Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex

Abstract

The observation that cyclin B1 protein and mRNAs are down-regulated in terminally dierentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-o of cyclin B1 promoter activity in dierentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation.

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

“…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). The NF-YA protein is not expressed in terminally differentiated C2C12 muscle cells and adult skeletal muscle tissues (Farina et al, 1999;Gurtner et al, 2003Gurtner et al, , 2008. These observations show that levels of NF-YA dictate the function of the NF-Y complex.…”

“…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.…”

Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression

“…In supershift experiments were used 100 ng of anti-Max rabbit polyclonal antibody, 2 mg of anti-Mxi1 rabbit polyclonal antibody, and 2 mg of anti-Mad2 goat polyclonal antibody (all provided by Santa Cruz Biotechnology). The following oligonucleotides were used as probe and competitor (consensus sites are underlined): B1 E-box 5'-GGGAGGCAGACCACGTGA-GAGCCTGG; B1up CCAAT: 5'-CCGCAGCCGCCAATGGGAAG-GGAGTGA (Farina et al, 1999).…”

Genome wide study of NF-Y type CCAAT boxes in unidirectional and bidirectional promoters in human and mouse

“…The boxes appear to recruit histone acetylase complexes such as p300 Salsi et al, 2003) and other TFs (Roder et al, 1999;Imbriano et al, 2005). Defects or malfunctioning of these CCAAT boxes have been linked to several diseases, including some forms of cancer (Farina et al, 1999;Pabst et al, 2001).…”