(Received for publication, June 12, 1995)
From the
Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide -672 to nucleotide -527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains.
Ribonucleotide reductase (EC 1.17.4.1) is a key enzyme in DNA
precursor synthesis reducing all four ribonucleotides to the
corresponding deoxyribonucleotides(1, 2) . Mouse
ribonucleotide reductase is a heterodimer composed of the two
homodimeric subunits, proteins R1 and R2, each inactive alone. Enzyme
activity is cell cycle regulated with low or undetectable levels in
G/G
and maximal activity in the S phase of the
cell cycle(3) .
The mouse R1 and R2 mRNA expression is S
phase specific with very low or undetectable levels in
G/G
cells, a pronounced increase as cells
progress into S phase, and a decline when cells progress into G
+ M(4) . Reporter gene constructs show that the R2
promoter is activated almost immediately after quiescent
G
/G
synchronized cells are released by serum
readdition. Promoter activity then increases steadily, reaching its
maximum at around 12 h after serum readdition(5) . From the
early promoter activation, the R2 gene could be classified as an
immediate early response gene. However, in vitro studies
demonstrated that this early activation only results in the synthesis
of immature short R2 mRNA transcripts due to a G
-specific
transcriptional block located in the first intron of the R2
gene(5) . This block is not released until cells reach S phase
when full-length transcripts are synthesized. Reporter gene constructs
containing the R2 promoter-1st exon/1st intron indicate that the
transcriptional block is active also in vivo, and S
phase-specific protein binding was identified to a DNA region just
upstream from the block. (
)
The mouse R2 promoter contains
a TTTAAA motif at position nt ()-24 and a CCAAT motif
at position nt -75 upstream from the transcription
start(6) . DNase I footprinting analyses revealed four
DNA-protein binding regions within the R2 promoter. The region most
proximal to the transcription start (nt -93 to -56)
includes the CCAAT box and is called
. The other three DNA-protein
binding regions, called
,
, and
, are located very close
to each other at positions nt -584 to -527, nt -623
to -597, and nt -672 to -637, respectively.
Experiments using R2 promoter-luciferase reporter gene constructs
suggested that the
,
, and
regions are required for the
early proliferation specific activation of the promoter. Only basal
transcription was observed in a construct lacking these regions but
retaining the
region.
Gel shift experiments with nuclear
extracts from synchronized BALB/3T3 mouse fibroblasts and an
oligonucleotide representing the region showed one specific, cell
cycle-independent DNA-protein complex.
The transcription
factor NF-Y, also called CBF, is a ubiquitous heteromeric
metalloprotein composed of three subunits: A, B, and C (7, 8, 9, 10) . The C subunit, which
was recently identified and cloned, is required together with the A and
B subunits to form an NF-Y-DNA complex, containing all three
subunits(10) . NF-Y was shown to bind to the promoters of the
major histocompatibility complex class II genes, the tissue-specific
-collagen gene, and the albumin
gene(11, 12, 13, 14) .
In this
study, we try to elucidate the functional importance of the
region within the R2 gene promoter and to identify the transcription
factor(s) binding to it. The results show that the
region is
required not only for basal transcription as suggested earlier but
plays a pivotal role for the continuous activity of the R2 promoter
through the S phase. Within the region, the CCAAT motif appears to be
of major functional importance by its interaction with the
transcription factor NF-Y.
Figure 1:
A,
structure of the p19lucR2 1.0- construct with the R2 promoter
,
,
, and
regions protected from DNase I cleavage
in footprinting assays. The thickline indicates the
deleted DNA fragment in footprint
. The numbering is from
the R2 gene transcription start, which is +1. B, R2
promoter-controlled luciferase expression in Balb/3T3 cells stably
transformed with the p19lucR2 1.0-
and synchronized by serum
starvation. C, cell cycle phase composition determined by DNA
flow cytometry.
, G
phase cells;
, S phase
cells; &cjs3648;, G
+ M phase
cells.
To construct the
plasmids p19lucR2 1.5J1 and -J2, the plasmid p19lucR2 1.5 was cut with SalI (a unique site located in the polylinker upstream from
the R2 promoter) and BssHII(-47), and the longer DNA
fragment representing the p19luc plasmid with the 3`-end of the R2
promoter was isolated on a low melting agarose gel. This fragment was
then ligated to the J1 or J2 oligonucleotides, both having a unique SmaI site close to the 5`-end and SalI and BssHII compatible ends. The intermediate constructs p19lucJ1
and p19lucJ2 were cleaved with SmaI, and each one was ligated
to a 1.4-kilobase BamHI (in the polylinker upstream from the
R2 promoter) to BstD102 I (-95) fragment isolated from
the p19lucR2 1.5 plasmid. The 1.4-kilobase fragment contained the
entire R2 promoter down to the 5`-end of the region, and the BamHI site was filled in by the Klenow fragment before
ligation. The right orientation of the fragment was checked by
restriction enzyme analyses. The final constructs p19lucR2 1.5J1 and
-J2 had the
region replaced by the J1 and J2 sequences ( Fig. 2and Fig. 3).
Figure 2:
A,
structure of the p19lucR2 1.5J1 construct where the substituted part of
the region is indicated by a thickline and its
new DNA sequence given below. The CCAAT motif is underlined. B, R2 promoter-controlled luciferase
expression in Balb/3T3 cells stably transformed with the p19lucR2 1.5J1
and synchronized by serum starvation. C, cell cycle phase
composition determined by DNA flow cytometry.
, G
phase cells;
, S phase cells; &cjs3648;, G
+ M phase cells.
Figure 3:
A, structure of the p19lucR2 1.5J2
construct where the substituted part of the region is indicated
by a thickline and its new DNA sequence given below. The mutated nucleotides are underlined. B, R2 promoter-controlled luciferase expression in Balb/3T3
cells stably transformed with the p19lucR2 1.5J2 and synchronized by
serum starvation. C, cell cycle phase composition determined
by DNA flow cytometry.
, G
phase cells;
, S
phase cells; &cjs3648;, G
+ M phase
cells.
The plasmid p19lucR2 TTTAAA was made by cleaving p19lucR2 1.5 with SalI and BssHII. The long fragment containing the p19luc vector and a short sequence of the R2 promoter was filled in by the Klenow fragment and religated. The final construct contained only the TTTAAA box and the transcription initiation site from the R2 promoter (nt -47 to +17) ligated to the luciferase reporter gene. All plasmid constructs were verified by double-stranded DNA sequencing.
To study if the
effects of deleting the region were caused by the shortening of
the promoter or a result of deleting specific DNA-protein binding
areas, we decided to replace the
region with nonspecific DNA. A
DNA fragment of the correct length was chosen from an upstream region
of the R2 promoter (nt -1380 to -1343), which seemed to be
of no importance for promoter activity (cf. the expression of
p19lucR2 1.0 and 1.5 as mentioned above). To our surprise, this
construct, p19lucR2 1.5J1, where the
region was replaced by the
J1 DNA sequence (Fig. 2A), showed almost the same
luciferase expression pattern as the intact R2 promoter constructs with
enzyme activity, increasing steadily up to 16 h after serum readdition (Fig. 2B). However, closer inspection showed that the
J1 sequence happened to contain a CCAAT motif like the native
region but was otherwise different.
We therefore made the p19lucR2
1.5J2 construct, which was identical to the J1 construct except that
the sequence just upstream from and within the CCAAT motif was mutated (Fig. 3A). Stable transformants carrying the J2
construct showed the same luciferase expression pattern as cells
transformed by the p19lucR2 1.0- (Fig. 3B), i.e. maximal luciferase activity at 4-5 h and back to
near basal values after 16 h. Taken together, these results indicate
that the
region is required for promoter activity through the S
phase but is dispensable for the early proliferation-specific
activation.
Figure 4:
Gel
electrophoresis mobility shift assays of the footprint region of
mouse R2 gene promoter. Nuclear extracts from G
/G
synchronized BALB/3T3 cells (10 µg/lane) were preincubated
with antibodies against NF-YA (500 ng) or NF-YB (100 ng) proteins for 1
h on ice (lanes3 and 4, 7 and 8, respectively) and then for 20 min at 30 °C or only for
20 min at 30 °C with end-labeled oligonucleotides representing
footprint
(lanes2-4), J1 (lanes6-8), or J2 (lane10) sequences
(10.000 cpm/lane). Lanes1, 5, and 9, no nuclear extract added.
To investigate the specificity of the binding, we also
made gel shift experiments with crude nuclear extracts and labeled
oligonucleotides corresponding to the J1 or J2 oligonucleotides. The J1
oligonucleotide contains a CCAAT motif located closer to the 3`-end of
the oligonucleotide than the CCAAT motif in the oligonucleotide,
while the J2 oligonucleotide lacks the CCAAT motif. The specific
DNA-protein complex formed with the J1 oligonucleotide had similar
mobility as the one formed with the
oligonucleotide (Fig. 4, lane6) and reacted in a similar way
with the NF-Y antibodies (Fig. 4, lanes7 and 8). The same pattern was obtained with nuclear extracts from
cells enriched in different cell cycle phases (data not shown).
No
specific DNA-protein complex was observed with the J2 oligonucleotide (Fig. 4, lane10). We conclude that the CCAAT
motif within the region is sufficient for binding the NF-Y
transcription factor, and no other transcription factor is binding
directly to the
region DNA.
The R2 promoter-reporter gene constructs clearly demonstrate
the importance of the region for S phase-specific expression of
the R2 gene. In the absence of the
region, the early
proliferation-induced activation of the promoter caused by protein
binding to the upstream regions would not result in any production of
mature R2 mRNA due to the G
transcriptional block. Protein
binding to the
region maintains a high promoter activity even
after the S phase-specific release from the block, and this is a
prerequisite of R2 mRNA expression.
It is obvious from the different
reporter gene constructs that a CCAAT motif is sufficient to
functionally replace the region DNA. The fact that the anti-NF-YB
antibodies completely inhibit the formation of any DNA-protein complex
using the
oligonucleotide and a nuclear extract strongly
indicates that NF-Y really binds to the
region and not some other
CCAAT binding protein. In this context, it is interesting that NF-Y
does not bind to the CCAAT motif present in the
footprint of the
mouse R1 gene promoter (15) or to a CCAAT motif present in an
inverted orientation in the
footprint of the R2 gene promoter,
indicating that the CCAAT motif by itself is not always sufficient for
NF-Y binding (data not shown). It has also been suggested that other
interactions with DNA in addition to the CCAAT element are important
for NF-Y binding(10) .
None of the three subunits of NF-Y shows any homology to known protein-protein binding motifs such as leucine zippers, coiled coils, or helix-loop-helix motifs(10) . This ubiquitous, heteromeric DNA binding protein, which binds to the proximal part of many eukaryotic promoters, may interact both with upstream DNA binding transcription factors and with proteins involved in the formation of the pre-initiation transcription complex. However, the precise role of NF-Y in transcription activation is still not known.
By studying the function of NF-Y in the transcription of
major histocompatibility complex class II genes, it was suggested that
NF-Y is involved in the very first stages of pre-initiation and in
re-initiation of transcription at the promoter(12) . NF-Y
carries glutamine-rich activation domains thought to be involved in
protein-protein interactions with other transcriptional activators. In
the activation of the R2 promoter, a close interaction between NF-Y and
the transcription factor(s) binding to the upstream ,
, and
regions may by required to maintain promoter activity into S
phase and achieve productive R2 mRNA expression. Such a role of NF-Y
would agree with the proposal that NF-Y bound to a promoter stabilizes
the pre-initiation complex. Without NF-Y, the complex formed by the
factors binding to the
,
, and
upstream regions may not
be stable enough to survive into S phase when DNA replication occurs.
On the other hand, the pre-initiation complex formed by NF-Y in the
absence of the upstream factors does not respond to proliferation but
only supports a low basal luciferase activity.
No measurable
luciferase activity was observed in cells stably transformed with an
R2-luciferase gene construct, lacking the region and only
containing the TTTAAA box and the transcription start of the gene. This
may be because the second nucleotide of the R2 TATA element does not
match the consensus TATAAA but represents a rare variant(19) .
Many promoters combine the function of a proximal element with that
of a more distant enhancer. In the human thymidine kinase gene, most of
the promoter activity is contributed by an upstream GC element and a
proximal CCAAT element. Competition studies indicated that the protein
binding to the CCAAT was NF-F(20) . In the thrombospondin 1
gene, serum-stimulated promoter activation is dependent on a serum
response element located at nt -1280 and NF-Y bound to a CCAAT
box at nt -65 (21) . A somewhat similar arrangement is
found in the serum-stimulated -actin gene promoter, where the
serum response element and the CCAAT box are closely located within a
50-base pair region centered at nt -75 with the NF-Y binding
situated upstream from the serum response
element(22, 23) . However, there are no direct
similarities between the reported serum response element consensus
sequence 5`-CC(A+T)
GG-3` and the R2 promoter
,
, and
sequences. Furthermore, in the thrombospondin case,
deletion of the upstream serum response element or the CCAAT both
resulted in decreased serum response. In contrast, deletion of the
CCAAT motif in the R2 promoter changes not only the level of activation
but also the cycle-dependent pattern of promoter activation. This shows
that alteration of a proximal promoter element can change the
qualitative response to upstream transcription activation domains (cf.(24) ). A more complete understanding of the
activation of the R2 promoter will require identification of the
transcription factor(s) binding to the upstream region.