NF-Y Dependent Epigenetic Modifications Discriminate between Proliferating and Postmitotic Tissue

Abstract

The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferation and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its effect on transcription through the modulation of the histone “code”. NF-Y colocalizes with nascent RNA, while RNA polymerase II is I phosphorylated on serine 2 of the YSPTSPS repeats within its carboxyterminal domain and histones are carrying modifications that represent activation signals of gene expression (H3K9ac and PAN-H4ac). Comparing postmitotic muscle tissue from normal mice and proliferating muscles from mdx mice, we demonstrate by chromatin immunoprecipitation (ChIP) that NF-Y DNA binding activity correlates with the accumulation of acetylated histones H3 and H4 on promoters of key cell cycle regulatory genes, and with their active transcription. Accordingly, p300 is recruited onto the chromatin of NF-Y target genes in a NF-Y-dependent manner, as demonstrated by Re-ChIP. Conversely, the loss of NF-Y binding correlates with a decrease of acetylated histones, the recruitment of HDAC1, and a repressed heterochromatic state with enrichment of histones carrying modifications known to mediate silencing of gene expression (H3K9me3, H3K27me2 and H4K20me3). As a consequence, NF-Y target genes are downregulated in this context. In conclusion, our data indicate a role of NF-Y in modulating the structure and transcriptional competence of chromatin in vivo and support a model in which NF-Y-dependent histone “code” changes contribute to the proper discrimination between proliferating and postmitotic cells in vivo and in vitro.

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.…”

“…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.…”

“…To further support the hypothesis that a stable NF-YA protein could influence the expression of NF-Y target genes, we followed the expression of cyclin B1, cyclin A, and cdk1 (Farina et al, 1999;Manni et al, 2001;Gurtner et al, 2003Gurtner et al, , 2008Imbriano et al, 2005;Di Agostino et al, 2006) after overexpression of wild-type NF-YA or mutant YA-R3 protein. As shown in Figure 11, in cells transfected with wildtype NF-YA, expression of these genes declined paralleling NF-YA decrease.…”

“…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).…”