Abstract
Cyclin B2 is a regulator of p34cdc2 kinase, involved in G2/M progression of the cell cycle, whose gene is strictly regulated at the transcriptional level in cycling cells. The mouse promoter was cloned and three conserved CCAAT boxes were found. In this study, we analysed the mechanisms leading to activation of the cyclin B2 CCAAT boxes: a combination of (i) genomic footprinting, (ii) transfections with single, double and triple mutants, (iii) EMSAs with nuclear extracts, antibodies and NF-Y recombinant proteins and (iv) transfections with an NF-YA dominant negative mutant established the positive role of the three CCAAT sequences and proved that NF-Y plays a crucial role in their activation. NF-Y, an ubiquitous trimer containing histone fold subunits, activates several other promoters regulated during the cell cycle. To analyse the levels of NF-Y subunits in the different phases of the cycle we separated MEL cells by elutriation, obtaining fractions > 80% pure. The mRNA and protein levels of the histone-fold containing NF-YB and NF-YC were invariant, whereas the NF-YA protein, but not its mRNA, was maximal in mid-S and decreased in G2/M. EMSA confirmed that the CCAAT-binding activity followed the amount of NF-YA, indicating that this subunit is limiting within the NF-Y complex, and suggesting that post-transcriptional mechanisms regulate NF-YA levels. Our results support a model whereby fine tuning of this activator is important for phase-specific transcription of CCAAT-containing promoters.
| Original language | English |
|---|---|
| Pages (from-to) | 1845-1853 |
| Number of pages | 9 |
| Journal | Oncogene |
| Volume | 18 |
| Issue number | 10 |
| Publication status | Published - Mar 11 1999 |
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Keywords
- Cell cycle
- Cyclin B2
- NF-Y
ASJC Scopus subject areas
- Molecular Biology
- Cancer Research
- Genetics
Cite this
The cyclin B2 promoter depends on NF-Y, a trimer whose CCAAT-binding activity is cell-cycle regulated. / Bolognese, Fabrizio; Wasner, Mark; Dohna, Christine Lange Zu; Gurtner, Aymone; Ronchi, Antonella; Muller, Heiko; Manni, Isabella; Mossner, Joachim; Piaggio, Giulia; Mantovani, Roberto; Engeland, Kurt.
In: Oncogene, Vol. 18, No. 10, 11.03.1999, p. 1845-1853.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The cyclin B2 promoter depends on NF-Y, a trimer whose CCAAT-binding activity is cell-cycle regulated
AU - Bolognese, Fabrizio
AU - Wasner, Mark
AU - Dohna, Christine Lange Zu
AU - Gurtner, Aymone
AU - Ronchi, Antonella
AU - Muller, Heiko
AU - Manni, Isabella
AU - Mossner, Joachim
AU - Piaggio, Giulia
AU - Mantovani, Roberto
AU - Engeland, Kurt
PY - 1999/3/11
Y1 - 1999/3/11
N2 - Cyclin B2 is a regulator of p34cdc2 kinase, involved in G2/M progression of the cell cycle, whose gene is strictly regulated at the transcriptional level in cycling cells. The mouse promoter was cloned and three conserved CCAAT boxes were found. In this study, we analysed the mechanisms leading to activation of the cyclin B2 CCAAT boxes: a combination of (i) genomic footprinting, (ii) transfections with single, double and triple mutants, (iii) EMSAs with nuclear extracts, antibodies and NF-Y recombinant proteins and (iv) transfections with an NF-YA dominant negative mutant established the positive role of the three CCAAT sequences and proved that NF-Y plays a crucial role in their activation. NF-Y, an ubiquitous trimer containing histone fold subunits, activates several other promoters regulated during the cell cycle. To analyse the levels of NF-Y subunits in the different phases of the cycle we separated MEL cells by elutriation, obtaining fractions > 80% pure. The mRNA and protein levels of the histone-fold containing NF-YB and NF-YC were invariant, whereas the NF-YA protein, but not its mRNA, was maximal in mid-S and decreased in G2/M. EMSA confirmed that the CCAAT-binding activity followed the amount of NF-YA, indicating that this subunit is limiting within the NF-Y complex, and suggesting that post-transcriptional mechanisms regulate NF-YA levels. Our results support a model whereby fine tuning of this activator is important for phase-specific transcription of CCAAT-containing promoters.
AB - Cyclin B2 is a regulator of p34cdc2 kinase, involved in G2/M progression of the cell cycle, whose gene is strictly regulated at the transcriptional level in cycling cells. The mouse promoter was cloned and three conserved CCAAT boxes were found. In this study, we analysed the mechanisms leading to activation of the cyclin B2 CCAAT boxes: a combination of (i) genomic footprinting, (ii) transfections with single, double and triple mutants, (iii) EMSAs with nuclear extracts, antibodies and NF-Y recombinant proteins and (iv) transfections with an NF-YA dominant negative mutant established the positive role of the three CCAAT sequences and proved that NF-Y plays a crucial role in their activation. NF-Y, an ubiquitous trimer containing histone fold subunits, activates several other promoters regulated during the cell cycle. To analyse the levels of NF-Y subunits in the different phases of the cycle we separated MEL cells by elutriation, obtaining fractions > 80% pure. The mRNA and protein levels of the histone-fold containing NF-YB and NF-YC were invariant, whereas the NF-YA protein, but not its mRNA, was maximal in mid-S and decreased in G2/M. EMSA confirmed that the CCAAT-binding activity followed the amount of NF-YA, indicating that this subunit is limiting within the NF-Y complex, and suggesting that post-transcriptional mechanisms regulate NF-YA levels. Our results support a model whereby fine tuning of this activator is important for phase-specific transcription of CCAAT-containing promoters.
KW - Cell cycle
KW - Cyclin B2
KW - NF-Y
UR - http://www.scopus.com/inward/record.url?scp=0033545567&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033545567&partnerID=8YFLogxK
M3 - Article
C2 - 10086339
AN - SCOPUS:0033545567
VL - 18
SP - 1845
EP - 1853
JO - Oncogene
JF - Oncogene
SN - 0950-9232
IS - 10
ER -