Cell cycle regulation of NF-YC nuclear localization

NF-Y is a trimeric activator with histone fold, HFM, subunits that binds to the CCAAT-box and is required for a majority of cell cycle promoters, often in conjunction with E2Fs. In vivo binding of NF-Y is dynamic during the cell cycle and correlates with gene activation. We performed immunofluorescence studies on endogenous, GFP- and Flag-tagged overexpressed NF-Y subunits. NF-YA, NF-YB are nuclear proteins. Unexpectedly, NF-YC localizes both in cytoplamatic and nuclear compartments and its nuclear localization is determined by the interaction with its heterodimerization partner NF-YB. Most importantly, compartmentalization is regulated during the cell cycle of serum restimulated NIH3T3 cells, accumulating in the nucleus at the onset of S phase. These data point to the control of HFM heterodimerization as an important layer of NF-Y regulation during cell cycle progression.     

NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.     

Complex interference in the eye developmental pathway by Drosophila NF-YA

The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB, and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. NF-YA contains a DNA binding domain in its C-terminal region. We established transgenic fly lines carrying the UAS-HA-dNF-YA or UAS-dNF-YAIR and showed over-expression or knockdown with various GAL4 drivers to be lethal at various developmental stages, suggesting that dNF-YA participate in various gene regulatory pathways during Drosophila development. Expression of dNF-YA with eyeless-GAL4 mainly resulted in lethality with a headless phenotype in pharate-adults. Reduction of the eyeless gene dose enhanced the dNF-YA-induced phenotype, while reduction of the Distal-less gene dose suppressed the phenotype. On the other hand, crossing the dNF-YA over-expressing flies with Notch mutant resulted in no apparent effect on the phenotype. These results suggest that dNF-YA can disturb eye disc specification, but not eye disc growth.     

Asymmetric evolution of duplicate genes encoding the CCAAT-binding factor NF-Y in plant genomes

NF-Y is a ubiquitous CCAAT-binding factor composed of NF-YA, NF-YB and NF-YC. Multiple genes encoding NF-Y subunits have been identified in plant genomes. It remains unclear whether the duplicate genes underwent different evolutionary patterns. Likelihood-ratio tests were used to examine whether the amino acid substitution rates are the same between duplicate genes. The influences of selection on evolution were evaluated by comparing the conservative and radical amino acid substitution rates, as well as maximum-likelihood analysis. Some NF-YB and NF-YC duplicates showed significant evidence of asymmetric evolution but not the NF-YA duplicates. Most amino acid replacements in the NF-YB and NF-YC duplicates result in changes in hydropathy, polar requirement and polarity. The physicochemical changes in the sequences of NF-YB seem to be coupled to asymmetric divergence in gene function. Plant NF-Y genes have evolved in different patterns. Relaxed selective constraints following gene duplication are most likely responsible for the unequal evolutionary rates and distinct divergence patterns of duplicate NF-Y genes. Positive selection may have promoted amino acid hydropathy changes in the NF-YC duplicates.     

Requirement for down-regulation of the CCAAT-binding activity of the NF-Y transcription factor during skeletal muscle differentiation

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.     

Recombinant AtNF-YA, a subunit of theArabidopsis CCAAT-binding transcription factor, forms a protein-DNA complex with mammalian NF-YB and NF-YC

The evolutionary conserved CCAAT binding protein NF-Y is a common regulatory DNA binding protein consisting of three distinct subunits. Unlike yeast and mammals, in which only a single copy of each subunit is encoded,Arabidopsis encodes a multi-gene family for each subunit in its genome. Compared with the NF-Y of mammals or yeast, very little is known about plant NF-Y homologs. HereArabidopsis NF-YA subunits were isolated to determine whether they could form a hete-rotrimeric NF-Y complex with mammalian NF-YB and NF-YC. This resultant chimeric NF-Y complex had DNA binding ability to the same CCAAT sequences as those of the other life systems. Therefore, it is possible that plant NF-Y homologs might have biochemical characteristics similar to mammalian NF-Y, thereby suggesting its functional conservation among organisms.