
cell lines were passaged in 200 µg/ml G418 to maintain expression of
the HA-tagged TAF1. Sf9 insect cells were propagated in Hanks'
TNM-FH insect media (JRH Biosciences) containing 10% FBS,
L-glutamine, and penicillin/streptomycin. Cultures were
grown in spinner flasks at 27 °C in the absence of
CO2.
Cloning of Hamster Cyclin D1 Promoter Fragment--
An upstream
fragment of the hamster cyclin D1 promoter was PCR amplified from ts13
genomic DNA using the following primer pair:
5'-AGGAAATAATGGCCACCATCTTG-3' and 5'-CTTTCAATTTCATCAACAGTG-3', designed
from alignment of rat, mouse, and human cyclin D1 promoter sequences
(GenBankTM accession numbers AF148946, AF212040, and
Z29078, respectively). A hamster-specific primer
5'-TCTTGAGCTGTTGCTGGGATTTT-3', based on the above PCR product, and a
primer (5"-GTCAGGGTACGCGCGGC-3') from alignment of downstream rat,
mouse, and human cyclin D1 gene sequences available in
GenBankTM (accession numbers AB042564, AF212040, and
Z29078, respectively) were used to PCR-amplify an 895-bp fragment
representing the hamster cyclin D1 promoter from ts13 genomic DNA. The
PCR fragment was subcloned into pCRII TOPO vector using the TOPO TA
cloning kit (Invitrogen), and the DNA sequence was determined. The
sequence of the hamster promoter fragment has been deposited into
GenBankTM under accession number AF539477.
Mapping of Hamster Cyclin D1 Transcription Start Sites by Primer
Extension--
The 32P-labeled oligonucleotide
(5'-TGCCTCGCGCTCTACTGCC-3'), complementary to the hamster cyclin D1
sequence, was used in reverse transcription reactions with 5 µg of
total RNA from either asynchronously growing ts13 cells or ts13 cells
synchronized in G1 ~ 8 h after release from serum
starvation. Reaction products were separated on denaturing 6%
polyacrylamide gels and visualized by autoradiography.
Reporter Plasmid Constructs--
Promoter fragments to be
examined in transient transfection assays were subcloned into
pGL2-basic (Promega), a reporter plasmid that contains the luciferase
coding region. Mouse c-fos reporter contains Transient Transfection Assays--
Before the introduction of
DNA, ts13 cells, seeded into 24-well plates, were maintained at
33.5 °C or shifted to 39.5 °C for 2 h. Cells were then
switched to Dulbecco's modified Eagle's medium containing no FBS and
no antibiotics. 0.1 µg of SV40- In Vivo Genomic Footprinting of the Hamster Cyclin D1
Promoter--
ts13 and ts13R3 cells were arrested in G0 by
serum starvation for 36 h at 33.5 °C, then released at either
33.5 °C or 39.5 °C by the addition of serum. Approximately 8 h post-serum addition, cells were treated with 0.01% DMS for 2 min.
Genomic DNA was isolated and methylated at guanine/adenine nucleotides
as described with the following minor modifications (33). For the
preparation of genomic DNA, the ethyl ether extraction was eliminated
from the protocol, and the isopropanol precipitation was replaced with an ethanol precipitation. Sites of guanine and adenine methylation were
chemically cleaved and sites of cleavage in the hamster cyclin D1
promoter were mapped using the technique of ligation-mediated PCR (34).
Briefly, 5 µg of genomic DNA served as template in a first-strand
synthesis reaction with the following primers: upper strand,
5'-CTGCCTCGCCGTCTACTGCC-3'; lower strand, 5'-TCCCGAGCCCCCTCCCCCT-3'. Linkers were then ligated onto the blunt-ended products. Next, a linker
and nested gene-specific primer (upper strand,
5'-GTCTCCGAGCGCGCGAATCT-3'; lower strand, 5'-GCGCCCGCCCAGTCCTTCC-3')
were used to amplify the cleavage products. The resulting PCR products
were end-labeled with the following 32P-labeled primers
(upper strand, 5'-ATCTGCCGCTCTCTGCTACGCG-3'; lower strand,
5'-CCCCTCCCTTTCTCTGCCTGGCT-3'). Reaction products were separated on
denaturing 6% polyacrylamide gels and visualized by autoradiography.
Preparation of Cell Lysates--
ts13 and ts13R3 nuclear
extracts were prepared as previously described (16) from synchronized
cells that had been serum-starved and released into the cell cycle for
8-12 h by serum addition. The preparation of HeLa nuclear extracts and
fractionation of the extracts on phosphocellulose to obtain the
TFIID-depleted extract and a partially purified TFIID fraction were
carried out following the protocol of Dignam et al.
(35).
Electrophoretic Gel Mobility Shift Assays--
ts13 or ts13R3
nuclear extracts to be used in binding reactions were equilibrated at
25 °C or 37 °C. Twenty micrograms of each extract was then
preincubated at the appropriate temperature for 20 min in 20 mM HEPES, 50 mM NaCl, 10% glycerol, 2%
polyethylene glycol 6000, 5 mM ammonium sulfate, 5 mM MgCl2, 3 mM Preparation of TAF1-TAF2 Heterodimers--
Recombinant
baculovirus vectors used to express HA-tagged TAF1 and FLAG-tagged TAF2
have been previously described (9, 38). For protein production,
Sf9 cells were seeded at ~7.5 × 106
cells/10-cm dish and infected with recombinant baculovirus at a
multiplicity of infection of 1-2. Approximately 48 h
post-infection, whole cell lysates were prepared by sonication in HEMG
buffer (25 mM HEPES, pH 7.6, 12.5 mM
MgCl2, 0.1 mM EDTA, 10% glycerol, 0.1%
Nonidet P-40) containing 0.4 M KCl. HA-TAF1 and FLAG-TAF2 monomers were immunoaffinity purified from Sf9 whole cell
extracts using the anti-HA (mAb 12C5) and anti-FLAG (M2 antibody)
antibodies, respectively. TAF1-TAF2 heterodimers were assembled
in vitro using the anti-HA and anti-FLAG antibodies as
previously described (39). For gel mobility shift assays, all proteins
were eluted under native conditions with peptides corresponding to the
appropriate antibody epitope (HA: YPYDVPDYA; FLAG: DYKDDDDK) in HEMG
buffer containing 0.1 M KCl.
Cloning and Characterization of Hamster Cyclin D1 Promoter
Fragment--
To investigate the function of TAF1 in the transcription
of cyclin D1 in ts13 cells, we cloned a fragment of the hamster cyclin D1 promoter using a PCR-based approach. Analysis of the endogenous promoter will avoid limitations that may be inherent to the study of a
human promoter in hamster cells. A short stretch of the hamster cyclin
D1 promoter was PCR-amplified from ts13 genomic DNA using primers
designed to regions of high sequence identity between the human, mouse,
and rat promoters. To eliminate errors generated during the
amplification reaction, several independent PCR products were subcloned
and subjected to DNA sequencing. The sequence information available in
GenBankTM and obtained from the PCR products were used to
design hamster-specific primers that amplified a single 895-bp fragment
from ts13 genomic DNA (data not shown). Comparison of this putative
hamster cyclin D1 promoter fragment with the previously described
human, rat, and mouse genes (27-29) revealed a remarkable 65.5%,
83.3%, and 83.7% sequence identity, respectively, in the noncoding
region, with even greater sequence identity between
We also mapped the sites of transcription initiation by primer
extension. Similar to the rat, mouse, and human promoters, hamster
cyclin D1 utilizes multiple transcription initiation sites in ts13
cells (Fig. 2). As a reference point, we
defined the most downstream transcriptional start site as +1, which is
124 nucleotides upstream of the translational start codon. This
numbering system places all identified transcriptional initiation sites
between +1 and
Despite nearly 100% sequence identity within the initiation region,
the start sites of transcription differs between species, although they
tend to cluster within the same region of the promoter. Only one of the
major clusters of transcriptional start sites in hamster cyclin D1
(between Hamster Cyclin D1 Transcription Requires TAF1 Function at the Core
Promoter--
We have previously reported that transcription from the
human cyclin D1 promoter is severely compromised when transiently transfected into ts13 cells at the nonpermissive temperature (20). To
determine if the hamster promoter also demonstrates a requirement for
TAF1, we cloned the 895-bp hamster cyclin D1 promoter fragment upstream
of the luciferase gene to generate the reporter construct
A series of hamster cyclin D1 promoter deletions was constructed to
determine what region is responsible for TAF1-dependent transcriptional activity (Fig. 3A). The successive removal
of transcription factor binding sites between
Unexpectedly, deletion of sequences between +21 and +109 led to ~50%
reduction in promoter activity (Fig. 3B). This region of the
cyclin D1 gene contains no known transcription factor binding sites or
apparent transcription initiation sites, whose loss could account for
the reduction in transcription levels. The contribution of this segment
of the cyclin D1 gene to promoter activity remains to be determined.
In Vivo Genomic Footprinting of the Cyclin D1 Core Promoter
Region--
We have demonstrated by deletion analysis that
transcription from the core promoter of cyclin D1 requires a function
of TAF1. In yeast, TAFs are dispensable for the transcription of many
genes (40, 41). However, yTAF1 is required at the core promoter for
transcription of a subset of genes (31, 42). Therefore, we investigated
if the decrease in cyclin D1 transcription at 39.5 °C is due to a
defect in transcription factor binding to the core region. To address
this issue, we utilized the technique of in vivo genomic
footprinting to map protein-DNA interactions at the cyclin D1 core
promoter in ts13 cells (Fig. 4). The
advantage of this approach is that it allows one to map protein-DNA
interactions that occur on the endogenous promoter assembled in native
chromatin with nucleotide resolution.
Cyclin D1 transcription occurs during the G1 phase of the
cell cycle. To maximize differences in transcription levels and thereby
DNA binding activity, we analyzed synchronized populations of ts13 and
ts13R3 cells at 33.5 °C and 39.5 °C. Approximately 8 h after
release from G0 by serum addition, a time of maximal cyclin
D1 transcription, cells were treated with 0.01% DMS to methylate
accessible base residues, and the in vivo modified genomic DNA was isolated by standard procedures. The position of methylated guanines and adenines was determined by chemical cleavage followed by
ligation-mediated PCR as described (34). The pattern of DNA methylation
in the absence of protein was also determined in vitro (Fig.
4, lanes 1 and 10). Both the upper and lower
strands were analyzed, because DMS methylates only adenine and guanine
residues present in each DNA strand. The ts13R3 cell line, which
expresses wild-type TAF1 at the higher temperature, was used in
parallel to identify changes that are simply due to the increase in
temperature (Fig. 4, lanes 4, 5, 8,
and 9). Only differences detected between the DNA
methylation patterns at the two temperatures in ts13 but not in ts13R3
cells can be attributed to changes in protein-DNA contacts dependent on
TAF1 activity. We focused on the cyclin D1 core promoter between
positions
Interestingly, the modification of cytosine residues was also observed
but only within the TDE1. Unpaired cytosines can be methylated by DMS.
Therefore, the modification of cytosines suggests the presence of
structured DNA, with these regions being single-stranded in nature.
TDE1 Is Sufficient to Confer a Requirement for TAF1 in Cyclin D1
Transcription--
We have defined protein-DNA contacts within a
segment of the cyclin D1 promoter or TDE1 that are altered at
39.5 °C in ts13 cells. Is this region of cyclin D1 sufficient to
confer a requirement for TAF1 function in gene transcription? To
address this question, the TDE1 of cyclin D1 was subcloned upstream of
a luciferase reporter gene and the resulting reporter construct
(
The TDE1 contains a major cluster of transcription initiation sites and
a putative NF- Temperature-sensitive DNA Binding Activity on the TDE1 of Cyclin
D1--
Our in vivo genomic footprinting results suggest
that protein binding to sequences within the core promoter of cyclin D1
is altered at 39.5 °C in ts13 cells. To identify the proteins
responsible for the change in DNA methylation protection, nuclear
extracts containing the mutant or wild-type allele of TAF1 were
prepared from permissively grown ts13 or ts13R3 cells, respectively,
and incubated with a radiolabeled DNA fragment containing the cyclin D1
TDE1. Binding reactions were carried out at either 25 °C or 37 °C, as these temperatures have been previously shown to be permissive and restrictive for in vitro transcription in
ts13 nuclear extracts, respectively (16). One predominant retarded protein-DNA complex was detected with both extracts when the reactions were incubated at 25 °C (Fig.
6B, left panel).
The formation of the shifted complex was disrupted when the reaction
temperature was increased to 37 °C but only in extracts containing
the mutant (ts13) TAF1 protein. The ts13 mutation in TAF1 decreases the
expression of multiple genes. Therefore, the lack of DNA binding
activity could be due to the absence of proteins responsible for
complex formation. We have ruled out this possibility, because TDE1
binding activity was also observed at 25 °C but not at 37 °C in
nuclear extracts prepared from ts13 cells grown at the nonpermissive
temperature (Fig. 6B, right panel). These data
suggest that TAF1 activity is required for efficient protein binding at
the cyclin D1 core promoter.
Transcription from only a subset of genes has been shown to be
defective at 39.5 °C in ts13 cells. If the lack of promoter DNA
binding is responsible for the decrease in transcription, we expect
that protein binding to the c-fos core promoter, which displays no reduction in transcriptional activity at 39.5 °C, will
be unaffected by temperature. On a DNA fragment containing the TATA box
and initiator of the c-fos promoter, comparable amounts of
complex formation was detected at 25 °C and 37 °C in ts13 and ts13R3 nuclear extracts (Fig. 6C). Therefore, the defect in promoter binding is specific to the cyclin D1 promoter and correlates with promoter activity as measured by transient transfection into ts13 cells.
Next, we assessed the sequence specificity of DNA binding by testing
the ability of different DNA molecules to function as effective
competitors. We found that increasing concentrations of the TDE1 as the
nonradioactive competitor efficiently blocked retarded complex
formation, whereas an unrelated double-stranded oligonucleotide had no
detectable effect (Fig. 6D, left panel). Although
the c-fos and cyclin D1 promoters have very little sequence similarity, we found that the c-fos TATA box and initiator
(c-fos core) and the initiator alone (c-fos INR)
competed for DNA binding activity at the cyclin D1 TDE1 (Fig.
6D, left panel). In fact, the c-fos
core promoter fragment was more effective than the TDE1 in the
competition assays. These data led us to speculate that the TATA box is
responsible for the dramatic decrease in complex formation. Because TBP
is known to bind the TATA box, we extended our results to suggest that
TFIID is binding to the cyclin D1 TDE1 in gel mobility shift assays.
The cyclin D1 promoter contains no TBP binding sites. Components of
TFIID have been reported to interact with DNA sequences that fit the
initiator consensus motif. The hamster cyclin D1 promoter contains one
region within the TDE1 that displays sequence homology to the initiator
consensus. To test the importance of this putative initiator sequence
in cyclin D1 promoter binding, we examined the ability of the cyclin D1
TDE1 fragment containing mutations in the initiator to function as a
competitor in mobility shift assays. The bases mutated were chosen
based on the consensus Inr motif YYAN(T/A)YY, with the last five bases
conserved in the cyclin D1 TDE1. We elected to mutate the (T/A)Y to GG,
because previous studies have reported that similar mutations disrupt initiator function (43). The resulting cyclin D1 initiator mutant was
found to be an ineffective competitor suggesting that the protein-DNA
interactions detected by gel mobility shift occur through the
"initiator" motif in the cyclin D1 TDE1 (Fig. 6D, right panel).
TFIID Binds to TAF1-dependent Element of Cyclin
D1--
Our finding that a promoter fragment containing a TATA box
functions as a strong competitor for TDE1 binding led us to investigate if the TAF1-dependent DNA binding activity in ts13 cell
extracts could be attributed to TFIID. TAFs originally identified in
TFIID have been subsequently found in many other complexes. They may even exist as free subunits. Therefore, we wanted to convincingly demonstrate that the mobility-shifted complexes dependent on TAF1 activity were indeed TFIID. First, the ability of HeLa nuclear extracts
and partially purified human TFIID to bind to the TDE1 of cyclin D1 was
examined (Fig. 7A, lanes
2 and 3). Both protein preparations resulted in
mobility shifts that migrated at similar positions on 1.2%
agarose/0.5× TBE gels. Complex formation was dramatically reduced upon
removal of TFIID by either phosphocellulose chromatography or
immunodepletion using a monoclonal antibody against the TAF4
(previously TAFII135) subunit of TFIID (Fig. 7A,
lanes 4 and 5). The addition of antibodies
against human TBP, TAF1, and TAF4 was also found to prevent complex
formation (data not shown). These findings place TFIID and thereby TAF1
at the cyclin D1 TDE1, suggesting that recruitment of TFIID and
assembly of the transcription machinery is compromised by the ts13
mutation at 39.5 °C.
Having demonstrated that TFIID in human extracts interacts with the
cyclin D1 TDE1, we then tested for the components of TFIID in the
mobility shifted complexes detected in ts13 nuclear extracts. The
addition of a TBP polyclonal antiserum supershifted the complexes formed in ts13 extracts (Fig. 7B). However, the existing
anti-TAF antibodies do not recognize the hamster subunits of TFIID. We found that addition of the anti-human TAF1 mAb 6B3 to gel mobility shift assays had no effect on the retarded complexes formed (Fig. 7B). To overcome this hurdle, we generated a ts13 cell line
that stably expresses an HA-epitope tagged version of the ts13 allele of human TAF1. A cell line that expresses an HA-tagged wild-type human
TAF1 was also established in parallel. Both subunits have been shown to
incorporate into the endogenous TFIID complex assembled in the cell
(13). Thus, the presence of the HA-epitope provides a means of tracking
the TAF1 protein in electrophoretic mobility shift assays.
Nuclear extracts prepared from the HA-tagged cell lines produced a
mobility shift at 25 °C that was indistinguishable from the shift
detected in the untagged cell extracts (Fig. 7C). Addition of the anti-HA mAb 12A5 retarded the migration of a portion of the HA
tag-shifted complexes. No such supershift was observed when the
antibody was added to the untagged ts13 and ts13R3 cell extracts. The
HA-tagged ts13 and ts13R3 cells continue to synthesize the endogenous
untagged version of the largest TFIID subunit. Therefore, TFIID
complexes containing the endogenously expressed TAF1 most likely
account for the significant amount of retarded complexes that are
unaffected by the presence of the anti-HA antibody. Consistent with
this explanation, the amount of supershifted complex appears to be
proportional to the expression levels of the HA-tagged proteins in the
two cell lines, with higher levels detected in the HA-ts13 than the
HA-ts13R3 cells. These data strongly suggest that TAF1 and TBP are
components of the retarded protein-DNA complexes that bind to the
initiator of the cyclin D1 core promoter and most likely contain
TFIID.
Binding of TAF1-TAF2 Dimers to the Cyclin D1 Initiator
Region--
A number of TAFs within the TFIID complex, in addition to
TAF1, have been shown to contact DNA, thereby providing multiple mechanisms for recruiting TFIID to a promoter. To rule out any contribution of these TAFs to the defect in DNA binding, we examined recombinant TAF1-TAF2 heterodimers, which preferentially bind DNA
sequences homologous to an initiator motif in gel mobility shift assays
(11). Heterodimers of TAF1 and TAF2 were assembled with either the
wild-type or mutant TAF1 protein (Fig.
8A), and their ability to bind
to the TDE1 was tested at 25 °C and 37 °C in vitro
(Fig. 8B). Although TAF1 and TAF2 alone displayed no DNA
binding activity, heterodimers containing wild-type TAF1 exhibited significant DNA binding independent of the reaction temperature. By
contrast, no detectable binding activity was detected with the ts13
mutant TAF1 containing heterodimers at 37 °C. We confirmed by
coimmunoprecipitation analysis that both the wild-type and mutant
heterodimers remain intact after incubation at 37 °C (data not
shown). Because cyclin D1 lacks a TBP binding site, our data suggest
that TAF1 plays a critical role in the efficient recruitment of TFIID
through the cyclin D1 initiator.
Recruitment of TFIID to the Cyclin D1 Promoter Restores Basal
Transcription in ts13 Cells--
Our transcription and DNA binding
studies collectively suggest that failure to recruit TFIID to the
cyclin D1 initiator is responsible for the transcription defect of
cyclin D1 detected in ts13 cells. Therefore we tested if providing an
alternative recruitment mechanism would restore cyclin D1 transcription
at 39.5 °C in ts13 cells. The strategy we took was to insert a
consensus TATA element upstream of the transcription initiation site at position
We have gathered preliminary evidence suggesting that transcription
mediated by the Sp1 sites, which are retained in The ts13 conditional mutation in TAF1 disrupts transcription from
a subset of protein encoding genes resulting in a G1 cell cycle arrest. It still remains unclear, at the molecular level, how the
mutation in TAF1 leads to the ts13 transcriptional defect. Chromatin
immunoprecipitation studies in yeast have demonstrated that the C
terminus of the yTAF1 protein is required for the association of TFIID
with promoters (44). Here we provide evidence in mammalian cells that
the recruitment of TFIID to the initiator requires a function of TAF1.
In addition, we present data suggesting that TAF1 carries out an
additional function required for activated transcription mediated by
the upstream Sp1 sites. By in vivo genomic footprinting, we
have defined a 33-bp DNA element in the cyclin D1 core promoter (TDE1
or TAF1-dependent element 1) that becomes more accessible
upon inactivation of a function of TAF1. The TDE1 displays weak
homology to a consensus initiator motif. It is also sufficient to
support RNA pol II-mediated transcription in a
TAF1-dependent manner suggesting this element contains a
functional initiator. Gel mobility shift assays suggest that TFIID
binds to the cyclin D1 TDE1 and that this binding event involves
contacts with the initiator and requires an activity of TAF1. These
data are the first pieces of evidence suggesting a molecular mechanism
for TAF1 action at the core promoter. Based on our results, we propose that a lack of TFIID recruitment to the initiator is responsible for
the decrease in cyclin D1 transcription in ts13 cells. This conclusion
is further supported by our finding that the insertion of a consensus
TBP binding site is able to fully restore basal but not activated
cyclin D1 transcription at 39.5 °C in ts13 cells.
Interestingly, insertion of a consensus TATA element only partially
restores transcription to a larger cyclin D1 promoter fragment
( The transcriptional control regions of eukaryotic promoters can be
divided into two distinct domains: 1) a core promoter consisting of the
transcriptional start site and flanking sequences that interact with
the general transcriptional machinery and 2) upstream activation
sequences (UAS) that are specific binding sites for transcriptional
activators. Studies in yeast have demonstrated that UAS selectively
recruits TAFs and thus TFIID to TAF-dependent promoters
(46, 47). However, the requirement for TAF1 activity appears to be
determined by core promoter elements. The UAS from a TAF-independent
promoter is unable to recruit TAFs and mediate efficient transcription
when inserted upstream of a TAF-dependent core promoter.
Therefore, efficient transcription requires a compatible UAS-core
promoter combination. It is the specific composition of the upstream
and core promoter sequences that will govern whether a promoter depends
on an activity of TAF1 for efficient transcription.
Whether or not transcription from a promoter will be compromised
appears to depend on the specific allele of TAF1 being characterized. For example, ts2 mutation, isolated by Shen and Green (31), impaired
RPS5 and CLN2 transcription. In contrast,
the CLN2 promoter functioned normally in yeast harboring the
T657K and N568 Another parallel between the ts13, T657, and N568 An emerging model is that distinct core promoter elements form an array
of binding sites for TFIID. The TATA box is bound by TBP (48, 49), the
Inr by TAF1-TAF2 (9, 11), and the downstream promoter element, or DPE,
by TAF6-TAF9 (TAFII60-TAFII40) (10). Many of
these core promoter elements have relatively flexible sequence
requirements. Therefore, the use of multiple correctly positioned
elements increases the specificity of TFIID binding. In the case of
TATA-less promoters, such as cyclin D1, RPS5, and TUB2, we predict that
the contribution of the Inr for efficient TFIID binding is
significantly increased compared with TATA-containing promoters. This
assumption has led us to propose the following model for the function
of TAF1 in RNA polymerase II transcription (see Fig.
10). The TAF1-independent
c-fos promoter contains a consensus TATA element such that
the primary mechanism for TFIID recruitment is mediated by TBP binding
to the TATA box. Upon shifting ts13 cells to 39.5 °C, the ability of
TAF1 to interact with the initiator region is disrupted. However, the
binding of TBP to the TATA element remains intact and is sufficient to
recruit TFIID and initiate transcription. In the cyclin D1 promoter, no
high affinity binding site for TBP is present. Recruitment
of TFIID depends on the interaction of TAF1 with the initiator.
Therefore, at the nonpermissive temperature, the ts13 mutation hinders
the primary mechanism by which TFIID can efficiently interact with the
core promoter region of cyclin D1 to initiate assembly of the
preinitiation complex. This model is based upon the assumption that
promoters affected by the ts13 mutation in TAF1 will lack a strong TBP
binding site. Although this criterion does not hold for all
TAF1-dependent promoters, it does appear that many of the
genes affected by the conditional mutations described in yeast and
mammalian cells, do not possess a consensus TATA element. The
requirement for TAF1 also appears to extend to upstream activation
elements. In the case of cyclin D1, TAF1 may serve a dual function, one
essential for efficient recruitment of TFIID to the initiation site of
transcription and the second to mediate DNA binding to Sp1 sites and
subsequent transcriptional activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
356 to +58 of
the mouse gene as previously described (16). The 895-bp PCR fragment
representing the hamster cyclin D1 promoter was restriction digested
with NotI to remove the translational start codon,
blunt-ended with Klenow, and subcloned into the SmaI site of
pGL2-basic to produce
714/+109 cycD1-luc. The following cyclin D1
promoter fragments were also introduced into the SmaI site
of pGL2-basic. The BstXI to NotI fragment from
the cyclin D1 promoter was used to construct
439/+109 cycD1-luc.
55/
22 cycD1-luc was generated by annealing
5'-AGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGC-3' and
5'-GCAGGACTTTGCAACTTCAACAAAACTCCCCT-3' and subcloning the double-stranded oligonucleotide into pGL2-basic. The remaining cyclin
D1 deletion constructs were generated by PCR. The fragment for
183/+109 cycD1-luc was amplified using
5'-CGCTTTGGGCTTGTCCCCCT-3' and 5'-GTCAGGGTACGCGCGGC-3' and
digested with NotI, and the ends were blunted with Klenow.
The fragment for
183/+21 cycD1-luc was amplified using the primers
5'-CGCTTTGGGCTTGTCCCCCT-3' and 5'-ATCTGCCGCTCTCTGCTACGCG-3'.
Primers 5'-TTCTCTGCCGGGCTTTGATC-3' and
5'-ATCTGCCGCTCTCTGCTACGCG-3' were used to synthesize the promoter fragment for
107/+21 cycD1-luc.
183/+21 wtTATA cycD1-luc and
183/+21 mtTATA cycD1-luc were constructed as follows. A
PstI site was introduced into
183/+21 cycD1-luc upstream
of the TDE1 by using the QuikChange site-directed mutagenesis kit
(Stratagene) and primers 5'-GTCACACGGACTGCAGGGGAGTT-3' and
5'-AACTCCCCTGCAGTCCGTGTGAC-3'. The wild-type TDE1 was dropped out
as a PstI fragment and replaced with a double-stranded
oligonucleotide derived from one of the following two primer sets: (for
wild-type TATA) 5'-GTATAAAGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGCA-3' and 5'-GCAGGACTTTGCAACTTCAACAAAACTCCCCTTTATACTGCA-3'; (for mutant TATA)
5'-GGAGAAAGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGCA-3'; and
5'-GCAGGACTTTGCAACTTCAACAAAACTCCCCTTTCTCCTGCA-3'.
107/+21
wtTATA cycD1-luc and
107/+21 mtTATA cycD1-luc were generated using
183/+21 wtTATA cycD1-luc and
183/+21 mtTATA cycD1-luc, respectively, and primers 5'-TTCTCTGCCGGGCTTTGATC-3' and
5'-ATCTGCCGCTCTCTGCTACGCG-3' in PCR reactions. The resulting PCR
products were subcloned into the SmaI site of
pGL2-basic.
-galactosidase expression plasmid,
and 0.25 µg of the cyclin D1 or c-fos reporter constructs
were transfected into cells using the LipofectAMINE and Plus reagents
as described by the manufacturer (Invitrogen) with the following
modification: the amount of Plus reagent used was reduced by 50%.
After 3 h, FBS to 10% final concentration and antibiotics were
added to the cell culture media. Whole cell lysates were prepared using
Report Lysis buffer (Promega) between 16 and 18 h post-serum
addition. The amount of luciferase activity was determined using the
Promega luciferase assay system and
-galactosidase activity was
measured as described (32).
-Galactosidase activity was used to
correct for differences in transfection efficiency.
-mercaptoethanol in the presence of 1 µg of dG-dC, and 300 µg/ml bovine serum
albumin. 32P-Labeled double-stranded oligonucleotides
encompassing the cyclin D1 TDE1
(5'-AGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGCA-3') or c-fos
core promoter
(5'-TTCTATAAAGGCACCGGCTGAGGCGCCTACTACTCCAACCGCGACTGCAG-3') were
added, and the reaction continued at 25 °C or 37 °C for an additional 20 min. Reaction mixtures containing the c-fos
probe were separated on high ionic strength 1.2%
Mg2+-agarose gels as described previously (36), and those
containing the cyclin D1 TDE1 were run on 1.2% agarose/0.5× TBE gels
(37). Agarose gels were run at 140 v for ~90 min.
Mobility-shifted complexes were detected by autoradiography after the
gels were dried onto DE81 filter paper. For the competition assays, 20 µg of extract was preincubated with the indicated molar excess of
competitor for 20 min at 25 °C. 32P-Labeled
double-stranded oligonucleotide was added, and the reaction continued
at 25 °C for an additional 20 min. The sequences of the competitor
DNAs are as follows: c-fos INR
(5'-GGCACCGGCTGAGGCGCCTACTACTCCAACCGCGACTGCAG-3'), c-fos core
(5'-TTCTATAAAGGCACCGGCTGAGGCGCCTACTACTCCAACCGCGACTGCAG-3'), NS
(5'-AGCTAGGCCGCGAGCGCTTTCATTGGTCCATT), and TDE1 mtINR
(5'-AGGGGAGTTTTGTTGAAGTTGCAAAGGGCTGCA-3'). Binding
reactions with recombinant HA-TAF1, FLAG-TAF2, and HA-TAF1/FLAG-TAF2 heterodimers were performed essentially as described above and separated on 1.5% agarose/0.5× TBE gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
183 and +1, the
region of transcriptional initiation (data not shown). The lower
percentage of sequence identity found for the human promoter is
primarily due to the insertion of short stretches of sequence unique to the human gene. Consistent with the cyclin D1 sequences isolated from
other species, the hamster promoter lacks a consensus TBP binding site
or TATA element. It also contains numerous binding sites for
transcription factors, including the E-box proteins, Oct-1, Sp1,
NF-
B, and CREB, most of which are conserved in other mammals,
although their exact placements vary slightly (Fig.
1).
View larger version (47K):
[in a new window]
Fig. 1.
Nucleotide sequence of the 5' regulatory
region of the hamster cyclin D1 gene. The 895-bp nucleotide
sequence of the putative hamster cyclin D1 promoter is provided. The
sequence lacks a canonical TATA box element or TBP binding site. The
positions of potential transcription factor binding sites, including
sites for the E-box protein, Oct-1, CREB/ATF, NF- B and Sp1, are
indicated. The hamster promoter has more putative Sp1 binding sites
than the mouse, rat and human promoters. The major sites of
transcription initiation, as determined by primer extension (see Fig.
2), are indicated by the asterisks. The ATG start codon for translation
is shown in large, bold type. The hamster cyclin D1 sequence has been
deposited in GenBankTM under accession no. AF539477.
32. The two clusters at positions
32 to
29 and
13 to
12 appear to be the preferred sites of transcriptional
initiation. We found that the same set of initiation sites are used at
the nonpermissive temperature in ts13R3 cells, a ts13 subclone that expresses wild-type human TAF1 and no longer cell cycle arrests at
39.5 °C (data not shown). This finding suggests that the mechanism of cyclin D1 transcription is not altered by the increase in
temperature and rules out any temperature-dependent shift
to less efficient start sites of transcription.
View larger version (51K):
[in a new window]
Fig. 2.
Hamster cyclin D1 promoter utilizes multiple
sites for transcription initiation. Reverse transcriptase-mediated
extension using a hamster cyclin D1 specific antisense primer
(5'-TGCCTCGCGCTCTACTGCC-3') annealed to mRNA isolated from
asynchronously grown (cycling) or G1 synchronized
(G1) ts13 cells was carried out to identify sites of
transcription initiation. Primer extension products were separated by
denaturing 6% polyacrylamide gel electrophoresis and detected by
autoradiography. Transcription initiation sites were found to cluster
into five groups and to span a 30-bp region, with the two groups at
31 to
29 and
13 to
12 the preferred sites of initiation. The
initiation site assigned the position of +1 is indicated.
32 and
29) displays any homology to the consensus
initiator motif of YYAN(T/A)YY, suggesting that other factors are
influencing the species-specific differences in initiation site
selection. According to the consensus motif, position
30 would be the
predicted site of transcriptional initiation. The two nucleotides
proceeding the
30 position are also adenines and are used as
initiation sites. Isolation of the hamster cyclin D1 promoter provides
us with the tools to further analyze the role of TAF1 at the endogenous
hamster promoter, by specifically investigating in vivo
protein-DNA interactions under permissive and restrictive conditions
for TAF1 function.
714/+109
cycD1-luc (Fig. 3A). When
transiently transfected into ts13 cells,
714/+109 cycD1-luc displayed
high levels of transcriptional activity at 33.5 °C (Fig.
3B, left panel). As expected, transcription decreased ~25-fold when the cells were shifted to 39.5 °C. The dramatic reduction in promoter activity is not due to heat shock effects, because in ts13R3 cells, which express wild-type human TAF1,
no decrease in promoter activity was observed at 39.5 °C (Fig.
3B, right panel). Using the identical
experimental approach, we confirmed our previous finding that
transcription from the TAF1-independent c-fos promoter is
not altered at 39.5 °C in both ts13 and ts13R3 cells (data not
shown). These results indicate that it is the inactivation of TAF1 and
not the increase in temperature that is responsible for the defect in
cyclin D1 transcription in ts13 cells.
View larger version (22K):
[in a new window]
Fig. 3.
Mapping of the TAF1-dependent cis-element in
the hamster cyclin D1 promoter. A, a schematic diagram
of hamster cyclin D1 promoter constructs used in transient transfection
assays is provided. Deletion constructs were engineered by either
restriction enzyme digestion or PCR amplification. The location of
putative transcription factor binding sites and the initiation sites of
transcription and translation are shown. B, the indicated
hamster cyclin D1 promoter constructs driving luciferase gene
expression were transiently transfected into ts13 or ts13R3 cells
incubated at either 33.5 °C or 39.5 °C, and luciferase activity
was measured after 16-18 h. The level of activity detected with
714/+109 cycD1-luc at 33.5 °C was given a value of 100 and used as
the reference point for normalizing the activity of all other reporter
constructs tested. The data represent the average of eight independent
experiments, each carried out in duplicate. Error bars
indicate ±S.E.
714 and
183 led to progressively greater increases in transcription levels, a
characteristic unique to the hamster promoter, but did not
significantly alter TAF1 dependence (Fig. 3B, left
panel). It wasn't until the Sp1 binding sites were deleted that a
significant decrease (>80%) in promoter activity was observed at
33.5 °C (Fig. 3B, left panel). This finding is
consistent with previously reported studies on the human and rat
promoters demonstrating that Sp1 sites are critical for maximal
activity. Despite the 4- to 5-fold decrease in promoter activity, the
remaining core promoter fragment (
107/+21 cycD1) retained
temperature-sensitive transcriptional activity and therefore a
requirement for TAF1 function. Because all deletion constructs displayed similar levels of activity at 33.5 °C and 39.5 °C in ts13R3 cells (Fig. 3B, right panel), our data
demonstrate that the core promoter of cyclin D1 is sufficient to confer
a requirement for a function of TAF1.
View larger version (62K):
[in a new window]
Fig. 4.
In vivo genomic footprint analysis
of the hamster cyclin D1 core promoter. A, the in
vivo DNA methylation pattern between nucleotides 79 and +3 of
the hamster cyclin D1 promoter is shown. ts13 and ts13 R3 cells were
subjected to DMS treatment at the indicated temperature. The modified
genomic DNA was chemically cleaved, and the position of methylated
nucleotide residues was mapped by ligation-mediated PCR using nested
cyclin D1 gene-specific primers. Both the upper and
lower strands were examined. The cleavage pattern obtained
from genomic DNA methylated in vitro in the absence of
cellular protein is shown in lanes 1 and 10. The
solid lines between the ts13 and ts13R3 samples indicate the
position of nucleotide residues (between nucleotides
55 and
22)
that demonstrated increased methylation at 39.5 °C in ts13 but not
in ts13R3 cells, in three independent experiments. Dashed
lines indicate sites of increased methylation observed in two out
of three experiments. The arrows indicate the position of
transcriptional start sites. B, alignment of nucleotides
79 to +3 of the hamster cyclin D1 promoter with the rat, mouse, and
human promoters shows nearly 100% sequence identity within this
region. The large asterisks indicate nucleotides that
repeatedly displayed increased methylation in ts13 but not in ts13R3
cells at 39.5 °C. The smaller asterisks indicate the
location of changes observed in two out of three experiments. Indicated
by the solid line underneath the nucleotide sequences is a
33-bp sequence that we defined as the TAF1-dependent
element 1 (TDE1), a region that repeatedly displayed changes in
methylation protection. Putative binding sites for cAMP-responsive
element binding protein (CREB) and nuclear factor kappa B (NF-
B) are
shown above the sequence. The sites of transcription initiation are
indicated by the arrows.
107 and +21, because this region is sufficient to confer
temperature-sensitive transcriptional activity in ts13 cells. Strong
protection of a guanine residue at position
69, located within the
CREB binding site, was detected at 33.5 °C and 39.5 °C,
suggesting that the CREB/ATF site is occupied at the two temperatures.
We found that the most predominant and reproducible changes localize to
a 33-bp segment encompassing one cluster of transcription initiation
sites for cyclin D1, which we termed the TAF1-dependent
element 1 or TDE1 (Fig. 4). The changes detected on both strands of the
TDE1 include enhancement of cleavage sites as well as the appearance of
new cleavage products at 39.5 °C. Specifically, guanine residues
between
55 and
49, a guanine at
29, and adenine residues at
positions
31,
30, and
22 become more accessible at 39.5 °C,
conditions at which TAF1 becomes partially inactive (Fig. 4, compare
lanes 2 and 3 and lanes 6 and
7). Such changes were not detected when parallel experiments
were carried out in ts13R3 cells (Fig. 4, compare lanes 4 and 5, and lanes 8 and 9). As an
additional control, we analyzed the core promoter of the
c-fos gene, which was strongly protected against DMS
modification, and detected no significant TAF1-dependent
changes in the pattern of DNA methylation (data not shown). Taken
together, these findings suggest that, upon inactivation of TAF1 by the
ts13 mutation at 39.5 °C, the efficient binding of one or more
transcription factors to TDE1 in the cyclin D1 core promoter is
compromised, thereby leading to a decrease in gene transcription.
55/
22 cycD1-luc) was transiently transfected into ts13 and ts13R3
cells (Fig. 5). Expectedly, the level of
transcription mediated by
55/
22 cycD1-luc was 20% of the
"full-length" hamster promoter fragment (
714/+109 cycD1)
at 33.5 °C. More importantly, the minimal construct still required
TAF1 activity, displaying almost 20 times more activity at 33.5 °C
than at 39.5 °C, which is similar to that observed for the
full-length cyclin D1 promoter.
View larger version (12K):
[in a new window]
Fig. 5.
TDE1 in the hamster cyclin D1 promoter is
sufficient to confer TAF1-dependent transcriptional
activity. The indicated cyclin D1-luc reporter constructs were
transiently transfected into ts13 or ts13R3 cells at the indicated
temperature. The amount of luciferase activity at 33.5 °C or
39.5 °C was measured after 16-18 h. The relative activity of each
promoter was determined by normalizing to the activity of 714/+109
cycD1-luc in ts13 cells at 33.5 °C, which was given a value of 100. The data presented are the average of eight independent experiments,
each carried out in duplicate. Error bars indicate
±S.E.
B binding site, both of which are conserved in the
human, mouse, and rat cyclin D1 promoters. The consensus NF-
B site
shows a large increase in DNA accessibility in vivo. However, we have been unable to demonstrate any involvement of NF-
B
or its binding site using a number of different experimental approaches, including binding site mutation and inhibition of NF-
B
activity. These data suggest that the transcription initiation site is
the important feature of the TAF1-dependent element.
View larger version (59K):
[in a new window]
Fig. 6.
TFIID binds to the initiator element in the
TDE1 of the hamster cyclin D1 promoter. A, nucleotide
sequence of one strand of double-stranded oligonucleotides used in gel
mobility shift assays is provided. The TATA element in the
c-fos core is indicated in bold italics. The
initiator elements in each sequence are boxed with the
consensus motif given below. The initiator adenine is indicated in
bold. B, electrophoretic gel mobility shift
assays were performed using a radiolabeled double-stranded
oligonucleotide that contains the 33-bp TDE1 from the hamster cyclin D1
promoter, as defined in Fig. 4. Twenty micrograms of nuclear extracts
prepared from either permissively (left panel) or
nonpermissively (right panel) grown ts13 or ts13R3 cells
were equilibrated to 25 °C or 37 °C then incubated with the
radiolabeled cyclin D1 TDE1 at the same temperature. Binding reactions
were separated by agarose gel electrophoresis as described under
"Experimental Procedures," and mobility shifted complexes were
detected by autoradiography. The formation of shifted complexes on the
cyclin D1 TDE1 was disrupted in ts13 nuclear extract at 37 °C but
not in ts13R3 extracts. This decrease in binding activity was observed
with both permissively and nonpermissively derived nuclear extracts.
C, mobility shift assays with permissively derived extracts
were carried out as described in B with a doubled-stranded
radioactive probe containing the TATA element and initiator from the
hamster c-fos promoter. No difference in binding activity
was observed on the c-fos probe at the two temperatures.
D, for competition assays, ts13 nuclear extracts were
preincubated with different amounts (given in molar excess) of the
indicated nonradioactive competitor DNA at 25 °C before the addition
of the 32P-labeled cyclin D1 TDE1. Reaction mixtures were
subjected to agarose gel electrophoresis followed by autoradiography.
Left panel, the competitor DNAs included the cyclin D1 TDE1
(TDE1), the initiator of c-fos (c-fos
INR), the TATA box and initiator of c-fos (c-fos
core), and a nonspecific DNA fragment (NS). Right
panel, competition assays were also carried out with the cyclin D1
TDE1 mutant containing base pair changes in the putative initiator
(mt INR) as unlabeled competitor DNA. The position of
shifted complexes is indicated by the arrows.
View larger version (48K):
[in a new window]
Fig. 7.
Binding of TFIID to the cyclin D1 TDE1
requires TAF1 activity. A, binding assays were
performed with the indicated source of human TFIID and the cyclin D1
TDE1 as probe. Lane 1, bovine serum albumin; lane
2, 20 µg of HeLa nuclear extract; lane 3, 100 ng of
partially purified human TFIID fraction; lane 4, 20 µg of
0.4 M unbound HeLa nuclear fraction from a phosphocellulose
column; lane 5, 20 µg of HeLa nuclear extract
immunodepleted with an anti-human TAF4 mAb 3A6. Binding reactions were
subjected to 1.2% agarose/0.5× TBE gel electrophoresis and
autoradiography. B, twenty micrograms of nuclear extracts
prepared from ts13 cells were preincubated with no antibody (lane
1) the anti-HA mAb 12CA5 (lane 2), anti-human TAF1 mAb
6B3 (lane 3), or anti-TBP polyclonal serum (lane
4) for 2 h on ice. Binding reactions were initiated by the
addition of the 32P-labeled cyclin D1 TDE1. Reaction
mixtures were incubated at 25 °C before agarose gel electrophoresis
followed by autoradiography to visualize complex formation. The
addition of the anti-TBP supershifted a portion of the protein-DNA
complexes in the ts13 extracts. C: Left panel, 20 µg of ts13 or ts13R3 nuclear extract was preincubated in the absence
( ) or presence (+) of the anti-HA monoclonal antibody for 2 h on
ice. Binding reactions were initiated by the addition of the
32P-labeled cyclin D1 TDE1 and analyzed as described in
B. Right panel, additional antibody supershift
experiments were performed in which the binding reactions contained
extracts from ts13 or ts13R3 cells that constitutively express an
HA-epitope tagged ts13 mutant or wild-type human TAF1 protein,
respectively. The addition of the anti-HA antibodies supershifted a
portion of the protein-DNA complexes in the HA-tagged but not the
un-tagged cell lysates.
View larger version (41K):
[in a new window]
Fig. 8.
TAF1-TAF2 dimer binding to the cyclin D1 TDE1
requires TAF1 activity. A, assembly of recombinant
TAF1-TAF2 dimers. FLAG-tagged TAF2 and HA-tagged TAF1 (wild-type and
ts13 mutant alleles) were expressed in Sf9 cells using the
recombinant baculovirus system and assembled into dimers as described
under "Experimental Procedures." An aliquot of the individual
immunopurified TAFs and the assembled wild-type and mutant
TAF1-containing heterodimers were analyzed by SDS-PAGE and visualized
by silver staining. The position of each TAF subunit is indicated.
B, DNA binding activity of TAF1-TAF2 dimers. The purified
wild-type and ts13 mutant TAF1-containing heterodimers and the
individual TAFs were examined in electrophoretic gel mobility shift
assays using a radiolabeled cyclin D1 TDE1 as probe. Reaction mixtures
were incubated at 25 °C or 37 °C, followed by agarose gel
electrophoresis and autoradiography. No DNA binding activity was
detected at 37 °C with the mutant heterodimer while the wild-type
form bound DNA.
29 (Fig. 9A). For
these studies we elected to use
183/+21 cycD1, the smallest fragment
of the cyclin D1 promoter that still retained high levels of
transcription and displayed a strong requirement for TAF1 activity. We
found that insertion of the TATA sequence into
183/+21cycD1-luc only
partially restored transcription at 39.5 °C (Fig. 9B).
Replacement of the TATA element with a mutant TBP binding site had no
effect on transcription levels or TAF1 dependence. Possible
interpretations of these data are that 1) TFIID is not being
effectively recruited to the promoter or that 2) recruitment of TFIID
is not sufficient to replace the activity of TAF1 disrupted by the
ts13 mutation.
View larger version (24K):
[in a new window]
Fig. 9.
TFIID recruitment to the cyclin D1 promoter
overcomes TAF1-dependent transcription in ts13 cells.
A, a schematic diagram of cyclin D1 reporter constructs used
in transient transfection assays is provided. The location at which a
WT or mutant TATA element was inserted into each construct is shown.
B, transcriptional activity in ts13 cells. The indicated
cyclin D1 reporter construct, containing either no ( ), a consensus
(WT), or a mutant (mt) TATA element were transiently transfected into
ts13 and ts13R3 cells. The amount of luciferase activity at 33.5 °C
or 39.5 °C was determined after 16-18 h and normalized to the level
of activity detected with
183/+21 cycD1-luc lacking a TATA element in
ts13 cells at 33.5 °C. The data are the average of three independent
experiments, each carried out in triplicate. Error bars
represent ±S.E. C, the experiments described in
B were also carried out with the
107/+21 cycD1-luc
reporter construct. The data presented are the average of two
independent experiments, each carried out in duplicate. ±S.E. is shown
by the error bars. Insertion of a properly positioned TATA
element restored basal transcription to the
107/+21 cycD1-luc
construct. By contrast, activated transcription from
183/+21
cycD1-luc was not recovered.
183/+21cycD1-luc, also requires TAF1 activity (data not shown). Thus, we repeated the
"artificial recruitment" experiments using a cyclin D1 reporter construct (
107/+21 cycD1-luc) that lacks the Sp1 sites (Fig. 9A). Under these conditions, we found that introduction of
the TBP binding site restored transcription at 39.5 °C to levels
nearly comparable to those observed at 33.5 °C (Fig. 9C).
As expected, the mutant TATA element had no effect on transcriptional
activity. The absolute amount of activity restored by TFIID recruitment to
183/+21 at 39.5 °C was approximately equal to the level of transcription detected with the
107/+21 cycD1-luc construct at 33.5 °C. These data suggest that basal transcription from the cyclin
D1 promoter requires TAF1 activity for efficient recruitment of TFIID
to the core promoter through an initiator motif. The observation that
artificial recruitment of TFIID was insufficient to overcome the
requirement for TAF1 activity at a promoter containing the Sp1 sites
and core region suggests that TAF1 serves two independent functions,
one in basal and one in activated transcription, both of which are
essential for maximal cyclin D1 expression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
183/+21 TATA cycD1-luc), whereas transcription from the basal
promoter (
107/+21 cycD1-luc) can be fully recovered at the
nonpermissive temperature. The only difference between the
183/+21cycD1-luc and
107/+21 cycD1-luc constructs is the presence
of Sp1 sites in the larger of the two promoters. These Sp1 sites are
critical for activated transcription of cyclin D1 in response to
several growth factors. Similar results have been reported for the
yeast RPS5 promoter, where transcription mediated by upstream
activation sequences also requires TAF1 function (45). We have obtained
in vivo footprinting data indicating that the interaction of
proteins with the Sp1 sites in the hamster cyclin D1 promoter is also
compromised at 39.5 °C in ts13 cells (data not shown). Therefore, it
appears that TAF1 has a second independent function that is essential
for recruitment of Sp1 or a related transcription factor and subsequent
activation of cyclin D1 transcription.
conditional mutations described by Tsukihashi
et al. (42). The sets of genes affected by the different
TAF1 mutations overlap but are not identical. Compared with the
independently isolated yeast TAF1 mutants, the mammalian
temperature-sensitive ts13 allele more closely phenocopies the T657 and
N568
mutants by compromising core promoter function at select genes.
More importantly, the introduction of a canonical TBP binding site into
a TATA-less promoter was sufficient to overcome the requirement for
TAF1 in cells harboring the T657, N568
, or ts13 mutation but not in
cells containing the ts2 allele. One caveat of all these experiments is
that the TATA sequence was inserted into different promoter constructs
for the mutant alleles analyzed.
alleles is that
all the mutations alter a single amino acid within the central histone
acetyltransferase domain of the protein. The proteins produced are
stably expressed at the restrictive temperature, and do not appear to
strongly affect the structure of TFIID. The structural consequence of
the glycine to aspartic acid missense mutation of ts13 cells has not
been demonstrated. Heterodimers of TAF1 and TAF2 bind to initiator-like
sequences and are thought to be important for the recruitment of TFIID
to select promoters (11). At 39.5 °C, the ts13-TAF1 protein may
undergo significant conformational changes resulting in the inability
of TFIID to efficiently associate with the initiator of cyclin D1. In
yeast, removal of the C terminus of TAF1 compromises the recruitment of
TFIID to promoter DNA (44). Therefore, a more biophysical analysis of
the consequences of different mutations in TAF1 will be required to
determine which portion of the protein is structurally compromised.
View larger version (37K):
[in a new window]
Fig. 10.
A model for TAF1 function in the recruitment
of TFIID. Recruitment of TFIID to the TATA-containing
c-fos promoter is via TBP binding to the consensus TATA
element. At 39.5 °C in ts13 cells, the disruption of TAF1 initiator
binding has no effect on TFIID binding and the initiation of
c-fos transcription. By contrast, the cyclin D1 promoter
lacks a TBP binding site such that recruitment of TFIID requires the
interaction of TAF1-TAF2 with the initiator region. At 39.5 °C, TAF1
initiator binding is abrogated. The lack of an alternative mechanism
for efficient TFIID binding compromises the transcriptional activity of
the cyclin D1 promoter.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to K. Lewis for providing excellent technical and laboratory support. The generation of the HA-tagged ts13 cell line by K. S. White and assistance by M. Gelbert with the cloning of the hamster cyclin D1 promoter are also greatly appreciated. We thank R. Tjian for kindly providing the anti-HA ascites fluid and S. Hahn and R. Tjian for critical reading of the manuscript. We especially thank members of the Wang laboratory, including R. M. Squillace and E. L. Dunphy, for valuable discussions and support of this research.
![]() |
FOOTNOTES |
---|
* This work was supported in part by research project Grant RPG-98-201-CCG from the American Cancer Society.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.
Supported by National Research Service Award GM7750 from NIGMS,
the National Institutes of Health.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF539477.
§ To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Health Sciences Center, Box 357280, Seattle, WA 98195-7280. Tel.: 206-616-5376; Fax: 206-685-3822; E-mail: ehwang@u.washington.edu.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M300412200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TFIID, transcription factor IID; TBP, TATA-binding protein; TAF, TBP-associated factors; yTAF, yeast TAF1; CRE, cAMP-responsive element; TDE1, TAF1-dependent element 1; HA, hemagglutinin; mAb, monoclonal antibody; FBS, fetal bovine serum; Inr, initiator; UAS, upstream activation sequence; DMS, dimethyl sulfate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Murray, A., and Hunt, T. (1993) The Cell Cycle , W. H. Freeman and Company, New York |
2. | Yam, C. H., Fung, T. K., and Poon, R. Y. (2002) J. Cell. Mol. Life Sci. 59, 1317-1326[CrossRef] |
3. | Donnellan, R., and Chetty, R. (1998) Mol. Pathol. 51, 1-7[Abstract] |
4. | Musgrove, E. A., Hui, R., Sweeney, K. J., Watts, C. K., and Sutherland, R. L. (1996) J. Mammary Gland Biol. Neoplasia 1, 153-162[Medline] [Order article via Infotrieve] |
5. | Nakamura, S., Yatabe, Y., and Seto, M. (1997) Pathol. Int. 47, 421-429[Medline] [Order article via Infotrieve] |
6. | Sutherland, R. L., and Musgrove, E. A. (2002) Breast Cancer Res. 4, 14-17[Medline] [Order article via Infotrieve] |
7. | Albright, S., and Tjian, R. (2000) Gene (Amst.) 242, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
8. | Sawadogo, M., and Roeder, R. G. (1985) Cell 43, 165-175[Medline] [Order article via Infotrieve] |
9. | Verrijzer, C. P., Chen, J.-L., Yokomori, K., and Tjian, R. (1995) Cell 81, 1115-1128[Medline] [Order article via Infotrieve] |
10. |
Burke, T. W.,
and Kadonaga, J. T.
(1997)
Genes Dev.
11,
3020-3031 |
11. |
Chalkley, G. E.,
and Verrijzer, C. P.
(1999)
EMBO J.
18,
4835-4845 |
12. | Talavera, A., and Basilico, C. (1977) J. Cell. Physiol. 92, 425-436[Medline] [Order article via Infotrieve] |
13. | Ruppert, S., Wang, E. H., and Tjian, R. (1993) Nature 362, 175-179[CrossRef][Medline] [Order article via Infotrieve] |
14. | Hisatake, K., Hasegawa, S., Takada, R., Nkatani, Y., Horikoshi, M., and Roder, R. G. (1993) Nature 362, 179-181[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hayashida, T., Sekiguchi, T., Noguchi, E., Sunamoto, H., Ohba, T., and Nishimoto, T. (1994) Gene (Amst.) 141, 267-270[CrossRef][Medline] [Order article via Infotrieve] |
16. | Wang, E. H., and Tjian, R. (1994) Science 263, 811-814[Medline] [Order article via Infotrieve] |
17. | Liu, H. T., Gibson, C. W., Hirschhorn, R. R., Rittling, S., Baserga, R., and Mercer, W. E. (1985) J. Biol. Chem. 260, 3269-3274[Abstract] |
18. |
Sekiguchi, T.,
Noguchi, E.,
Hayashida, T.,
Nakashima, T.,
Toyoshima, H.,
Nishimoto, T.,
and Hunter, T.
(1996)
Genes Cells
1,
687-705 |
19. | Suzuki-Yagawa, Y., Guermah, M., and Roeder, R. G. (1997) Mol. Cell. Biol. 17, 3284-3294[Abstract] |
20. |
Wang, E. H.,
Zhou, S.,
and Tjian, R.
(1997)
Genes Dev.
11,
2658-2669 |
21. | Walker, S. S., Shen, W.-C., Reese, J. C., Apone, L. M., and Green, M. R. (1997) Cell 90, 607-614[Medline] [Order article via Infotrieve] |
22. | Holstege, F. C. P., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., and Young, R. A. (1998) Cell 95, 717-728[Medline] [Order article via Infotrieve] |
23. |
O'Brien, T.,
and Tjian, R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2456-2461 |
24. |
Yan, G. Z.,
and Ziff, E. B.
(1997)
J. Neurosci.
17,
6122-6132 |
25. |
Sabbah, M.,
Courilleau, D.,
Mester, J.,
and Redeuilh, G.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11217-112122 |
26. | Perry, J. E., Grossmann, M. E., and Tindall, D. J. (1998) Prostate 35, 117-124[CrossRef][Medline] [Order article via Infotrieve] |
27. | Herber, B., Truss, M., Beato, M., and Muller, R. (1994) Oncogene 9, 1295-1304[Medline] [Order article via Infotrieve] |
28. |
Kitazawa, S.,
Kitazawa, R.,
and Maeda, S.
(1999)
J. Biol. Chem.
274,
28787-28793 |
29. | Eto, I. (2000) Cell. Prolif. 33, 167-187[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Nagata, D.,
Suzuki, E.,
Nishimatsu, H.,
Satonaka, H.,
Goto, A.,
Omata, M.,
and Hirata, Y.
(2001)
J. Biol. Chem.
276,
662-669 |
31. | Shen, W.-C., and Green, M. R. (1997) Cell 90, 615-624[CrossRef][Medline] [Order article via Infotrieve] |
32. | Herbomel, P., Bourachot, B., and Yaniv, M. (1984) Cell 39, 653-662[Medline] [Order article via Infotrieve] |
33. | Mueller, P. R., Wold, B., and Garrity, P. A. (1992) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 15.5.8-15.5.13, John Wiley and Sons, Inc., Boston, MA |
34. | Strauss, E. C., and Orkin, S. H. (1997) Methods 11, 164-170[CrossRef][Medline] [Order article via Infotrieve] |
35. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
36. | Mantovani, R., Tora, L., Moncollin, V., Egly, J. M., Benoist, C., and Mathis, D. (1993) Nucleic Acids Res. 21, 4873-4878[Abstract] |
37. | Zerby, D., and Lieberman, P. M. (1997) Methods 12, 217-223[CrossRef][Medline] [Order article via Infotrieve] |
38. | Chen, J.-L., Attardi, L. D., Verrijzer, C. P., Yokomori, K., and Tjian, R. (1994) Cell 79, 93-105[Medline] [Order article via Infotrieve] |
39. | Chen, J.-L., and Tjian, R. (1996) Methods Enzymol. 273, 208-217[CrossRef][Medline] [Order article via Infotrieve] |
40. | Moqtaderi, Z., Bai, Y., Poon, D., Weil, A. P., and Struhl, K. (1996) Nature 383, 188-191[CrossRef][Medline] [Order article via Infotrieve] |
41. | Walker, S. W., Reese, J. C., Apone, L. M., and Green, M. R. (1996) Nature 383, 185-188[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Tsukihashi, Y.,
Miyake, T.,
Kawaichi, M.,
and Kokubo, T.
(2000)
Mol. Cell. Biol.
20,
2385-2399 |
43. |
Roy, A. L.,
Du, H.,
Gregor, P. D.,
Novina, C. D.,
Martinez, E.,
and Roeder, R. G.
(1997)
EMBO J.
16,
7091-7104 |
44. |
Mencia, M.,
and Struhl, K.
(2001)
Mol. Cell. Biol.
21,
1145-1154 |
45. |
Tsukihaski, Y.,
Kawaichi, M.,
and Kokubo, T.
(2001)
J. Biol. Chem.
276,
25715-25726 |
46. | Mencia, M., Moqtaderi, Z., Geisberg, J. V., Kuras, L., and Struhl, K. (2002) Mol. Cell 9, 823-833[Medline] [Order article via Infotrieve] |
47. | Li, X.-Y., Bhaumik, S. R., Zhu, X., Li, L., Shen, W.-C., Dixit, B. L., and Green, M. R. (2002) Curr. Biol. 12, 1240-1244[CrossRef][Medline] [Order article via Infotrieve] |
48. | Hoey, T., Dynlacht, B. D., Peterson, M. G., Pugh, B. F., and Tjian, R. (1990) Cell 61, 1179-1186[Medline] [Order article via Infotrieve] |
49. | Peterson, M. G., Tanese, N., Pugh, B. F., and Tjian, R. (1990) Science 248, 1625-1630[Medline] [Order article via Infotrieve] |