The cyclin B2 promoter depends on NF-Y, a trimer whose CCAAT-binding activity is cell-cycle regulated

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 dierent phases of the cycle, we separated MEL cells by elutriation, obtaining fractions 480% 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 con®rmed 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 ®ne tuning of this activator is important for phase-speci®c transcription of CCAATcontaining promoters.

Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

“…In transfection assays, pcDNA3-p300 (gift from M. Levrero, University of Rome La Sapienza, Rome, Italy) was used. In cotransfection assays, pCCAAT-B2LUC and pmutCCAAT-B2LUC (Bolognese et al, 1999) were used. LUC activity was assayed on whole cell extract, as described (Brasier et al, 1989).…”

“…The NF-Y complex supports the basal transcription of a class of regulatory genes responsible for cell cycle progression, among which are mitotic cyclin complexes (Zwicker et al, 1995a,b;Bolognese et al, 1999;Farina et al, 1999;Korner et al, 2001;Gurtner et al, 2003Gurtner et al, , 2008Di Agostino et al, 2006). Knock-out mice clearly demonstrates that NF-Y dependent transcription is essential during early mouse development (Bhattacharya et al, 2003).…”

“…These observations show that levels of NF-YA dictate the function of the NF-Y complex. Interestingly, control of NF-YA accumulation is posttranscriptional, because NF-YA mRNA is relatively constant in growing and differentiated cells (Bolognese et al, 1999;Farina et al, 1999). Yet, the mechanisms regulating the stability of the NF-YA protein remain unknown.…”

“…The differential expression of NF-YA, altering NF-Y CCAAT-binding activity, has been observed in several cell lines and tissues both during cell cycle progression and under specific conditions. NF-YA expression is modulated during the cell cycle, being high in G1, further increasing in S, and then decreasing in the G2/M phase (Bolognese et al, 1999). Reduction of NF-YA expression has been reported in IMR-90 fibroblasts after serum deprivation (Chang et al, 1994) and in human monocytes (Marziali et al, 1997).…”

Gene expression of cyclin‐dependent kinase subunit Cks2 is repressed by the tumor suppressor p53 but not by the related proteins p63 or p73

“…The wild-type and mutant expression constructs of p73a, p73b, TAp63a, TAp63c, TA * p63a, TA * p63c, DNp63a and DNp63c have been described previously [17]. The plasmid YA13m29 expresses a dominant-negative mutant of transcription factor subunit NF-YA and has been described previously [18]. The control plasmid pRL-null (Promega) contains a cDNA coding for Renilla luciferase.…”