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

Abstract

Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity. INTRODUCTIONThe CCAAT-binding transcription factor NF-Y is a heteromeric protein composed of three subunits, NF-YA, -YB, and -YC, all necessary for CCAAT binding (Mantovani, 1999). 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). The NF-Y function in proliferation is also demonstrated by the inhibition of DNA binding by endogenous NF-Y, resulting in retardation of fibroblast growth, which is brought about by overexpression of a dominant negative mutant of the NF-YA subunit (Hu and Maity, 2000). 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). 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. 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 covalent addition of ubiquitin, a 76-amino acid protein highly conserved among eukaryotes, is an increasingly common posttranslation modification that controls both the expression and the activity of numerous proteins in the eukaryotic cell (Hershko and Ciechanover, 1998;Ciechanover et al., 2000). The ubiquitin-proteasome pathway is composed of the ubiquitin-conjugating system and the 26S proteasome; the latter contain...

Arabidopsis Defense against Botrytis cinerea: Chronology and Regulation Deciphered by High-Resolution Temporal Transcriptomic Analysis

“…During senescence, NF-YA genes also show coordinated expression; however, they are overrepresented for upregulated genes (Breeze et al, 2011). The activity of the NF-Y complex could be regulated by the expression of either A, B, or C subunits; however, the coordinate regulation of NF-YA genes following both B. cinerea infection and during senescence suggests expression of A subunits may be an important control mechanism, as is the case in mammals (Manni et al, 2008).…”

The Short Isoform of NF‐YA Belongs to the Embryonic Stem Cell Transcription Factor Circuitry

“…There are two major drawbacks in conducting these experiments in mESCs: (a) NF-YA half-life upon transfections is short, with the overexpressed protein disappearing within 24 hours [26]; (b) attempts at stable expressions were repeatedly unsuccessful. An alternative, appealing approach was transfecting a recombinant TAT-fusion protein, a strategy used in HSCs by the Emerson's group: a functional GST-TAT-NF-YAs was delivered in the nucleus thanks to the TAT epitope [20].…”

NF-Y affects histone acetylation and H2A.Z deposition in cell cycle promoters

“…53,60 Given the histone-like structure of NF-Y, we are currently working on the hypothesis that regulation is fine tuned by specific NF-Y PTMs, which might be structurally related to those of core histones of the H2A/H2B type. If this is the case, a long genetic and biochemical road lies ahead to study the molecular details.…”