Here we study initi-ation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene as
Trang 1Sequences downstream of the transcription initiation site are
important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription
Irina Kotova, Anna L Chabes*, Sergei Lobov, Lars Thelander and Stefan Bjo¨rklund
Department of Medical Biochemistry and Biophysics, Umea˚ University, Sweden
Ribonucleotide reductase is essential for the synthesis of all
four dNTPs required for DNA replication The enzyme is
composed of two proteins, R1 and R2, which are both
needed for activity Expression of the R1 and R2 mRNAs is
restricted to the S-phase of the cell cycle, but the R1 and R2
promoters show no obvious sequence homologies that could
indicate coordination of transcription Here we study
initi-ation of transcription at the natural mouse R2 promoter,
which contains an atypical TATA-box with the sequence
TTTAAA, using a combination of in vivo reporter gene
assays and in vitro transcription Our results indicate that in
constructs where sequences from the R2 5¢-UTR are present,
the mouse R2 TATA-box is dispensable both for unregu-lated, basal transcription from the R2 promoter and for S-phase specific activity Instead, initiation of R2 transcrip-tion is directed by sequences downstream from the tran-scription start We report that this region contains a conserved palindrome sequence that interacts with TAFIIs This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box
Keywords: in vitro transcription; ribonucleotide reductase; TAFs; TATA-box; transcription regulation
Efficient transcription initiation at a eukaryotic
protein-encoding gene requires assembly on promoter DNA of a
protein complex containing RNA polymerase II and five
general transcription factors (GTFs) IIB, IID, IIE, IIF, and
IIH (reviewed in [1–3]) TFIID refers to a multiprotein
complex composed of the TATA-binding protein (TBP)
and a set of proteins called TBP-associated factors (TAFs)
Transcription of most eukaryotic genes is initiated around
20–30 base pairs downstream from a conserved sequence
called the TATA-box, which binds TBP as a first step in a
sequence of events leading to formation of a functional
preinitiation complex [4] The TATA-box sequence is
conserved through evolution [consensus sequence:
TATA(A/T)A(A/T)] but it is known that TBP can interact
with sequences that differ considerably from this consensus
sequence However, at most natural promoters, TBP is not
solely responsible for promoter recognition Rather, a more
extended sequence around the TATA-box interacts with
both TAFs and other GTFs, such as TFIIB Mapping of
interactions between the human TFIID subunits and the
adenovirus major late promoter using a photocrosslinking method showed that TFIID interacts with promoter sequences both upstream and downstream of the TATA-box, but also that TFIID–DNA interactions are formed downstream from the position for transcription initiation [5] In contrast to the TATA-box, these upstream and downstream sequences show no obvious homology when different promoters are compared
Regulation of transcription is normally explained by signaling from regulatory proteins binding to specific promoters DNA sequences located upstream from the TATA-box These signals are then transferred to the general transcription machinery via coactivators or core-pressors such as the Mediator complex [6,7] However, considering that the sequences surrounding the TATA-box, which interact with components of the general transcription machinery, show little or no homology between different promoters, it is likely that transcription
is also regulated at this level This type of regulation would then both be dependent on the strength of interactions between different promoter DNA and the GTFs depend-ing on the sequence surrounddepend-ing the TATA-box, but also
by formation of preinitiation complexes that contain different general transcription factors For example, it is known that TAF-complexes exist in multiple forms, some even lacking TBP [8,9] It is therefore not surprising that expression from specific promoters is regulated by recruit-ment of different TAF-complexes for example in different tissues, at different developmental stages or at different phases of the cell cycle [reviewed in 10] Furthermore, the fact that TBP is also required for transcription of genes that lack a TATA-box, shows that the requirement of TBP for initiation is uncoupled from the requirement of a consensus TATA-box
Correspondence toS Bjo¨rklund, Department of Medical Biochemistry
and Biophysics, Umea˚ Univ ersity, SE-901 87 Umea˚, Sweden.
Fax: +46 907869795, Tel.: + 46 907866788,
E-mail: stefan.bjorklund@medchem.umu.se
Abbreviations: 5¢-UTR, 5¢-untranslated region; GTFs, general
trans-cription factors; TBP, TATA-binding protein; TAFs, TBP associated
factors; DPE, downstream promoter element; Py, pyrimidine;
Im, imidazole.
*Present address: Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724, USA.
(Received 23 December 2002, revised 21 February 2003,
accepted 26 February 2003)
Trang 2Ribonucleotide reductase catalyzes the formation of
deoxyribonucleotides from the corresponding
ribonucleo-tides, which is the rate-limiting step in the production of
precursors for DNA synthesis [11] The mouse ribonucleotide
reductase is composed of two subunits, proteins R1 and R2
Both subunits are required for enzyme activity and it could be
reasonable to assume that the expression of the R1 and R2
mRNAs should be coordinated at the transcription level
Accordingly, both the R1 and R2 mRNAs show an S-phase
specific expression with low or undetectable levels in G1 cells,
and a synchronized increase as cells enter the S-phase of the
cell cycle [12] However, analysis of the mouse R1 and R2
promoter sequences show that they differ, not only from each
other, but also from the common promoter structure found
in the majority of eukaryotic genes The mouse R1 promoter
lacks TATA-box and instead contains an initiator sequence
that has been shown to interact with the transcription factor
TFII-I [13], and a downstream sequence called c that shows
no homology to previously identified downstream promoter
elements [14] The protein(s) that interacts with the c
se-quence is so far unidentified In contrast, the mouse R2
promoter contains no initiator element-sequence or c
ele-ment, but instead has an atypical TATA-box with the
sequence TTTAAA, located approximately 30 base pairs
upstream from the mapped major transcription start sites in
the R2 promoter [15] The transcriptional efficiency for this
variant of the TATA-box has been studied in the adenovirus
major late promoter context [16] Compared to the consensus
TATA-box sequence, the R2-type sequence resulted in a
75% reduction in transcriptional efficiency However, no
analysis of how these mutations affected the transcription
start position was presented
Here we examine the mouse ribonucleotide reductase R2
TATA-box and sequences downstream of it for their
function in transcription from the R2 promoter Our data
indicate that the R2 TATA-box is dispensable for basal and
S-phase-specific expression from the R2 promoter, but that
it is important for the position of transcription initiation
However, the R2 TATA-box is required for maximal
promoter strength in the presence of sequences downstream
from the R2 transcription start and the availability of
proteins binding to these sequences
Experimental procedures
Plasmids
The internal control plasmid pML311 contains a shorter
(311 bp) G-less cassette fused with the adenovirus major late
promoter It was created from pML(CAT)19 [17] by PCR
using one primer complementary to the polylinker upstream
from the AdML promoter and a second primer
comple-mentary to nucleotide 311–292 bp downstream from the
start of the G-less sequence, followed by cleavage of the
PCR product with EcoRI and BamHI and ligation into
pML(CAT)19 digested with the same enzymes The
R2-luciferase reporter construct (pAC 10, here also called
TATAWt UTRWt) and the R2 promoter construct with
mutation in the CCAAT box have been described previously
[18] The R2 TATAmutUTRWt-luciferase reporter construct
is analogous to the pAC10/TATAWtUTRWtexcept that the
TATA-box (TTTAAA) at the position)29 bp relative to
the mouse R2 transcription start site was substituted by the sequence GCGCGC by overlap extension PCR
The TATAWt UTRmut and the TATAmut UTRmut constructs fused to the G-cassette were made by overlap extension PCR using pML(CAT)19 and the pAC10/ TATAWtUTRWtor the TATAmutUTRWt-luciferase repor-ter constructs as templates Creation of a TATAWtUTRWt -G-less cassette construct with exactly the same 5¢-UTR sequence as the analogous luciferase construct failed prob-ably because primers specific for the mouse R2 5¢-UTR sequence must include a 10-bp perfect palindrome which might cause secondary primer structures Instead we used primers further downstream from the R2 transcription start site, nucleotides +43 to +22 relative to the major transcrip-tion start site and introduced an NcoI site between the end of the R2 5¢-UTR and the G-less cassette The TATAmut UTRWt-G-less cassette construct was created from the TATAmutUTRWt-luciferase construct in the same way as the TATAWtUTRWt-G-less cassette construct Therefore, these constructs contain a longer sequence from the R2 5¢-UTR compared to the corresponding luciferase constructs (42 instead of 17 nucleotides) The TATAWtUTRmut- and the TATAmutUTRmut-luciferase reporter constructs were made from the corresponding G-less cassette constructs As these constructs, in contrast to the corresponding G-less constructs need to be efficiently translated, we decided to replace the R2 5¢-UTR with a nonrelated 5¢-UTR instead of deleting it Therefore, these constructs were made by amplification of the R2 promoter from corresponding G-less cassettes constructs using a downstream primer that included
23 nucleotides from the luciferase 5¢-UTR
Synthesis of the mouse R2-specific polyamide The R2 TATA-specific Py-Im polyamide (Fig 1A) was synthesized by solid-phase methods as described [19] The purity and identity of the polyamide was verified by analytical high-pressure liquid chromatography,1H nuclear magnetic resonance, and matrix-assisted laser desorption ionization-time of flight mass spectrometry The polyamide was dissolved in distilled water and the concentration of the stock solution was calculated using an extinction coefficient
of e¼ 78 000M )1Æcm)1at 310 nm The binding affinity of the polyamide to the R2 promoter was determined to
Kd¼ 1 nm by quantitative DNase footprinting under equilibrium conditions [20]
Treatment of stably transformed cells with polyamide Balb/3T3 cells (5· 105per 10 cm dish) stably transformed with the pAC10 full-length R2 promoter-luciferase reporter gene construct [21] were grown overnight in 10 mL Dulbecco’s modified Eagle’s medium (DMEM) + 10% heat-inactivated horse serum After 24 h, 5 and 50 lMR2 polyamide (in DMEM) was added to the plates After 6 h, cells were harvested and assayed for luciferase activity as described [21]
Gel shift experiments Nuclear extracts used in gel shift experiments with TBP were prepared from logarithmically growing Balb/3T3 cells
Trang 3[22] Gel shift experiments were made using the
oligonu-cleotides 5¢-GCGGTTGGGTGGCTCTTTAAAGGGCG
CG-3¢ and 5¢-CGCGCCCTTTAAAGAGCCACCCAA
CCGC-3¢ In oligonucleotides with mutated TATA-box
the sequence TTTAAA (shown in bold) is substituted by
GCGCGC Single-stranded oligonucleotides were
end-labeled using T4 polynucleotide kinase and [c-32P]ATP
(specific activity 3000 CiÆpmol)1, Amersham Biosciences)
as previously described [23], annealed by heating to 65C
followed by cooling down slowly to the room temperature
and purified by gel filtration on Sephadex G-50
(Amer-sham Biosciences)
A typical binding reaction contained 5 fmol labeled
oligonucleotide and 10 ng (26 fmol) recombinant human
TBP (Promega) in 10 lL of binding buffer [10% glycerol,
20 mM Tris/HCl (pH 8.0), 80 mM KCl, 10 mM MgCl,
2 mM dithiothreitol] After 15 min incubation at room
temperature, 5 lL loading buffer [40% glycerol, 250 mM
Tris/HCl (pH 8.0)] was added to the reactions and
DNA-protein complexes were resolved by electrophoresis through
6% polyacrylamide gels in electrophoresis buffer
(0.5· Tris/borate/EDTA, 4 mM MgCl2, 0.02% NP-40)
The gels were run at +4C, dried and subjected to
autoradiography
Nuclear extracts used in gel shift experiments with the
palindrome sequence in the R2 5¢-UTR were prepared from
Ehrlich–Lettre ascites mouse carcinoma cells (ATCC No
CCL77) as previously described [24] The gel shift
experi-ments were performed essentially as described in [25] using
the oligonucleotides: wild-type: 5¢-CAGTCGGCGGTGC
ACCGGATTCCAGCTGTTT-3¢; mutation 1: 5¢-CAGT
muta-tion 2: 5¢-CAGTCGGCGGTGCACCGTAATCCAGCT GTTT -3¢; mutation 3: 5¢-CAGTCGGCGGTGCACCG GATTCCGAGTGTTT-3¢ Nucleotides in bold represent mutated nucleotides and the palindrome sequence is underlined After labeling and annealing, the probes were
Fig 1 A polyamide specific for the mouse R2 TATA-boxinterferes with
TBP-binding to the R2 promoter in vitro but has no effect in vivo (A)
Structure of the Im-b-ImPyPy-c-ImImPyPyPy-b-Dp R2
TATA-spe-cific polyamide and the sequence in the mouse R2 promoter to which it
binds Im-Py targets GÆC base pairs and Py-Im targets CÆG base pairs
and the Py-b-alanine pair recognizes both AÆT and TÆA base pairs [38].
The boxed sequence represents the atypical R2 TATA-box Black and
white circles, Im and Py rings, respectively; curved line, hairpin
junc-tion, which is formed with c-aminobutyric acid; diamonds, b-alanine;
parenthesis with plus sign, 3-(dimethylamino)propylamine (B) Gel
shift experiments with a32P-labeled oligonucleotide representing the
R2 TATA-box Lane 1, only labeled oligonucleotide; lane 2, labeled
oligonucleotide incubated with 10 ng (26 fmol) recombinant human
TBP; lanes 3–6, as in lane 2 but with decreasing amounts (0.15 pmol,
0.075 pmol, 0.03 pmol, 0.0075 pmol respectively) of unlabeled R2
TATA-box oligonucleotide added to the binding reactions Lanes 7
and 8, as lanes 1 and 2, respectively; lanes 9–11, as lane 8 but with
increasing amounts of polyamide (540 fmol, 3.38 pmol, 33.8 pmol,
which is a 20-, 130-, and 1300-fold excess compared to TBP,
respect-ively) added to the binding reactions Arrows to the right indicate the
position for free probe (lower) and the TBP-probe complex (upper).
Numbers below the figure represent quantifications of the TBP-probe
complexes in each lane The quantifications were made using the Scion
Image software (C) Addition of the R2-polyamide to Balb/3T3 cells,
which are stably transfected with a reporter gene construct where the
wild-type R2 promoter controls the luciferase gene (TATA Wt UTR Wt
-luciferase).
Trang 4purified by electrophoresis in 5% polyacrylamide gel under
nondenaturing conditions In competition assays, the
annealed unlabeled oligonucleotides were mixed with
labe-led probe prior to the addition of nuclear extracts In
experiments with antibodies, antibodies were preincubated
with nuclear extracts for 15 min on ice in the binding buffer
before adding the labeled probe
Transfection of cells, serum starvation and luciferase
assays
Transient transfection, isolation of stably transformed cells,
synchronization of Balb/3T3 cells by serum starvation and
luciferase assays were done as described previously [18] The
relative luciferase value was determined as firefly luciferase
activity normalized against Renilla luciferase activity
mul-tiplied by 1000
Preparation of nuclear extracts andin vitro
transcription assays
Nuclear extracts were prepared from logarithmically
grow-ing Ehrlich–Lettre ascites cells as previously described [24]
In vitro transcription assays with purified transcription
factors were performed as described in [18] In vitro
transcription assays with crude nuclear extracts were made
in a similar way but with the following modifications Each
transcription reaction contained a total amount 35–45 lg of
nuclear extract For experiments using templates containing
the mutated 5¢-UTR upstream from the G-less cassette, the
2· transcription mixture contained 1.6 mMGTP in
addi-tion to the other three nucleotides Stop mixture contained
10 mM Tris/HCl (pH 7.5), 0.3MNaCl, 5 mM EDTA,
0.1 mgÆmL)1 glycogen (Boerhinger Mannheim) and
130 UÆmL)1T1 ribonuclease After incubation for 30 min
at 25C the reaction was stopped by addition of 200 lL
stop mixture and incubated for 30 min at 37C prior to
proteinase K treatment When an internal control was
included in the reactions, each reaction contained 60 fmol of
the DNA template to be studied, and 60 fmol of pML 311
In the experiments with TAFII135 antibodies, the antibodies
were added to the reaction mixtures at different
concentra-tions and the reacconcentra-tions were incubated on ice for 15 min
prior to addition of the 2· transcription mixture
Primer extension
Primer extension reactions were carried out essentially as
described in [26] Avian myeloblastosis virus reverse
tran-scriptase, T4 polynucleotide kinase and /X174 DNA/HinfI
dephosphorylated marker were purchased from Promega
RNAs to be used as template for the primer extension
reactions were synthesized by in vitro transcription reactions
using nuclear extracts and the different R2
promoter-luciferase constructs The oligonucleotide used for primer
extension was complementary to the nucleotides 65–90 in
the coding strand of the luciferase cDNA (5¢-CTCTTCATA
oligonucleotide (0.1 pmol, specific activity 2 · 106
cpmÆpmol)1) was added to the in vitro transcribed RNA,
the volume was adjusted to 20 lL with water and the
oligonucleotide was annealed to the RNA by heating to
65C followed by slow cooling to room temperature After the primer extension reaction, the products where denatured for 5 min at 95C the reaction products were resolved by electrophoresis on 7Murea, 10% polyacrylamide gels and subjected to autoradiography
Results TBP binding to the mouse R2 promoter TATA-box
is not required for transcription from the R2 promoter
in vivo
In order to study initiation of transcription from the R2 promoter, we obtained a polyamide specific for a sequence
at the 5¢-end of, and immediately upstream from, the atypical mouse R2 TATA-box (TTTAAA; Fig 1A) DNase1 footprinting analysis showed that the synthesized polyamide bound specifically to this sequence with a Kdof
1 nM Gel shift experiments using an end-labeled oligo-nucleotide corresponding to the region around the R2 TATA-box (Experimental procedures), recombinant human TBP and the polyamide showed that human TBP binds specifically to the mouse R2 TATA-box and that the polyamide interferes with this binding (Fig 1B) To study if the polyamide also inhibits transcription from the R2 promoter in vivo, polyamide was added to Balb/3T3 cells stably transfected with a reporter gene construct where the luciferase gene is under the control of the full-length mouse R2 promoter [21] To our surprise we found that the polyamide had no effect on R2-promoter-luciferase expres-sion even when cells are incubated with high concentrations (50 lM) of polyamide (Fig 1C)
Importance of the R2 TATA-box and TBP binding
to the R2 promoter
We next studied the importance of TBP and the R2 TATA-box in an in vitro transcription system reconstituted from recombinant mouse TBP, TFIIB, TFIIE, TFIIF and highly purified mouse TFIIH and RNA polymerase II [18]
As templates for these experiments we used constructs where a G-less cassette is ligated directly downstream from the nucleotide that has been mapped as the major transcription initiation site in the mouse R2 gene The only difference between the two templates is that they either contain the natural R2 TATA-box or a mutation of it to GCGCGC (Fig 3D; G-less templates TATAWtUTRmut and TATAmutUTRmut) We found that both TBP (Fig 2A, compare lanes 4 and 5) and the R2 TATA-box (Fig 2A, compare lanes 2 and 3) were absolutely required for transcription from the R2 promoter in this system As a positive control for these experiments, we included a reaction using a template where the G-less cassette is controlled by the adenovirus major late (AdML) promoter (lane 1) We found that the activity of the natural R2 promoter is 64% of the AdML promoter strength in this basal, unregulated in vitro transcription system (Fig 2A, compare lanes 1 and 2)
In contrast to the results obtained in vitro, analysis of the importance of the R2 TATA-box in vivo using the R2-luciferase reporter genes showed that a mutation of the R2 TATA-box only resulted in a limited decrease of the
Trang 5R2 promoter strength (Fig 2B) Finally, we also performed transient transfection experiments in synchronized Balb/3T3 cells using full-length R2 promoter-luciferase constructs with wild-type or mutated TATA-box (Fig 2C; TATAWtUTRWt and TATAmutUTRWt) These results showed that a mutation of the R2 TATA-box does not effect the S-phase specific expression from the mouse R2 promoter
The discrepancy between the in vitro results described in Fig 2A, which show that the R2 promoter is dependent both of TBP and its TATA-box, and the in vivo results presented in Figs 1C and 2B, which indicate that the R2 TATA-box has a very limited effect on transcription from the R2 promoter, could possibly be explained by the lack of
an essential transcription factor in the reconstituted in vitro transcription system In order to study transcription from the mouse R2 promoter in vitro in a more complex context,
we therefore used an in vitro transcription system based on a crude nuclear extract In line with the results obtained using the defined in vitro transcription system reconstituted from pure general transcription factors, also this crude system showed that the mouse R2 promoter is highly dependent on its TATA-box for full transcription (Fig 3A, compare lanes 1 and 2) As indicated, transcription from the TATA-mutated R2-promoter construct is fourfold lower compared
to the natural R2 promoter
Sequences downstream of the R2 transcription initiation site are important for proper initiation
of mouse R2 transcription Our results so far indicate a difference in the requirement of the R2 TATA-box when comparing results obtained in vitro and results obtained in vivo This could still reflect a difference between the two systems, either because the
in vitro systems lack an essential transcription factor or because of interactions between the template and chromatin components in the in vivo system However, also the templates used for the in vitro transcription and in vivo luciferase experiments differed from each other Our wild-type R2-luciferase template (TATAWtUTRWt-luciferase) contains a sequence from the mouse R2 5¢-UTR while the corresponding wild-type R2 promoter G-less template (TATAWtUTRmut-G-less) lacks this sequence In order to study if this difference was important, we made additional luciferase reporter gene constructs where the native or TATA-mutated R2 promoters were fused to an unrelated 5¢-UTR (Fig 3D summarizes all template constructs)
We then tested the new luciferase constructs in vivo in transient transfection experiments Comparison of these two new R2-luciferase constructs, which contain a mutated 5¢-UTR, shows that they differ in expression levels in a way that indicates a requirement for the R2 TATA-box also
in vivo(Fig 3B) As shown, a mutation of the TATA-box in combination with a mutated 5¢-UTR causes a threefold reduction in transcription also in vivo In conclusion, the experiments presented show that the R2 TATA-box is important for initiation both in vitro (Fig 3A) and in vivo (Fig 3B) when the R2 5¢-UTR is either deleted or mutated
In contrast, the in vivo results suggest that when the R2 5¢-UTR is included in the template, the R2 TATA-box becomes redundant
Fig 2 Both the R2 TATA-boxand TBP are required for transcription
from the R2 promoter in a defined basal in vitro transcription system but
the R2 TATA-boxis dispensable for basal and S-phase specific
trans-cription from the R2 promoter in vivo (A) In vitro transtrans-cription
experiments using highly purified mouse GTFs and the G-less cassette
reporter gene under the control of: lane 1, the AdML promoter;
lane 2, the full-length mouse R2 promoter (TATAWtUTRmut); lane 3,
the full-length R2 promoter with a mutation in the TATA-box
(TATAmutUTRmut); lane 4, same as lane 2; lane 5, same as lanes 2 and
5 but with TBP omitted from the in vitro transcription reaction.
(B) Transient transfection experiments using the indicated amounts of
the full-length mouse R2 promoter (TATA Wt UTR Wt ) ligated to the
luciferase gene (black bars) and of the full-length R2 promoter with a
mutation in the TATA-box (TATA mut UTR Wt ), ligated to the
luciferase gene (white bars) (C) A mutation of the TATA-box does not
effect the S-phase specific expression from R2 promoter Balb/3T3 cells
were transiently transfected with the full-length R2 promoter-luciferase
constructs, TATA Wt UTR Wt (m) and TATA mut UTR Wt (j) The graph
shows relative luciferase values at the indicated time points after release
from serum starvation.
Trang 6In reciprocal experiments we also made new G-less
templates for in vitro transcription that included the R2
5¢-UTR and either contained the natural R2 TATA-box
or a mutation of it to GCGCGC (G-less templates
TATAWtUTRWt and TATAmutUTRWt in Fig 3D) The
results from these experiments confirm the in vivo results As seen in Fig 3C, we found that transcription from these templates, which include the R2 5¢-UTR, was almost independent of the R2 TATA-box (compare to Fig 2B) Similarly to the experiments with the mutation of the R2 TATA-box (Fig 2C), we also performed transient trans-fection experiments in synchronized Balb/3T3 cells using full-length R2 promoter-luciferase constructs with wild-type or mutated 5¢-UTR (Fig 3E TATAWtUTRWt and TATAWtUTRmut) We found that a mutation of the R2 5¢-UTR had no effect on the S-phase specific expression from the mouse R2 promoter
Mutation of the R2 TATA-box affects the position for transcription initiation
It was possible that the mutation of the TATA-box and the different 5¢-UTR could result in a change of transcription start that might in turn influence the observed transcription levels We therefore performed primer extension assays on
Fig 3 Importance of the R2 TATA-boxfor promoter activity in the absence of the R2 5¢-UTR (A) In vitro transcription experiments using the R2-promoter G-less templates TATA Wt UTR mut (lane 1) and TATAmutUTRmut(lane 2) (described in Fig 3D) Two templates were used in each experiment: the R2 promoter construct ligated to a longer G-less cassette (product indicated by the upper arrow to the left of the autoradiograph) and the adenovirus major late promoter ligated to a shorter G-less cassette (product indicated by the lower arrow to the left
of the autoradiograph) All bands were quantified using the Scion Image program and the ratio between the upper and lower bands for each assay is presented below the autoradiograph (B) Transient transfection experiments with increasing amounts of DNA Black bars: the TATAWtUTRmut R2-luciferase construct; white bars: the TATAmutUTRmut R2-luciferase construct The values on the y-axis indicate the relative luciferase values after normalization of the R2 promoter luciferase to the cotransfected SV40-driven Renilla luciferase values for each transfection experiment (C) In vitro transcription experiments using the R2-promoter G-less templates 1, TATA Wt UTR Wt ; 2, TATA mut UTR Wt (described in Fig 3D) The numbers below the autoradiograph represent the ratio between the upper and lower bands (D) Overview of the reporter gene constructs used in experiments All constructs start at the Pvu II restriction site located at nucleotide )1500 relative to the R2 transcription start site TATAWt indicates that the construct contains the wild-type R2 TATA-box, TATA mut indicates that the construct carries a TTTAAA fi GCGCGC mutation, UTR Wt
indicates that the con-struct contains 17 base pairs (luciferase concon-structs) or 42 base pairs (G-less cassette constructs) from the wild-type mouse R2 5¢-UTR Finally, UTR mut indicates that the construct either lacks a 5¢-UTR (the G-less cassette constructs) or that it contains a 5¢-UTR composed of 21 nucleotides from the 5¢-end of the G-less cassette fused to 18 nucleo-tides from the 5¢-UTR of the luciferase reporter gene Arrows indicate the corresponding position for the wild-type R2 transcription start site (E) A mutation of the R2 5¢-UTR does not effect the S-phase specific expression from R2 promoter Balb/3T3 cells were transiently trans-fected with the full-length R2 promoter-luciferase constructs, TATAWtUTRWt(d) and TATAWtUTRmut (r) The graph shows relative luciferase values at the indicated time points after release from serum starvation.
Trang 7RNA synthesized in vitro using all four luciferase constructs
in nuclear extract-based transcription assays The luciferase
templates were used in these experiments as it was difficult
to find suitable sequences for synthesis of a primer in the
G-less cassette Similarly to the previously reported
map-ping of the transcription start in the wild-type R2 gene [15],
we found that all four constructs used here initiate
transcription on two adjacent nucleotides (Fig 4) We also
found that both constructs that contain the wild-type
TATA-box (TATAWtUTRWtand TATAWtUTRmut)
initi-ate transcription at the positions used by the native R2 gene
In contrast, the two constructs that contain the mutated
TATA-box (TATAmutUTRWtand TATAmutUTRmut) both
initiate transcription 2–3 base pairs upstream from the
normal R2 promoter initiation sites However, this change
in transcription start site does not correlate to the expression
levels from the different promoters (compare to Figs 3A–C
and 4) Please observe that the UTR of the UTRmut
templates is longer than the UTRWt templates, which
explains the differences in length of the primer extension
products between these two types of constructs
Protein interaction with the R2 5¢-UTR Our data presented above suggest that the R2 5¢-UTR contains sequences that bind protein(s) which assist TBP
in the formation of a functional preinitiation complex However, the R2 5¢-UTR showed no homologies to other 5¢-UTR sequences that previously have been identified as important for expression from different promoters, for example downstream promoter element (DPE) [14] We therefore analyzed the sequence of the R2 promoter and the 5¢-UTR, and compared it to the corresponding region
in the human R2 gene We could identify a potentially interesting palindrome in the R2 5¢-UTR sequence that covers 10 base pairs and overlaps with the position for transcription initiation in the mouse R2 promoter (Fig 5A, palindromes in boxes) Interestingly, this se-quence is also conserved in the human promoter except for the second and the last base pairs In general, the sequence from the conserved atypical TATA-box into the first 30 nucleotides of the 5¢-UTR shows a much higher homology between mouse and human (66% identity) compared to either the sequence upstream or downstream from this sequence
To study if the sequence downstream from the mouse R2 transcription start site interacts with proteins, we performed gel shift assays using a labeled oligonucleotide correspond-ing to base pairs)8 to +23 in the R2 gene relativ e to the major transcription start site, and nuclear extracts prepared from logarithmically growing Ehrlich–Lettre ascites mouse cells Incubation of nuclear extract with this oligonucleotide resulted in formation of a DNA-protein complex (Fig 5B, lanes 2 and 4), which could be competed specifically by an unlabeled oligonucleotide with the same sequence as the labeled oligonucleotide (Fig 5B, lane 3) However, it could not be competed by unrelated oligonucleotides (data not shown) We also performed similar experiments using an oligonucleotide that included the R2 TATA-box (nucleo-tides)34 to +23) The results from these experiments were identical to the results obtained with the shorter oligo-nucleotide (data not shown)
TAF subunits have been shown to interact to sequences both upstream and downstream from the TATA-box of promoters In addition, mapping of interactions between different TAF subunits and the adenovirus major late promoter also showed interactions between TAF250 and TAF135 and sequences even downstream from the tran-scription start [5] We therefore wanted to study if the sequence downstream from the R2 transcription start site could interact with TFIID, but all commercially available TAF antibodies are specific for human TAFs and show no cross-reactivity with the corresponding mouse proteins according to the manufacturers We could however, obtain
a monoclonal antibody that recognizes mouse TAFII135 in Western blots and which also can immunoprecipitate the mouse TFIID-complex (W S Mohan II, E Scheer,
O Wendling, D Metzger & L Tora, personal communica-tion) Incubation of a nuclear extract with monoclonal antibodies against TAF135 abolished formation of the complex (Fig 5B, lanes 5–7), whereas incubation with the same amounts of monoclonal antibodies against the ribo-nucleotide reductase R1 protein had no effect (Fig 5B, lanes 8–10)
Fig 4 Determination of the transcription start site in the different
R2-luciferase constructs The indicated R2 promoter luciferase
con-structs were used as templates in in vitro transcription experiments and
the resulting RNA products were used for primer extension
experi-ments The same primer, specific for the luciferase open-reading frame
was used for all constructs Arrows indicate the position for the two
major transcription start sites for each template.
Trang 8In order to map the position in the R2 5¢-UTR where the
TAF135 protein interacts, we synthesized three different
oligonucleotides corresponding to nucleotides)8 to +23 in
the R2 gene Each oligonucleotide carried mutations in
discrete regions (Experimental procedures) Gel shift
experi-ments using these mutated oligonucleotides and nuclear
extracts showed the only mutations within the palindrome
sequence (mutation 1) resulted in a shift that differed from
the one observed using the wild-type oligonucleotide
(Fig 5C and data not shown) As seen in Fig 5C, the
oligonucleotide with mutations in the palindrome sequence
resulted in two gel shift bands compared to the single band
observed with the wild-type oligonucleotide However,
neither of these two bands could be competed by addition
of TAF135 antibodies
We next included the TAF135 antibody in in vitro
transcription experiments using nuclear extracts and the
G-less cassette constructs that contain the native R2
TATA-box and either the natural or the deleted R2 5¢-UTR (TATAWtUTRWtand TATAWtUTRmut, respectively) We found that addition of increasing amounts of the TAFII135 antibody resulted in an up to 5.1-fold increase in transcrip-tion from the TATAWtUTRWt template (Fig 5D, lanes 1–3) A corresponding, 4.8-fold increase was also found when comparing the wild-type (TATAWtUTRWt) and the template that lacks the R2 5¢-UTR (TATAWtUTRmut; compare lanes 1 and 4) In contrast, addition of the TAFII135 antibody to reactions using the TATAWtUTRmut template resulted in a much less pronounced increase in transcription (1.6-fold, compare Fig 5D, lanes 4–6) In control experiments, monoclonal antibodies against the ribonucleotide reductase R1 protein had no effect on transcription efficiency in similar in vitro transcription
Fig 5 Sequences downstream from the R2 transcription start site interact with proteins and affect R2 transcription (A) Comparison of the mouse and human R2 DNA sequences around their transcription start sites Capital letters in bold style indicate nucleotides that are conserved between human and mouse; arrows indicate the mapped major transcription start at each promoter and boxes represents the partially conserved palindrome sequence described in the text (B) Gel shift experiments using nuclear extracts from logarithmically growing Ehrlich–Lettre ascites cells and a 32 P-labeled wild-type oligonucleotide including the transcription start and the downstream sequence from of the mouse R2 gene The protein-DNA complex is indicated by the arrow to the right of the autoradiograph Lanes 2–10, 32 P-labeled R2 5¢-UTR oligonucleotide with nuclear extracts (10 lg) from exponen-tially growing Ehrlich–Lettre ascites cells; lane 3, a 100-fold molar excess of unlabeled R2 5¢-UTR oligonucleotide was added; lanes 5–7, nuclear extract was preincubated with different amounts (0.43 lg, 0.85 lg and 1.7 lg, respectively) of monoclonal antibody towards human TAF II 135 for 10 min on ice prior to addition of the probe; lanes 8–10, the same as 5–7, but with monoclonal antibodies against mouse R1 The numbers under the gel represent the intensity of each band normalized to the band in lane 2 (C) Gel shift experiment using nuclear extracts from logarithmically growing Ehrlich–Lettre ascites cells, wild-type and mutated oligonucleotides including the transcrip-tion start and the downstream sequence from of the mouse R2 gene Lanes 1–3: 32
P-labeled R2 5¢-UTR wild-type oligonucleotide; lanes 2 and 3,32P-labeled R2 5¢-UTR oligonucleotide with nuclear extracts (10 lg) from exponentially growing Ehrlich–Lettre ascites cells; lane 3, nuclear extracts were preincubated with 1.7 lg of the TAF II 135 monoclonal antibody; lanes 4–6, the same as lanes 1–3, but using a labeled oligonucleotide containing mutations in the palindrome sequence (mutation 1, experimental procedures); lanes 7–9, as lanes 1–3 but using a labeled mutation 2 oligonucleotide; lanes 10–12, as lanes 1–3 but using a labeled mutation 3 oligonucleotide (D) In vitro transcription experiments with nuclear extracts isolated from log-arithmically growing Ehrlich–Lettre ascites cells, the TATA Wt UTR Wt
R2-G-less cassette reporter gene (lanes 1–3), the TATAWtUTRmut R2-G-less cassette reporter gene (lanes 4–6) and monoclonal anti-TAF II 135 antibodies Lanes 1 and 4, no antibody added to the in vitro transcription reaction; lanes 2 and 5, 1 lL of 300-fold diluted antibody was added to the in vitro transcription reactions; lanes 3 and 6, 1 lL of 60-fold diluted antibody was added to the in vitro transcription reac-tions All bands were quantified using the Scion Image program The upper numbers represent the intensity of each band normalized to the band in lane 1 The lower numbers represent the intensity of the bands
in lanes 5 and 6 normalized to the band in lane 4.
Trang 9experiments (data not shown) The fact that both a deletion
of the R2 5¢-UTR or addition of the monoclonal TAFII135
antibodies to the transcription reactions results in a similar
fivefold increase in transcription shows that TFIID binds to
the mouse R2 5¢-UTR and causes down-regulation of
transcription from the R2 promoter
Finally, we also performed primer extension experiments
on in vitro transcription reactions were the TAFII135
antibody was included We found that inclusion of the
TAFII135 antibodies in the in vitro transcription reaction
had no effect on the position for the R2 transcription start
site (data not shown)
Discussion
Previous and present experiments on the regulation of
expression of the ribonucleotide reductase R2 subunit are
focused on sequences located upstream from the R2
transcription start site, and on regulatory proteins that bind
to these sequences The aim of these studies is both to
understand how transcription from a natural promoter is
regulated in detail, but also to identify potential targets for
interference with the expression of the R2 gene Inhibition of
ribonucleotide reductase activity by hydroxyurea, which is a
specific inhibitor of the R2 subunit, or by peptidomimetics
that interfere with the interaction between the herpes
simplex virus R1 and R2 subunits, has previously proven
to be useful for antiproliferative therapy [28,29]
In order to extend these studies to also include the
function of sequences around the R2 transcription start
site, we obtained a polyamide that bound specifically to
the sequence immediately upstream from the mouse R2
TATA-box Polyamides are described as potent inhibitors
of protein–DNA interactions and they penetrate the
plasma membrane efficiently [30] Our initial experiments
presented here showed that the R2-specific polyamide
bound efficiently to its target sequence in the R2 promoter
but we found no inhibition of expression from an R2
promoter-luciferase reporter gene in stably transformed
cells, even at high concentrations of polyamide We
realized that we had taken for granted that the R2
TATA-box is essential for transcription initiation at the
R2 promoter and had overseen the possibility that the R2
TATA-box could be redundant Our results presented
here show that the polyamide could compete with
recombinant TBP for binding to the mouse R2
TATA-box and that both TBP and the R2 TATA-TATA-box are
required for transcription from the R2 promoter in an in
vitrotranscription system reconstituted from recombinant
or highly purified mouse general RNA polymerase II
transcription factors In these assays we used naked
plasmid DNA templates where the G-less cassette is fused
directly downstream from the mapped transcription start
of the full-length R2 promoter, which either contained the
normal R2 TATA-box, or a mutation of the TATA-box
to GCGCGC However, these results do not reflect the
situation in vivo, as our reconstituted in vitro transcription
system lacks both TAFs and coactivators like mediator
Initially, we therefore made corresponding R2-promoter
constructs fused to the luciferase reporter gene to study
the importance of the R2 TATA-box in vivo Similar to
the experiments using the polyamide in vitro, that
indicated redundancy of the R2 TATA-box, we found
no requirement for the R2 TATA-box in vivo
We had first neglected that the two types of templates used here, the R2-promoter coupled to either the G-less cassette or to the luciferase reporter gene, differed as the luciferase constructs also contained 17 base pairs of the mouse R2 5¢-UTR We therefore made new reporter gene constructs, both G-less cassette constructs including the R2 5¢-UTR, and luciferase constructs where the R2 5¢-UTR was replaced by sequences from the G-less cassette and the luciferase 5¢-UTR For each construct we also made a corresponding version with a mutated R2 TATA-box
By comparison of all constructs we could now find a common theme for the dependency of the R2 TATA-box (Table 1) Both in vivo and in vitro, a mutation of the R2 TATA-box had a very limited effect on transcription from the R2 promoter in the presence of the R2 5¢-UTR In contrast, the R2-TATA box was required for full expression from R2-promoter templates lacking the 5¢-UTR However, the requirement of the R2 TATA-box was not absolute in the later situation Rather, either the lack of a 5¢-UTR (G-less cassette controlled by the TATAWtUTRmutR2 promoter) or
a mutation of the R2 5¢-UTR (luciferase reporter controlled
by the TATAWtUTRmutpromoter) results in an up-regula-tion of transcripup-regula-tion In this background, an addiup-regula-tional mutation of the R2 TATA-box brings transcription down
to the levels observed for the constructs that contain the R2 5¢-UTR (G-less cassette and luciferase under the control of the TATAWtUTRWtor TATAmutUTRWtpromoters) The results presented above led us to focus on the R2 5¢-UTR Sequences downstream from the transcription start site have previously been identified as essential for transcription from different promoters, especially from those lacking a TATA-box The most well studied example
is the downstream promoter element (DPE) which was identified as a sequence present in many TATA-less promoters, and which interacts with the TFIID-complex [14] However, more recent studies showed that DPE interacts with the Drosophila homolog of the transcriptional repressor known as NC2 or Dr1-Drap1 and that purified recombinant dNC2 activates DPE-containing promoters and represses TATA-containing promoters [31] Detailed studies of protein–DNA interactions between the human TFIID complex and the adenovirus major late promoter using cross-linking have shown that TFIID subunits contact DNA both downstream and upstream of the TATA-box, but also that the hTAFII135 and hTAFII250 interact with sequences even downstream of the transcription start site Several recent reports also show that TFIID or specific TAF subunits are involved in repression of transcription rather
Table 1 Comparison of the relative transcription levels from the dif-ferent R2-promoter gene constructs used in vivo and in vitro.
Relative values
in vivo
Relative values
in vitro TATA Wt -UTR Wt 1 1 TATAmut-UTRWt 0.7 0.73 TATAWt-UTRmut 2.90 6.14 TATA mut -UTR mut 1.03 1.46
Trang 10than activation For example, human TAFII130 interacts
with heterochromatin protein 1 (HP1) to mediate
transcrip-tional repression, and TAFII250 has been shown both to
bind to the DNA-binding domain of TBP to inhibit
TBP:DNA interactions and to be involved in repression
of MHC class I expression by the HIV protein Tat [32–34]
While these results would fit with the regulation of the
mouse R2 promoter as we have presented it here, we were
unable to find a consensus DPE-sequence [(A/G)G(A/T)
CGTG] in the mouse R2 5¢-UTR Instead, comparison of
the mouse and human R2 promoters from the TATA-box
to 30 base pairs downstream from the R2 transcription start
showed that this region is highly conserved from mouse to
human In this region 39 out of 59 basepairs (66%) are
identical, without including any gaps, between the mouse
and human sequences (Fig 5A) This is in contrast to the
sequences immediately upstream or downstream from this
region, which shows <20% sequence identity Interestingly,
we also found a perfect palindrome (CGGTGCACCG)
located immediately downstream from the mouse R2
transcription start Except for the second and last
nucleo-tides, the palindrome is completely conserved in the human
R2 5¢-UTR
Our results support a model where the palindrome
sequence downstream of the mouse R2 transcription start
interacts with TFIID subunits This interaction functions
both as a core promoter element, as it eliminates the
requirement for the atypical R2 TATA-box, and also as a
repressor of transcription, as a deletion or a mutation of
this sequence or addition of TAFII135 antibodies leads to a
three- to fivefold up-regulation of transcription from the
mouse R2 promoter TAFII135 has been shown to be a
subunit of two different TAF-containing complexes, TFIID
and TFTC [35] TFTC comprises most of the TAF subunits
present in TFIID However, TFTC lacks TBP and has been
shown to promote both basal and activated transcription
from TATA-containing as well as TATA-less promoters
but with three times lower efficiency compared to TFIID or
free TBP [36] It is therefore possible that the fivefold
repression of R2 transcription that we detect from templates
containing the R2 5¢-UTR is due to TFTC-dependent
transcriptional initiation In R2 templates lacking the
downstream sequence, or when anti-TAFII135 antibodies
are included in the in vitro transcription assays,
transcrip-tional initiation would be TBP-dependent and thus more
efficient However, genome-wide expression analyses using
temperature-sensitive mutations in different TAFII-subunits
show that expression of many genes are up-regulated
when cells are shifted to the nonpermissive temperature,
thus indicating that expression of these genes are
negat-ively regulated by TFIID or other TAFII-containing
complexes [37]
Acknowledgements
We thank Irwin Davidson for the 32 TA monoclonal antibody towards
TAF II 135, and Peter Dervan for synthesis of the polyamide used in our
experiments This work was supported by grants from the Swedish
Research Council, the Swedish Cancer Society and the Swedish
Foundation for Strategic Research to both L.T and S.B, by grants from
the Human Frontier Science Program to S.B and from the Kempe
foundation to I.K and A.L.C.
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