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J Biol Chem, Vol. 273, Issue 45, 29816-29821, November 6, 1998
From the Department of Medical Biochemistry and Biophysics, Umeå
University, S-901 87 Umeå, Sweden
Ribonucleotide reductase is essential for DNA
synthesis. In mammalian cells, the enzyme consists of two non-identical
subunits, proteins R1 and R2. The expression of the mouse R1 and R2
genes is strictly correlated to S phase. Using promoter-reporter gene constructs, we have defined a region of the TATA-less mouse
ribonucleotide reductase R1 gene promoter that correlates reporter gene
expression to S phase. This is demonstrated in stably transformed cells
both synchronized by serum starvation and separated by centrifugal elutriation, suggesting that the R1 gene expression during the cell
cycle is mainly regulated at the transcriptional level. The region
contains four protein-binding DNA elements, The enzyme ribonucleotide reductase is essential for de
novo synthesis of deoxyribonucleotides. In this perspective,
ribonucleotide reductase plays a central role in providing the
precursors for DNA synthesis. Mammalian ribonucleotide reductase
consists of two non-identical homodimeric subunits, proteins R1 and R2
(1). Protein R1 (large subunit) contains the active site and allosteric sites for regulating enzyme activity. Protein R2 (small subunit) carries a tyrosyl free radical essential for enzyme activity. In
quiescent or differentiated cells, none of the two subunits can be
detected. In proliferating cells, the expression of protein R2 is S
phase specific, whereas protein R1 shows a constant level throughout
the cell cycle (2-4). However, both the R1 and R2 mRNA levels vary
in parallel during the cell cycle with negligible levels during
G0/G1 and maximal levels during S phase (5, 6). The R1 and R2 promoters are also activated in resting cells upon UV
irradiation leading to nucleotide excision repair (7).
Genomic clones covering the mouse R1 and R2 genes have been isolated
(8, 9). The R2 promoter contains a TATA-box and a proximal element,
called The R1 gene consists of 19 exons that covers 26 kb.1 In contrast to the R2
promoter, the R1 promoter is TATA-less. DNase I footprinting assays
proximal to the transcription start revealed two protected regions, A functional analysis of the R1 promoter using promoter-reporter gene
constructs in stably transformed cells indicated that Promoter-Luciferase Reporter Gene Constructs--
The luciferase
reporter gene plasmid pGL3-basic (Promega) was opened with
BglII and HindIII, and a new linker containing
unique EcoRI and Esp3I sites was introduced
upstream from the luciferase gene. Next, a 177-bp fragment from the
mouse R1 5'-UTR (EcoRI-Esp3I) was introduced into
the linker. This construct, pGL3R1 177, was then opened with
SacI and EcoRI, and a 5.5-kb
SacI-EcoRI R1 promoter fragment was inserted to
complete the pGL3R1 5.7 construct (Fig. 1). Shorter constructs, pGL3R1
3.7 and pGL3R1 420 (Fig. 1), were made by digesting the pGL3R1 5.7 with
SacI-BglII and
EcoICRI-EcoRV, respectively, followed by filling
in sticky ends with the Klenow fragment and religation. The constructs
pGL3R1 420-Inr(
The pGL3-control plasmid contains a firefly luciferase gene under the
control of SV40 promoter and enhancer. This plasmid was used to
construct a hybrid gene that express luciferase mRNA containing R1
mRNA in the 3'-UTR. This construct (pGL3 +49) was designed as
follows. Two 49-mers were annealed (13),
5'-CTAGACAAACTTCTATAAGTCATTTTGAAATAAACATTTCTAAGTGATA-3' and
5'-CTAGTATCACTTAGAAATGTTTATTTCAAAATGACTTATAGAAGTTTGT-3'. The overhangs were compatible with the opened XbaI site in
the pGL3-control plasmid, and the 49-mer was subsequently ligated into
the firefly luciferase 3'-UTR, upstream from the luciferase gene
poly(A) signal.
Analyses of R1 Promoter Activity in Stably Transformed Cell
Lines--
Balb/3T3 fibroblasts were cotransfected by electroporation
as described previously (14) with R1 promoter-reporter gene constructs and a pSV2neo plasmid. Several independent clones of stable
transformants were selected in the presence of Geneticin (G418) and
assayed for luciferase activity, expressed as light units per microgram of protein (14, 15). Cells were synchronized by serum starvation as
described earlier (5). At least two independent clones were analyzed
for each construct. Logarithmically growing cells were separated on the
basis of different cell size by centrifugal elutriation using a Beckman
JE-6B elutriation system (2). A sample of each fraction was analyzed by
flow cytometry to give cell cycle distribution and cell concentration.
The remaining cells were lysed and assayed for luciferase activity,
which in this experiment is given as light units per cell.
Transient Transfection Assays--
To measure the promoter
strength, Balb/3T3 cells were transiently cotransfected with R1
promoter-reporter gene constructs and a control plasmid, pRLSV40
(Promega), by the calcium-phosphate precipitation method (16). A
typical precipitate contained 2-3 µg of control plasmid, 2-8 µg
of R1 promoter-reporter gene construct, and pUC18 to a total amount of
20 µg of DNA. This precipitate was incubated with 0.5 × 106 cells for 14 h. Then, the cells were washed twice
with phosphate-buffered saline, and fresh medium was added. This medium
contained 10% heat-inactivated horse serum when exponentially growing
cells were harvested 24 h later and 0.6% fetal bovine serum when
the cells were synchronized by serum starvation. After 48 h of
starvation, medium containing 20% fetal bovine serum was added back,
and the cells were harvested at different time points. One-half of the cells were used for flow cytometry analyses and the other half was used
for enzyme assays. Firefly luciferase expressed by the pGL3R1 plasmids
and Renilla luciferase expressed by the pRLSV40 plasmid were assayed by
the dual luciferase assay kit (Promega). Firefly luciferase activity
was then divided by Renilla luciferase activity to normalize for
differences in transfection efficiency between plates. This value
reflects the R1 promoter activity.
Gel Shift Assays--
Nuclear extracts were prepared from
Balb/3T3 cells as described (17), and the protein concentration was
measured by the Bradford method (15). The reaction mixtures were
composed as described (11), and the antibodies against YY1, SRF, DP-2
(Santa Cruz Biotechnology), NF-YA, and NF-YB (kindly provided by Dr. Roberto Mantovani, Universita di Milano) were added last to the reaction mixture, followed by an incubation on ice for 1-2 h before the 15-min incubation at 30 °C. The following oligonucleotides annealed with a complementary oligonucleotide were used in the competition experiments: Screening with the Yeast One-hybrid System--
The yeast
one-hybrid system and a mouse T-cell lymphoma Matchmaker cDNA
library were purchased from CLONTECH. The screening was essentially performed as recommended by the manufacturer. Five
copies of the R1 Transcriptional Regulation of the R1 Promoter during the Cell
Cycle--
Using the improved luciferase vector, pGL3, we made a
plasmid containing 5.7 kb of the R1 promoter in front of the luciferase gene. This construct, pGL3R1 5.7, and two shorter versions, pGL3R1 3.7 and pGL3R1 420 (Fig. 1), were all
regulated in the same way when independent clones of stably transformed
cells were analyzed after serum starvation and readdition (Fig.
2 and data not shown). Now, the increase
in luciferase activity showed a good correlation to the increase in S
phase cells like the expression of R1 mRNA (5), indicating that the
R1 gene is regulated mainly at the level of transcription. The pGL3
control plasmid, containing the firefly luciferase gene under the
control of the SV40 promoter, did not show an S phase correlated
expression (data not shown).
The observed increase in R1 promoter activity as cells entered S phase
could be due to a serum proliferation response and not represent a true
cell cycle regulation. To exclude this possibility, we elutriated
logarithmically growing stably transformed cells containing the pGLR1
420 construct (Fig. 3). The fractions
enriched in S phase cells clearly showed a higher promoter activity
compared with fractions dominated by G1 cells.
Possible Control of R1 Gene Expression during the Cell Cycle by
Posttranscriptional Mechanisms--
It was earlier demonstrated that
the addition of phorbol 12-myristate 13-acetate to mouse cells causes a
rapid increase in the levels of R1 and R2 transcripts. This was
reported to be the result of mRNA stabilization caused by protein
binding to a specific regulatory 49-nucleotide element in the 3'-UTR of
the mRNA. However, no studies on cell cycle-regulated R1 gene
expression were reported (13). To study mRNA stabilization as a
mechanism for regulating the R1 mRNA levels during the cell cycle,
we introduced the putative regulatory 49-mer from the R1 gene into the
luciferase reporter gene. The 49-mer was introduced upstream from the
luciferase poly(A) signal in the 3'-UTR region of the luciferase gene;
therefore, it is included in the luciferase mRNA. Our mosaic
luciferase gene was under the control of the SV40 promoter and
enhancer. This construct and a control plasmid without the 49-mer was
introduced into Balb/3T3 cells, and stably transformed clones were
synchronized by serum starvation. An excess of mRNA containing the
49-mer might lead to loss of regulation due to exhaustion of regulatory
proteins. Therefore, clones with very different levels of mRNA
(luciferase activity ranging from 151 LU/µg to 144499 LU/µg) were
analyzed. Cells containing the control plasmid showed the same
expression pattern as five different clones containing the 49-mer
plasmid, and no clones showed any S phase-specific luciferase
expression (data not shown).
The
After this crude functional characterization of the promoter regions,
we wanted to make a more detailed study on the
The different regions of the The Transcription Factor YY1 Binds to the
The mutations inserted in the Yeast One-hybrid Screening to Identify Transcription Factors
Binding to the Our data from promoter-reporter gene constructs in stably
transformed cells indicate that all elements required for a correct cell cycle regulation of the mouse R1 gene expression can be found within the proximal 420 bp of the R1 promoter. No indication for post-transcriptional regulation of cell cycle-dependent R1
gene expression was observed. Earlier data suggested that the Gel shift assays with supershift antibodies, competition experiments,
and yeast one-hybrid screening showed that the transcription factor YY1
binds to the YY1 activates or represses transcription by binding to DNA elements
that often show a very complex structure. Repression may occur as a
result of interaction with histone deacetylases (22). There are often
several factors that can bind to the element, sometimes with
overlapping binding sites. YY1 can inhibit the binding of SRF (23, 24)
but also facilitate the binding of SRF (25). It has been shown that NF1
and YY1 both can activate the human p53 promoter by alternative binding
to a composite element (26). To make these elements even more
complicated, c-Myc has been shown to regulate YY1 activity via
protein-protein interactions to DNA-bound YY1 (27, 28).
A comparison with different YY1-binding elements showed that the R1 We can only speculate on how YY1 activates the R1 promoter at this
stage. One mechanism can be to facilitate the formation of the S
phase-specific complex C. However, it is interesting that YY1 is
required for cell cycle-regulated transcription from E2F-1-dependent promoters (29). At the same time, NF-Y was
reported to have a similar function as YY1. We did not find that NF-Y
binds to the We thank S. Skog, B. Tribukait, and K. Persson for flow cytometry measurements.
*
This work was supported by the Swedish Natural Sciences
Research Council, Knut och Alice Wallenbergs Stiftelse, Cancerfonden, Lion's Cancer Research Foundation, Umeå University, and Svenska Sällskapet för Medicinsk Forskning.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 46-90-7866742;
Fax: 46-90-136310; E-mail: lars.thelander{at}medchem.umu.se.
The abbreviations used are:
kb, kilobase(s); nt, nucleotide(s); bp, base pair(s); UTR, untranslated region; PCR, polymerase chain reaction; LU, light unit(s).
Two YY-1-binding Proximal Elements Regulate the Promoter Strength
of the TATA-less Mouse Ribonucleotide Reductase R1 Gene*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(nucleotides 
189 to
167), 
(98 to 76), Inr (4 to +16), and 
(+34 to +61),
together regulating promoter activity. The nearly identical upstream
elements, and , each form three DNA-protein complexes in gel
shift assays. We have identified YY1 as a component in at least one of
the complexes using supershift antibodies and a yeast one-hybrid
screening of a mouse cDNA library using the element as a
target. Transient transfection assays demonstrate that the and elements are mainly important for the R1 promoter strength and suggest
that YY1 functions as an activator.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, that contains an NF-Y binding CCAAT motif (10).
(nt 98 to 76) and (nt 189 to 167) (8). These two regions
are each 23 nt and identical except for one nucleotide. Three
DNA-protein complexes, A, B, and C, are formed when oligonucleotides
corresponding to the or footprints are used in gel shift
assays. Complexes A and B are present at constant levels during the
cell cycle, whereas complex C increases in S phase cells. A comparison
between the human and mouse R1 promoters revealed four conserved
regions (11). These were , , an initiator element (Inr), and one
downstream region, . The initiator element did not fully conform to
the Inr consensus YYAN(T/A)YY (12), but a functional analysis showed
that both the Inr and the regions were essential for cell
cycle-regulated R1 promoter activity (11). Furthermore, it was shown
that TFII-I binds to the R1 Inr, whereas the transcription factors YY1
or USF did not recognize the Inr.
and were
not essential for cell cycle-regulated transcription (11). However,
these assays only allowed a qualitative analysis of R1 promoter
activity. Furthermore, in these experiments the major increase in R1
promoter activity came about 4 h after cells entered S phase,
indicating that we were lacking an important regulatory DNA element. We
have now made new promoter-reporter gene constructs defining an R1
promoter region that faithfully regulates S phase expression of the
reporter gene. Using a transient transfection assay for a quantitative
analysis of R1 promoter activity, we demonstrate that and are
important for R1 promoter strength. Finally, our data suggest that YY1
functions as an activator in the R1 promoter by binding to the and
footprints.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4/+6) and pGL3R1 420-(+33/+43) were made by
digesting the pGL3R1 420 with EcoRI-XbaI and
EcoRI-HindIII, respectively. The primer pairs, 5'-GACGGACGGAATTCCTTGATATCACTTGACGTTTCAACG-3' and
5'-GTCTCCCGGGCTTTGCCCACACTC-3', were used in a PCR reaction to
introduce a 10-nt mutation in the Inr element. The resulting PCR
product was cut with EcoRI-XbaI and ligated into
the opened pGL3R1 420 construct. The primer pairs, 5'-ATTCGAATTCCGTCCGTCGCGTTGCTCTGCAACGAGATATCGGCGGGAGCGGGAA-3' and
5'-CTTATGCAGTTGCTCTCCA-3', were used in a PCR reaction to introduce a 10-nt mutation in the region. The resulting PCR product
was cut with EcoRI-HindIII and ligated into the
opened pGL3R1 420 construct. The pGL3R1 420-Inr(4/+6)-(+33/+43)
was created by subcloning the EcoRI-HindIII
fragment from the pGL3R1 420 -(+33/+43) into the pGL3R1
420-Inr(4/+6) construct, opened with
EcoRI-HindIII. The pGL3R1 420-Inr(4/+6)
construct was digested with EcoRV and HindIII,
and then the ends were filled in and religated, creating the pGL3R1
/ construct. A series of constructs, pGL3R1 287 with or without
mutations in the footprint, were designed in the following way
(Fig. 4B). pGL3R1 177 was opened in XbaI and
EcoRI. A region from XbaI nt 106 to nt +61 was
amplified from pGL3R1 3.7 by a polymerase chain reaction using five
different 5' primers, each one starting 4 nt upstream from the
XbaI site and containing the mutations shown in Fig.
4B and 5'-CTCCTTCCCGCTCCCGCCACCCGTGACG-3' at the 3'-end. The
resulting fragment was digested with XbaI and EcoRI, purified on an agarose gel, and ligated into the
opened pGL3R1 177 bp.
footprint,
5'-TTGCCCACACCCAATATGGCGGC-3'; (YY1),
5'-TTGCCCACACCCttTtaccCGGC-3'; c-Myc 260 YY1 oligonucleotide (18), 5'-GAAGAGAAAATGGTCGGGCG-3'. Oligonucleotides with the sequence
shown in Fig. 4B annealed with a complimentary
oligonucleotide were used in gel shift experiments to determine how
different mutations in the footprint effect the protein-DNA interaction.
footprint were inserted into the polylinker of the
three plasmids pHisi, pHisi-1, and placzi just upstream from the
minimal promoter of the HIS3 and lacz genes. A dual reporter yeast
strain (YMEJ2) was established by integrating pHisi-1 and placzi into
the genome. YMEJ2 grew poorly on SD(His)+15 mM
3-aminotriazole and not at all on SD(His)+45 mM
3-aminotriazole agar plates. The Matchmaker cDNA library,
expressing mouse proteins fused to the GAL4 activation domain, was
transfected into YMEJ2, and the yeast was plated on SD(His)+45
mM 3-aminotriazole agar plates. The expression plasmid was
isolated from the positive yeast clones and transfected into
Escherichia coli, JM109. A portion of the mouse cDNA was
sequenced in each plasmid and used to search Genbank with Blastsearch.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Functional assays of the R1 promoter
region. To the left, the R1 promoter-luciferase
constructs used to characterize the functions of different regions in
the R1 promoter are shown. The filled-in boxes show the
different known protein binding elements, , , Inr, and . An
open box in such an element indicates where a mutation has
been made. The large open box to the right represents the
luciferase gene. The 5'-ends of the pGL3R1 5.7 and pGL3R1 3.7 are not
drawn to scale. S, B, E, X, and 3I denote the restriction endonucleases
SacI, BglII, EcoRV, XbaI,
and Esp3I, respectively. To the right, results
from transient transfection assays to measure promoter strength are
given. The firefly luciferase activity controlled by the R1 promoter
was divided by the Renilla luciferase control plasmid activity to
quantify the R1 promoter activity. The promoter activity of the
different constructs in logarithmically growing cells were normalized
against the 3.7-kb R1 promoter activity (pGL3R1 3.7). The result is the
mean value (± standard deviation) from two to six independent
experiments.
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Fig. 2.
Cell cycle-regulated luciferase activity in
synchronized Balb/3T3 cells stably transformed with the pGL3R1 3.7 or
pGL3R1 420 constructs. Cells were first synchronized by serum
starvation and then harvested at different time points after serum
readdition (5). At each time point, one fraction of the cells was
assayed for luciferase activity and protein concentration (top
panel), and the values were normalized to the luciferase activity
at 0 h (pGL3R1 3.7 ( 
), 1628 LU/µg protein and pGL3R1 420 (
), 906 LU/µg protein). The remaining cells from each time point
were analyzed by flow cytometry (lower panel). The two
experiments showed the same degree of synchrony, and for simplicity,
only the values from the experiment with the pGL3R1 420 are plotted.
, G0/G1 cells; , S phase cells; ×,
G2 + M cells.
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Fig. 3.
Cell cycle-regulated luciferase activity in
elutriated logarithmically growing Balb/3T3 cells stably transformed
with the pGL3R1 420 construct. The upper panel shows
the luciferase activity in each fraction of elutriated cells, and the
lower panel shows the distribution of cells in different
growth phases. , G0/G1 cells; , S-phase
cells; ×, G2+M cells.
and Footprints Are Important for R1 Promoter
Strength--
Earlier R1 promoter-reporter gene constructs indicated
that cell cycle-specific expression of the R1 gene required the Inr and
elements, whereas there seemed to be no requirement for the /
elements (11). This puzzled us since we could demonstrate S
phase-specific binding to the / oligonucleotide (complex C) in
gel shift assays (8). To analyze if the and footprints were
important for the R1 promoter strength, we performed a series of
transient transfection assays in logarithmically growing cells. First,
we made a deletion analysis of the R1 promoter. These constructs were
cotransfected with an SV40-driven Renilla luciferase gene to normalize
for transfection efficiency. No significant difference in promoter
strength was seen as the promoter was shortened from 5.7 kb to 420 bp
in our R1 promoter-reporter gene constructs (Fig. 1). The deletion of
the footprint (pGL3R1 287) did not cause any dramatic reduction in
promoter strength; in contrast, a mutation in the remaining footprint caused a 50-60% decrease in promoter strength (pGL3R1 287 (85/81) compared with pGL3R1 287). The importance of the Inr and
regions was again clearly demonstrated as the promoter activity
decreased to about 20%, when either region was mutated in the presence
of the and footprints. Interestingly, a construct containing
only the and footprints, pGL3R1 /, completely lacking the
Inr element and region, showed a higher promoter activity than the
empty pGL3 vector. In contrast, a promoter lacking both functional
/ footprints and Inr element, pGL3R1 177, was as inactive as the
empty vector.
footprint. Therefore, additional constructs were made with point mutations within
the footprint (Fig. 4B).
As in the previous experiments, the constructs were cotransfected with
a plasmid containing an SV40-driven Renilla luciferase gene to
normalize for transfection efficiency. The results from a series of
transient transfection experiments, summarized in Fig. 4, show that all
our point mutations in the footprint decreased the promoter
activity in logarithmically growing cells to about 40-55% of
wild-type activity with mutations in the center of the footprint being
somewhat less active than mutations in the 5'or 3'-ends.
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Fig. 4.
Transient transfection assays analyzing the
function of the footprint in exponentially growing cells.
A, the firefly luciferase activity controlled by the R1
promoter was divided by the Renilla luciferase control plasmid activity
to quantify the R1 promoter activity. The promoter activity of the
mutant constructs was normalized against the wild-type promoter
activity. The bars show the mean value (± standard
deviation) from two independent experiments. B, the pGL3 287 construct is defined as the wild-type promoter, carrying the correct
DNA sequence in the footprint. The different mutations in the footprint are listed below the wild-type sequence.
footprint may bind different proteins
important under different cell cycle phases. Therefore, we synchronized
the cells by serum starvation after the transient transfection (Fig.
5 at 0 h). Again, mutations in the
center of the footprint (91/86 and 85/81) lowered the
promoter activity more than mutations in the 5'- or 3'-ends. The
synchronized cells were then released by serum readdition and harvested
at different time points. The promoter activity of the wild-type
construct was low in G0/G1 cells and started to
increase in S phase cells (Fig. 5). A maximal promoter activity was
reached after 20 h when there was a maximal number of S phase
cells. This value was 3.4 times higher than the 0-h value. The mutated
footprint constructs all showed the same pattern, but maximal
promoter activity was only comparable with the wild-type
G0/G1 value (Fig. 5). This indicates that the
footprint is important for total R1 promoter activity.
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Fig. 5.
Transient transfection assays analyzing the
function of the footprint in synchronized cells. The top
panel shows the firefly luciferase activity divided by the Renilla
luciferase in cells harvested at different time points after serum
readdition. The mean value (± standard deviation) of two independent
plates are illustrated. , pGL3R1 287 wild type; , 79/77; ×,
85/81; 
, 91/86; 
, 97/93. The bottom panel
shows the distribution of cells in different growth phases at the
different time points for one of the parallel cultures (pGLR1 287 wild
type). , G0/G1 cells; , S phase cells;
×, G2+M cells. The remaining cultures were also analyzed
by flow cytometry and showed a similar synchrony.
and Footprints--
A data bank search was performed for sequences in the
or footprints that could be recognized by already known
transcription factors. The result suggested that YY1 should bind to
both the and footprints, since they both contain the YY1
consensus binding sequence AANATGG. This was tested using a labeled
oligonucleotide corresponding to the footprint and a crude nuclear
extract from mouse fibroblasts in gel shift assays (Fig.
6). All three previously reported
complexes, A, B, and C, were competed with unlabeled footprint
oligonucleotide. An unlabeled oligonucleotide corresponding to a YY1
binding DNA element in the c-Myc promoter clearly competed complexes B
and C, but only to a smaller extent complex A. The sequence of the
putative YY1 binding site in the footprint was mutated in an
oligonucleotide (R1 (YY1)). This mutated footprint competed
complex A but not complexes B and C. Next, we used a supershift
antibody that recognizes YY1. This antibody specifically inhibited the
formation of complexes B and C, indicating that YY1 is involved in the
formation of these complexes (Fig. 7). Antibodies recognizing SRF or DP-2 did not inhibit the formation of or
supershifted any complex (Fig. 7). The antibodies recognizing the CCAAT
binding transcription factor NF-Y, subunit a or b, did not affect any
of the three complexes (data not shown).
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Fig. 6.
Competition experiments in gel shift assays
indicating that YY1 participates in complex B and C. Lane
1, R1 oligonucleotide; lanes 2-10, oligonucleotide
with nuclear extracts (10 µg) from exponentially growing cells;
lanes 3-5, a 100-, 200-, and 500-fold molar excess,
respectively, of unlabeled c-Myc 260 oligonucleotide was added;
lanes 6-8, a 100-, 200-, and 500-fold molar excess,
respectively, of unlabeled oligonucleotide was added; lanes
9 and 10, a 250- and 500-fold molar excess,
respectively, of unlabeled (YY1) oligonucleotide was added.
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Fig. 7.
A gel shift assay using labeled footprint
oligonucleotide and supershift antibodies specific for YY1, SRF, or
DP-2. Lane 1, labeled oligonucleotide; lanes
2-5, oligonucleotide with nuclear extract (10 µg) from
exponentially growing cells; lane 3, supershift antibody
recognizing YY1 was added; lane 4, supershift antibody
recognizing SRF was added; lane 5, supershift antibody
recognizing DP-2 was added.
footprint in the previously described
functional analyses were also analyzed in gel shift assays.
Oligonucleotides corresponding to the different mutated footprints
(Fig. 4B) were labeled and mixed with crude nuclear extracts
from logarithmically growing cells. The 79/77, 85/81, and
91/86 mutations all overlap the known consensus binding sequence of
YY1. As expected, complex B could not be formed or was severely
affected by all these mutations (Fig. 8).
Complex A was only affected in the mutation 91/86. Complex C (the S phase-specific complex) was affected by all mutations, but only to a
small extent by the 97/93 mutation. The different DNA-protein complexes most likely consist of several different proteins, since a
new complex b' appeared with all the four mutated oligonucleotides (Fig. 8).
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Fig. 8.
A gel shift assay using labeled wild-type or
mutant footprint oligonucleotide to analyze the effects of the
mutations on the DNA-protein complexes. Lanes 1-3,
labeled oligonucleotide; lanes 4-6, labeled (79/77)
oligonucleotide; lanes 7-9, labeled (85/81)
oligonucleotide; lanes 10-12, labeled (91/86)
oligonucleotide; lanes 13-15, labeled (97/93)
oligonucleotide; lanes 2, 5, 8,
11, and 12, 5-µg nuclear extracts were added;
lanes 3, 6, 9, 12, and
15, 10-µg nuclear extracts were added. Complex b' is
located between A and B in lanes 5,
6, 8, 9, 11, 12,
14, and 15.
Footprint--
In an attempt to identify other
transcription factors that participate in complexes A, B, and C, we
performed a yeast one-hybrid screening using five copies of the footprint as the target element. After establishing a yeast strain
containing both the pHis-1 and pLacz reporter genes, 238,000 independent clones were screened, and 13 clones survived on the
selective medium and expressed -galactosidase. The plasmids
expressing the DNA binding activity in these yeast clones were
isolated, used to transfect E. coli and subsequently sequenced. Four of the clones expressed the DNA binding domain of YY1
fused to GAL4AD.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
and footprints were not required for the cell cycle regulation of the
R1 promoter (11). Our transient transfection assays now clearly show
that the and footprints are important for the overall R1
promoter activity. In the transient transfection assays in synchronized
cells, the mutations in the footprint did not significantly alter
the expression pattern during G1-S transition, but the
maximal level of mutant promoter activity in S phase was only
comparable with the activity of the wild-type construct in quiescent
cells. This level of promoter activity does not give rise to measurable
levels of R1 mRNA in resting cells (5).
and footprints and forms complex B. This complex
was earlier shown not to vary during the cell cycle (8). YY1 is a
ubiquitous transcription factor with many functions. It can activate,
repress, and initiate transcription (19). In the R1 promoter, YY1
normally does not initiate transcription at the and footprints,
since S1 nuclease analysis has shown that the transcription start is at
the R1 Inr and not located to the two footprints (8). Furthermore, YY1
does not bind to the R1 Inr (11). However, there is a possibility that
in our transient transfection assays, YY1 may initiate transcription at
the / footprints in the R1 promoter when the Inr and elements are deleted. It has been shown in in vitro transcription
experiments that YY1 can initiate transcription on supercoiled plasmids
in the presence of only TFIIB and RNA pol II (20, 21). It is not
unlikely that this can occur inside cells as they are transfected with
supercoiled plasmids.
and footprints are nearly identical to a serum response element in
the skeletal -actin promoter (SRE1 nt 93 to nt 77) (23). This
element contains a consensus binding site, 5'-CCAAATATGG-3', for SRF.
However, this site is not present in the R1 and footprints, and
SRF does not bind to the / footprints (Fig. 7). We took advantage
of linker-scanning mutations in the SRE1 of the skeletal -actin
promoter in an attempt to characterize the functional role of YY1
binding to the R1 promoter. There are three different mutations in the
SRE1 that binds only SRF, only YY1, or neither YY1 nor SRF (23). The
mutation that inhibits only YY1 from binding to the SRE1 was also found
to inhibit the binding of YY1 to the R1 footprint (79/77) (Fig.
8). In contrast, the mutation in the SRE1 that only blocks SRF from
binding to the element not only inhibited complex A but also YY1 from
binding to the footprint (91/86). From these results, we cannot
draw any conclusions regarding the function of complex A. However, the
results suggest that YY1 functions as an activator in the R1 promoter.
Complex C appeared to be severely affected by any mutation across the footprint except for (97/93). Both the competition gel shift experiments and the gel shift with the YY-1 antibody suggest that the
formation of complex C is dependent on complex B (YY1). The appearance
of a new DNA-protein complex b' in all the gel shifts using a mutated
oligonucleotide indicates the participation of more than one
protein in the complexes or multiple DNA-protein interactions.
or footprints, ruling out the possibility of a
common factor between the mouse R1 and R2 promoters. A further
characterization of the proteins that bind to the and footprints may clarify how YY1 activates the R1 promoter.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. Biochemistry and Molecular Biophysics,
Washington University School of Medicine, 660 S. Euclide, St. Louis, MO 63110.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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