The chromatin of eukaryotic genomes is compacted into several levels. Nucleosomes, which form the lowest level of compaction, are made up of ~147 bp of DNA wrapped around a histone protein complex and interspersed by ~50 bp of exposed linker DNA. In recent years, the occupancy of nucleosome positions in yeasts has been investigated by using different approaches (such as tiling arrays and parallel sequencing), which employs micrococcal nuclease (MNase) digestion [1–3]. The results show that about 70-80% of the yeast genome is occupied by nucleosomes [4–6]. The intrinsic mechanisms that determine the nucleosome locations have long been of interest to researchers. Studies of budding yeast have discovered dinucleotides (AA/TT/AT) periodicity along nucleosome positioning sequences [7, 8]; and that nucleosome depleted regions (NDRs) are characterized by positioned stretches of poly (dA:dT) tracts [9, 10]. In addition, a number of patterns of nucleosome occupancy have been observed. For example, a ~140 bp NDR is often found upstream of the transcription start site flanked by -1 and +1 nucleosomes, with the +1 nucleosome located ~13 bp downstream from the transcription start site [11, 12]. It has also been found that, near the 5' end of genes, a uniform 165 bp spacing of nucleosomes (18 bp linker) extends to as many as nine nucleosomes [5–8, 13–15]. Importantly, many of these features are evolutionary conserved [7, 16].
It is known that the transcription mechanism in eukaryotes functions at different levels, e.g. at the DNA sequence level, transcription factors interact with cis-regulatory sequences; and at the chromatin level, where the chromatin allows the chromosomal segments to switch between activated state and suppressed states of transcription [17, 18]. The interplay of changes in nucleosome occupancy and transcriptional machinery at each level suggests a strong association between nucleosome positioning and transcription mechanism [19, 20]. For example, TATA-less promoters, which are characterized by NDRs, are frequently linked to basal transcription. Conversely, the promoters of TATA-containing genes tend to be occupied by nucleosomes and are stress responsive [13, 21, 22]. Moreover, it has been demonstrated that nucleosomes could facilitate the recognition of transcription factor binding sites (TFBSs), and guide transcription factors to their target sites in a DNA sequence [22, 23]. As an example, Maffey et al.  characterized the constraints imposed by well positioned nucleosomes on the interaction of androgen receptors with their binding sites, which are located in the proximal promoters of murine probasin genes. The above evidence confirms the importance of the association between nucleosome positioning and transcriptional regulation. Such evidence in turn raises the interesting issue of the role of nucleosomes in constraining evolutionary changes in TFBSs.
Recent studies have identified the evolutionary features related to nucleosome organization in yeasts [9, 25]. For example, it has been found that nucleosome free linker regions have a lower evolution rate than nucleosome occupied regions (NRs) [9, 25]. In an another study, a large-scale comparative genomic analysis of distantly related yeasts found that gene expression divergence is coupled with the evolution of DNA-encoded nucleosome organization . Further, by analyzing the nucleosome position of two closely related yeast species, Tirosh et al.  indicated that the major contribution towards divergence of nucleosome positioning is through mutations in the local sequences (cis-effects). Moreover, the sequences that quantitatively affect nucleosome occupancy were found to evolve under compensatory dynamics while maintaining heterogeneous levels of AT content . Considering the fact that significant fraction of regulatory variation can be attributed to changes in cis-regulatory elements [29–32], understanding the evolutionary process requires the investigation of all the factors that contribute to TFBS evolution . With the availability of the whole genome nucleosome map in yeast species , it is thus desirable to extend existing studies on regulatory regions from an evolutionary perspective while considering the presence of chromatin structure. In this paper, we have attempted a more comprehensive analysis to demonstrate that nucleosome occupancy in yeast promoters plays an important role in the evolutionary changes in TFBSs.
To determine the evolutionary features of TFBSs constrained by nucleosome occupancy, we first investigated the distribution of TFBSs in NRs and NDRs that regulate 1) orthologous genes of Saccharoymyces cerevisiae, Candida glabrata, and Kluyveromyces lactis (Saccharomycetaceae); and 2) those that specifically regulate S. cerevisiae (Saccharomyces specific) genes, which represent young genes. We found that TFBS locations in orthologous genes are dominant in NDRs, but those in Saccharomyces specific genes appear more frequently in NRs. To further validate this evolutionary tendency, we investigated the distribution of TFBSs in NRs and NDRs in duplicate gene pairs of yeast that might have undergone relaxation of selection pressure. Since TFBS variations are due to difference in consensus sequences and nucleotide substitutions can promote diversification of regulatory elements [35, 36], these interesting findings motivated us to estimate the evolution of TFBSs by position-specific evolution rates . The evolution rates of TFBSs were found to be higher at NRs than their depleted counterparts (NDRs). Finally, the impact of TFBS changes on gene expression at NRs and NDRs were evaluated using site-directed mutagenesis of TFBS and real-time PCR analysis. Our findings on the evolutionary events in TFBSs suggest that 1) NRs can accommodate more changes that contribute to the variation in TFBSs, and 2) the selection constraints of NRs and NDRs are different. Future analyses of data across different biological conditions can reflect on the role of variations in TFBSs.