Requirement for Down-Regulation of the CCAAT-binding Activity of the NF-Y Transcription Factor during Skeletal Muscle Differentiation

Abstract

NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC, all required for DNA binding. All subunits are expressed in proliferating skeletal muscle cells, whereas NF-YA alone is undetectable in terminally differentiated cells in vitro. By immunohistochemistry, we show that the NF-YA protein is not expressed in the nuclei of skeletal and cardiac muscle cells in vivo. By chromatin immunoprecipitation experiments, we demonstrate herein that NF-Y does not bind to the CCAAT boxes of target promoters in differentiated muscle cells. Consistent with this, the activity of these promoters is down-regulated in differentiated muscle cells. Finally, forced expression of the NF-YA protein in cells committed to differentiate leads to an impairment in the down-regulation of cyclin A, cyclin B1, and cdk1 expression and is accompanied by a delay in myogenin expression. Thus, our results indicate that the suppression of NF-Y function is of crucial importance for the inhibition of several cell cycle genes and the induction of the early muscle-specific program in postmitotic muscle cells.

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.…”

“…As shown in Figure 4A, the FK2 antibody recognized a smeared ladder in the anti-NF-YA immunoprecipitates. This antibody does not recognize the 43-kDa NF-YA protein that correspond to the posttranslational unmodified form (Farina et al, 1999;Gurtner et al, 2003). A similar ladder and unmodified NF-YA protein were recognized by anti-NF-YA antibody ( Figure 4B).…”

“…After treatment, the NF-YA protein accumulated both in the cytoplasm and in the nucleus, whereas the protein levels of NF-YB and -YC were only slightly modulated. We have previously demonstrated that in terminally differentiated C2C12 muscle cells, myotubes, NF-YA expression is abrogated (Farina et al, 1999;Gurtner et al, 2003Gurtner et al, , 2008. Thus, we asked whether regulation of NF-YA expression occurs at posttranslational level in this cell system.…”

“…These results support the hypothesis that acetylated NF-YA protein, acting as a transcription factor in the nucleus, is the active form, whereas the nonactive ubiquitylated form is prevalently stored in the cytoplasm. If this hypothesis is correct, we would expect that in postmitotic cells, where we have previously demonstrated absence of NF-Y activity (Farina et al, 1999;Gurtner et al, 2003;Gurtner et al, 2008), only ubiquitylated NF-YA species are expressed. As already shown in Figure 1A, myotubes express NF-YA only after treatment with LLnL.…”

“…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).…”