©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene (*)

(Received for publication, June 12, 1995)

Dmitri Filatov (§) Lars Thelander

From the Department of Medical Biochemistry and Biophysics, Umea University, S-901 87 Umea, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(0)/G(1) 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(0)/G(1) cells, a pronounced increase as cells progress into S phase, and a decline when cells progress into G(2) + M(4) . Reporter gene constructs show that the R2 promoter is activated almost immediately after quiescent G(0)/G(1) 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(1)-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. (^1)

The mouse R2 promoter contains a TTTAAA motif at position nt (^2)-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 alpha. The other three DNA-protein binding regions, called beta, , and , are located very close to each other at positions nt -584 to -527, nt -623 to -597, and nt -672 to -637, respectively.^1 Experiments using R2 promoter-luciferase reporter gene constructs suggested that the beta, , 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 alpha region.

Gel shift experiments with nuclear extracts from synchronized BALB/3T3 mouse fibroblasts and an oligonucleotide representing the alpha region showed one specific, cell cycle-independent DNA-protein complex.^1

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 alpha-collagen gene, and the albumin gene(11, 12, 13, 14) .

In this study, we try to elucidate the functional importance of the alpha region within the R2 gene promoter and to identify the transcription factor(s) binding to it. The results show that the alpha 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.


MATERIALS AND METHODS

Cell Culture, Synchronization, Transfection, and Selection of Stable Transformants

Balb/3T3 cells (ATCC CCL 163) were grown as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum. Synchronization of cells by serum starvation and readdition and flow cytometry to check the cell cycle distribution was described earlier(5) . Transfection of cells by electroporation was made using reporter gene DNA and pSV2neo, and stable transformants were selected in the presence of Geneticin base (Sigma)(5) . Resistant clones were expanded, and the presence of transfected plasmid DNA was verified by PCR using specific primers for the R2 promoter and luciferase cDNA (see below). Usually, two to three independent clones were tested for each particular construct.

Oligonucleotides

All oligonucleotides were synthesized on an Applied Biosystems 392 RNA/DNA synthesizer and purified by chromatography on Sephadex G-25 columns. Oligonucleotides used for construction of p19lucR2 1.0-alpha were 5`-CCTGCGGCGCAACTCAAATCTCCCGCGCTGGGCGGCGAGCGGGGTCAGCGG-3` and 3`-GGACGCCGCGTTGAGTTTAGAGGGCGCGACCCGC CGCTCGCCCCAGTCGCCGCGC-5`; oligonucleotides used for construction of p19lucR2 1.5J1 were 5`-TCGACCCCGGGAGACTCTTGAAGTTGAACCAACCAATCACAGAGCAGAGGTTACG-3` and 3`-GGGGCCCTCTGAGAACTTCAACTTGGTTGGTTAGTGTCTCGTCTCCAATGCGCGC-5`; oligonucleotides used for construction of p19lucR2 1.5J2 were 5`-TCGACCCCGGGAGACTCTTGAAGTTGATCCTAC TAGTCACAGAGCAGAGGTTACG-3` and 3`-GGGGCCCTCTGAGAACTTCAACTAGGATGATCAGTGTCTCGTCTCCAATGCGCGC-5`; and oligonucleotides used in gel shift experiments representing footprint alpha were 5`-CGGGGTCAGCGGCGTCCAATGGGAGGCGAGGCCACGCCCC-3` and 3`-CCCCAGTCGCCGCAGGTTACCCTCCGCTCCGGTGCGGGG-5`. EJ1 oligonucleotide for PCR corresponding to the 5`-end of the luciferase reporter gene (minus strand) was 5`-CTTATGCAGTTGCTCTCCA-3`; anti-EJ1 oligonucleotide for PCR corresponding to the 5`-end of the luciferase reporter gene (plus strand) was 5`-TGGAGAGCAACTGCATAAG-3`; EJ2 oligonucleotide for PCR corresponding to the 3`-end of the luciferase reporter gene (minus strand) was 5`-CGCTGAATACAGTTACATT-3`; R2p1 oligonucleotide for PCR corresponding to the DNA sequence of the R2 promoter from position nt -725 to nt -704 (plus strand) was 5`-GGTATCTTGGACCCCGTAGGGC-3`; and TTTAAA oligonucleotide for PCR (nt -47 to -22, plus strand) was 5`-CGCGCGGTTGGGTGGCTCTTTAAAGG-3`.

PCR Reactions

Geneticin-resistant stable transformants were expanded, and then cells were harvested in 1 ml of phosphate-buffered saline by scraping using a rubber policeman. The cells were washed in 10 ml of phosphate-buffered saline, spun down and re-suspended in PCR lysis buffer (50 mM KCl, 10 mM Tris-Cl, pH 8.3, 2.5 mM MgCl(2), 0.45% Nonidet P-40, 0.45% Tween 20, 0.6 mg/ml Proteinase K; 6 times 10^6 cells/ml), and incubated at 60 °C for 1 h and at 95 °C for 10 min. From the resulting cell extract, 10-20 µl were used in a 100-µl PCR reaction mixture. The reactions were run as described(15) , except that the incubation time was reduced to 1 min at each temperature and a mixture of 1 mM dGTP and 1 mM 7-deaza dGTP was used in place of 2 mM dGTP when the R2 promoter was analyzed. To verify the presence of an intact luciferase gene, the anti-EJ1 and EJ2 primers were used, and to verify the presence of the R2 promoter linked to the luciferase gene, the R2p1 and EJ1 primers were used. Finally, the presence of p19lucR2 TTTAAA was verified using the TTTAAA and EJ2 primers.

Plasmid Constructs

The plasmid p19lucR2 1.5 contains a PvuII-PvuII fragment of the mouse R2 promoter (nt -1497 to +17, according to (6) and Footnote 1, where +1 indicates the position of the transcription start) ligated into the unique SmaI site in the polylinker of the vector p19luc (16) . The plasmid p19lucR2 1.0 contains a ClaI-PvuII (nt -1006 to +17) fragment of the R2 promoter ligated into the polylinker of the p19luc vector cleaved with HindIII (made blunt end by filling in using the Klenow fragment) and SmaI. The plasmid p19lucR2 1.0-alpha was made by cleaving p19lucR2 1.0 with BssHII (3 sites within the R2 promoter with the most proximal site at position -47 and no sites within the p19luc) and SphI (one unique site within the luciferase cDNA in the p19luc). A 950-base pair BssHII-SphI fragment representing the 3`-end of the R2 promoter and the 5`-end of the luciferase cDNA was isolated on a low melting agarose gel. Then, the p19lucR2 1.0 plasmid was cleaved again but with StuI (nt -133) and SphI (both unique), and the longer fragment representing the 5`-end of the R2 promoter and the rest of the p19luc plasmid was isolated on a low melting agarose gel. The two DNA fragments were ligated to the oligonucleotide ending with StuI and BssHII sites in a triple ligation reaction. The resulting plasmid, p19lucR2 1.0-alpha, had 33 base pairs deleted from the alpha region (Fig. 1).


Figure 1: A, structure of the p19lucR2 1.0-alpha construct with the R2 promoter alpha, beta, , and regions protected from DNase I cleavage in footprinting assays. The thickline indicates the deleted DNA fragment in footprint alpha. 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-alpha and synchronized by serum starvation. C, cell cycle phase composition determined by DNA flow cytometry. bullet, G(1) phase cells; circle, S phase cells; &cjs3648;, G(2) + 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 alpha 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 alpha 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 alpha 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. bullet, G(1) phase cells; circle, S phase cells; &cjs3648;, G(2) + M phase cells.




Figure 3: A, structure of the p19lucR2 1.5J2 construct where the substituted part of the alpha 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. bullet, G(1) phase cells; circle, S phase cells; &cjs3648;, G(2) + 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.

Luciferase Assay

Harvesting of cells and preparation of cell extracts for luciferase assays were preformed as described(5) . Luciferase activity was measured as the number of light units emitted during a 10-s period per µg of total protein, as determined by the Bradford assay(17) .

Gel Shift Analysis and Antibodies

Crude nuclear extracts from BALB/3T3 cells were prepared at different time points after serum stimulation as described(18) . Binding reactions were performed as described(15) , and DNA-protein complexes were resolved through native low ionic strength 4% polyacrylamide gels (6.7 mM Tris-Cl, pH 7.9, 1 mM EDTA, 3.3 mM sodium acetate, 2.5% glycerol; acrylamide:bisacrylamide, 80:1). Antibodies against the NF-YA and NF-YB proteins were kindly supplied by Dr. Roberto Mantovani (Universitadi Milano, Italy). The NF-YA antibodies were the monoclonal YA7 made against the complete recombinant NF-YA protein, and the NF-YB antibodies were a polyclonal YB antiserum made in rabbits against the complete recombinant NF-YB protein(12) . The monoclonal antibodies were shown to have lower affinity for NF-Y than the polyclonal antiserum.


RESULTS

The alpha Region of the R2 Gene Promoter Is Required for Continuous Promoter Activity during the S Phase of the Cell Cycle

To investigate the functional role of the alpha region for R2 promoter activity, three R2 promoter-luciferase reporter gene constructs were made. In the first one, most of the alpha region including the CCAAT box was deleted, while the rest of the promoter containing the beta, , and upstream regions was left intact. After electroporation of Balb/3T3 cells, stable transformants were selected, and the presence of the appropriate construct was verified by PCR. Cells synchronized by serum starvation were harvested at different time points after serum readdition and were subjected to luciferase activity measurements and flow cytometry (Fig. 1). There was a rapid increase in luciferase activity when cells passed from quiescence to proliferation much like the earlier observed increase with the intact R2 promoter-luciferase gene constructs p19lucR2 1.0^1 and p19lucR2 1.5(5) . However, the p19lucR2 1.0-alpha reached its maximum already after 5-6 h and then declined to reach near basal levels at 16 h, unlike the intact promoter constructs, where the luciferase activity reached its maximum 12 h after serum readdition and remained almost constant at this level until 16 h. Therefore, the decline in promoter activity started at about the same time as the cell entered the S phase.

To study if the effects of deleting the alpha region were caused by the shortening of the promoter or a result of deleting specific DNA-protein binding areas, we decided to replace the alpha 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 alpha 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 alpha 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-alpha (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 alpha region is required for promoter activity through the S phase but is dispensable for the early proliferation-specific activation.

The alpha Region Is Also Required for Basal Non-proliferation-dependent Transcription from the R2 Promoter

Deletion of the beta, , and upstream regions from an R2 promoter-luciferase reporter gene construct abolished the early, proliferation-specific activation of the promoter. However, a proliferation-independent low basal activity was still detectable.^1 To test if this activity was dependent on the alpha region, an R2 promoter-luciferase reporter gene construct was made containing only the TTTAAA box and the transcription start of the promoter (nt -47 to +17). The presence of the R2 promoter fragment linked to an intact luciferase gene in the stably transformed cells was confirmed by PCR. However, no detectable luciferase activity was observed in synchronized G(0)/G(1) or S phase-enriched cells. Therefore, the alpha region is required for basal promoter activity.

The NF-Y Transcription Factor Binds to the alpha Region within the Mouse R2 Gene Promoter

Our functional studies of the alpha region of the R2 promoter strongly underlined the importance of the CCAAT motif for promoter activity. Gel shift analyses using a crude nuclear extract from Balb/3T3 cells and an oligonucleotide representing the alpha region showed, as demonstrated by Björklund et al.,^1 one major retarded DNA-protein complex (Fig. 4, lane2). Pre-incubation of the nuclear extract with antibodies against the two non-identical subunits of the CCAAT binding NF-Y transcription factor A and B clearly affected the DNA-protein complex (Fig. 4, lanes3 and 4). The anti-NF-YA monoclonal antibodies super shifted the complex (Fig. 4, lane3), while the polyclonal anti NF-YB serum completely inhibited its formation (Fig. 4, lane4). This pattern is similar to that reported by Mantovani et al.(12) and may reflect a different specificity of the antibodies. The results positively identify that NF-Y binds to the alpha region.


Figure 4: Gel electrophoresis mobility shift assays of the alpha footprint region of mouse R2 gene promoter. Nuclear extracts from G(0)/G(1) 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 alpha (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 alpha 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 alpha 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 alpha region is sufficient for binding the NF-Y transcription factor, and no other transcription factor is binding directly to the alpha region DNA.


DISCUSSION

The R2 promoter-reporter gene constructs clearly demonstrate the importance of the alpha region for S phase-specific expression of the R2 gene. In the absence of the alpha 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(1) transcriptional block. Protein binding to the alpha 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 alpha region DNA. The fact that the anti-NF-YB antibodies completely inhibit the formation of any DNA-protein complex using the alpha oligonucleotide and a nuclear extract strongly indicates that NF-Y really binds to the alpha 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 beta footprint of the mouse R1 gene promoter (15) or to a CCAAT motif present in an inverted orientation in the beta 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 beta, , 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 beta, , 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 alpha 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 beta-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)(6)GG-3` and the R2 promoter beta, , 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.


FOOTNOTES

*
This work was supported by grants from the Swedish Natural Science Research Council and Lion's Cancer Research Foundation, Umea University, S-901 87 Umea, Sweden. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institute for Molecular Biology and Biotechnology, Cancer Research Group, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada.

(^1)
S. Björklund, D. Filatov, E. Skogman, M. Thelander, and L. Thelander, manuscript in preparation.

(^2)
The abbreviations used are: nt, nucleotide; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. S. Skog and Dr. B. Tribukait for the flow cytometric analyses. We also thank Dr. Roberto Mantovani for the antibodies against NF-Y.


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