**2. The CCAAT** *cis***-element promoter**

Eukaryotic genes contain numerous *cis*-regulatory elements that mediate their induction, repression or basal transcription (Dynan and Tjian, 1985; Myers *et al.*, 1986; Maity and de Crombrugghe, 1998). These regulatory elements can be found in the proximity of transcribed genes, such as the promoter region and/or in distant regions of the genes where they may act as enhancers (de Silvio *et al.*, 1999).

The transcriptional regulation of several eukaryotic genes is coordinated through sequencespecific binding of proteins to the promoter region located upstream of the gene. During evolution, many of these protein-binding sequences, which are found in a wide variety of organisms, have shown a high degree of conservation (Edwards *et al.*, 1998).

The CCAAT box is one of the most common upstream elements, found in approximately 25–30% of eukaryotic promoters (Bucher, 1990; Mantovani, 1998). It is typically located between 60–100 bp upstream of the transcription start site and it can function in direct or in inverted orientations (Dorn *et al.*, 1987b; Bucher, 1990; Edwards *et al.*, 1998; Mantovani, 1998; Stephenson *et al.*, 2007) with possible cooperative interactions between multiple boxes (Tasanen *et al.*, 1992) or other conserved motifs (Muro *et al.*, 1992; Rieping and Schoffl, 1992;

The Evolutionary History of CBF Transcription Factors:

(Matuoka and Yu Chen, 1999).

Gene Duplication of CCAAT – Binding Factors NF-Y in Plants 199

NF-Y binding sites (Bucher, 1990; Testa *et al.*, 2005), and chromatin immunoprecipitation

Suggesting the importance of binding context, NF-Y-regulated gene expression can be tissue specific, developmentally regulated, or constitutive (Maity and de Crombrugghe, 1998; Siefers *et al.*, 2009).The transcriptional activity of NF-Y can be regulated by differential expression, alternative splicing, protein–protein interactions, and cellular redox potential

NF-Y has been shown to be involved in the regulation of some G1/S genes whose expressions are attenuated during the senescence process (Matuoka and Yu Chen, 1999). NF-Y plays a pivotal role in the cell cycle regulation of the mammalian cyclin A, *cdc25C*, and *cdc2* genes, in the S-phase of the cell cycle (Currie, 1998). Additionally, there are a number of genes involved in the cellular response to damage and stress, including the phospholipid hydroperoxide glutathione peroxidase genes (Huang *et al.*, 1999), which are regulated by NF-Y, indicating its pivotal role in the removal of damaging agents from cells (Matuoka and Chen, 2002). Although NF-Y functions basically as a transactivator of gene expression, it is also involved, directly or indirectly, in the downregulation of transcription. For instance, NF-Y binds to the mouse CCAAT box renin enhancer and blocks the binding of positive regulatory elements (Shi *et al.*, 2001). In this case, NF-Y dysfunction would lead to the

NF-Y is composed of three different subunits named NF-YA (also known as HAP-2 or CBF-B), NF-YB (HAP3 or CBF-A), and NF-YC (HAP5 or CBF-C) that interact to form a complex that can bind CCAAT DNA motifs and control the expression of target genes (Figure 1). Each subunit is required for DNA binding, subunit association and transcriptional regulation in both vertebrates and plants (Sinha *et al.*, 1995; Stephenson *et al.*, 2007). Yeast possesses a fourth subunit, called HAP4, which provides a transcriptional activation domain to the complex (Forsburg and Guarente, 1989; Lee *et al.*, 2003). The yeast HAP4 protein is not needed for DNA-binding but contains an acidic domain that is essential to promote transactivation when associated with the HAP2/HAP3/HAP5 complex (Olesen and Guarente, 1990; Serra *et al.*, 1998). In vertebrates, the function of this fourth domain was incorporated into other subunits (Forsburg and Guarente, 1989; Yazawa and Kamada, 2007). Despite the wide cellular distribution and functional variability of NF-Y-regulated genes, most eukaryotic genomes have only one or two genes encoding each NF-Y subunit (Maity and de Crombrugghe, 1998; Riechmann and Ratcliffe, 2000). Fungi and animals, for example, present single genes encoding each protein subunit. Thus, there is minimal combinatorial diversity in the subunit composition of the heterotrimeric NF-Y in these organisms (Siefers *et al.*, 2009). In contrast, the NF-Y complex in vascular plants is generally

NF-Y is the only transcription factor thus far identified for which the interaction of three heterologous subunits creates the DNA binding domain (Maity and de Crombrugghe, 1992; McNabb *et al.*, 1995; Sinha *et al.*, 1995). All three NF-Y subunits are essential for the DNA binding activity and one molecule of each subunit forms the NF-Y-DNA complex (Maity and De Crombrugghe, 1996)**.** Each NF-Y subunit contains a conserved domain with identities greater than 70% across species. This highly conserved domain is located at the *C*terminus of NF-YA; in the central part of NF-YB; and at the *N*-terminus of NF-YC (Li *et al.*,

data have demonstrated additional widespread NF-Y binding in nonpromoter sites.

damage of systems that control blood pressure (Matuoka and Chen, 2002).

encoded by gene families (Riechmann and Ratcliffe, 2000).

**3.1 NF-Y subunits** 

1992).

Edwards *et al.*, 1998). CCAAT boxes are highly conserved within homologous genes across species in terms of position, orientation, and flanking nucleotides (Mantovani, 1998). In addition, the spacing between the CCAAT box and other promoter-specific *cis*-elements is also conserved among species (Dorn *et al.*, 1987a; Chodosh *et al.*, 1988; Maity and de Crombrugghe, 1998).The expression of genes under the control of promoters that contain CCAAT boxes may be ubiquitous or tissue/stage specific, suggesting that the gene expression pattern is also determined by other *cis* and *trans* elements (Stephenson *et al.*, 2007).

In *Sacharomyces cerevisiae*, CCAAT boxes are found in the promoters of cytochrome genes, in genes coding for proteins that are activated by non-fermentable carbon sources (McNabb *et al.*, 1995) and in genes involved in nitrogen metabolism (Dang *et al.*, 1996). In the filamentous fungus *Aspergillus nidulans*, CCAAT boxes are present in genes involved with penicillin biosynthesis (Steidl *et al.*, 1999). In higher eukaryotes, a multitude of promoters contain CCAAT boxes, including those of developmentally controlled and tissue-specific genes (Berry *et al.*, 1992), housekeeping and inducible genes (Roy and Lee, 1995) and cellcycle regulated genes (Mantovani, 1998). In addition, many cell-cycle regulated promoters lack a recognizable TATA-box, but contain more than one CCAAT box in a position close to and sometimes overlapping with the start site of transcription (Zwicker and Muller, 1997).
