De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation

Nuclear factor Y (NF-Y) is a highly conserved trimeric activator that recognizes with high specificity and affinity the widespread CCAAT box promoter element. We previously cloned the genes of 23 NF-Y genes of Arabidopsis thaliana (Gene 264 (2001) 173). Now that the Arabidopsis genome sequencing project is complete, we present the cloning, alignments and expression profiles of the remaining six genes coding for the three NF-Y subunits. Consistent with our previous reports, most of the new members of the three subunits show a unique tissue-specific pattern, while another AtNF-YC9 is rather ubiquitous.     

Regulation of the CCAAT-Binding NF-Y subunits in Arabidopsis thaliana

NF-Y is a CCAAT-specific binding factor composed of three distinct subunits. In vertebrates and fungi all three subunits are encoded by evolutionary conserved single copy genes. In this report we have cloned twenty-three NF-Y genes in A. thaliana, assessed their mRNA expression levels in a large number of tissues and confirmed that indeed multiple CCAAT-binding activities are present. Alignments of the genes coding for the three NF-Y subunits yield a considerable amount of information concerning the divergence/conservation of protein subdomains and of single residues within the conserved parts. Careful evaluation of mRNA expression levels by sensitive RT-PCR assays provide evidence that all three subunits have members that are ubiquitous and others that are tissue-specific and induced only after the switch to reproductive growth phase, in flowers and siliques.     

Dissection of the NF-Y transcriptional activation potential.

NF-Y is a trimeric CCAAT-binding factor with histone fold subunits (NF-YB/NF-YC) and bipartite activation domains located on NF-YA and NF-YC. We reconstituted the NF-Y activation potential in vivo with GAL4 DBD fusions. In the GAL4-YA configuration, activation requires co-expression of the three subunits; with GAL4-YB and GAL4-YC, transfections of the histone fold partners are sufficient, provided that the Q-rich domain of NF-YC is present. Combinations of mutants indicate that the Q-rich domains of NF-YA and NF-YC are redundant in the trimeric complex. Glutamines 101 and 102 of NF-YA are required for activity. We assayed NF-Y on different promoter targets, containing single or multiple GAL4 sites: whereas on a single site NF-Y is nearly as powerful as VP16, on multiple sites neither synergistic nor additive effects are observed. NF-Y activates TATA and Inr core elements and the overall potency is in the same range as other Q-rich and Pro-rich activation domains. These results represent the first in vivo evidence of subunit interactions studies and further support the hypothesis that NF-Y is a general promoter organizer rather than a brute activator.     

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.     

Expression of the CCAAT-binding factor NF-Y in Caenorhabditis elegans

NF-Y is a conserved trimer with histone-like subunits that binds and activates the common CCAAT promoter element.C.elegansNF-Y genes present two CeNF-YAs, a unique feature in kingdoms other than plants, one CeNF-YB and one CeNF-YC. The expression of both CeNF-YAs is restricted to the gonads and developing embryos, whereas the histone-like CeNF-YB- and CeNF-YC are also present in the pharyngeal bulb, in the neurons of ganglia surrounding the pharynx and in sensory organs of the head. Moreover, in infertile, 12-day-old worms, expression of the three subunits falls dramatically in the gonads. Our data indicate that NF-Y is not ubiquitously expressed.     

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.