BMC Genomics 2009, 10:576 http://www.biomedcentral.com/1471-2164/10/576 lian genomes [10-13]. The aquatic vertebrates, the teleost fish lineage, also have a similar odorant receptor gene family [14]. However, the odorant receptor (OR) family of the fish lineage consists of a much smaller number of genes than that of the mammals, and these OR genes are also much more diverse in sequence identity than those of mammals. Mammalian OLFR genes are divided into Class I and Class II groups based on sequence identity [15]. Class II genes make up ~90% of OLFRs and are thought to have expanded during the transition to land-based living. In mammals these olfactory receptors presumably expanded due to the selective advantage conferred by a well developed sense of smell [15]. While mice and other mammals retain function and expression of almost all OLFRs, the majority of these are pseudogenized in humans [16]. The mammalian OLFR genes are highly tis- sue-specific and are expressed primarily in the olfactory epithelium though a subset expresses in a chemosensory role in other tissues such as kidney and sperm [17-19]. Furthermore, only one copy (allele) out of all 1,000 OLFR genes is selected and expressed in each neuron cell of the olfactory tissue [20]. The unusual transcriptional control of the OLFR gene family is likely mediated through unknown trans-acting factors [21]. The tissue-specific nature of their expression coupled with their widespread duplication requires a mitotically-stable global silencing mechanism in all cell types. According to recent studies, potential cis-regulatory elements recruiting these trans fac- tors are hypothesized to be located within the protein- coding regions of the OLFR genes rather than their sur- rounding genomic regions [22,23]. While performing genome-wide searches of the DNA- binding motifs of YY1, we discovered that the mamma- lian OLFR genes contain unusual clusters of YY1 binding sites within their protein-coding regions, whereas most YY1 binding sites are solitary and upstream of a regulated gene. In this study, we further analyzed the significance of this discovery with several bioinformatic and statistical measures, which will be described below. Specifically we test whether the presence of the YY1 binding sites could be explained by DNA sequence or amino acid motif conser- vation. Results YY1 DNA-binding motifs in the mammalian OLFR genes YY1 is predicted to be a global epigenetic regulator based on its ubiquitous expression and interaction with many histone-modifying enzymes [4]. As part of the efforts exploring this possibility, we have performed several series of YY1 binding motif searches using the genome sequences of mammals (human, mouse, and cow) [24- 26]. Here we first scanned the genome sequences of human and mouse using a Position Weight Matrix (PWM)-based Perl script. Repetitive elements are known to contain YY1 binding sites so the RepeatMasked genome was used [27]. Later, the results of these searches were vis- ualized using the repeat-masked Custom Track of the UCSC genome browser [28]. While inspecting global localization patterns of YY1 binding motifs in each genome, we noticed that clusters of YY1 binding motifs are co-localized with the genomic regions harboring olfac- tory receptor (OLFR) genes. The mammalian OLFR genes show a single coding exon structure, and they are also localized as gene clusters in specific regions of mamma- lian chromosomes. One such example is shown using the 100-kb genomic region from Mmu 7 chromosome (Fig- ure 1). This figure shows a representative sample of 5 OLFR genes, and the locations of these genes coincide with those of YY1 binding motifs. Each OLFR gene appears to contain a range of 4 to 8 YY1 binding motifs within its 1-kb-long Open Reading Frame (ORF). As expected, the coding regions correlate well to the placental mammal conservation plot, a default track available on the UCSC genome browser. Also, the identified YY1 bind- ing motifs appear to be random in location within the coding regions but do show a bias in orientation with respect to the direction of OLFR gene transcription, which will be described later. The unusual clustering of YY1 binding motifs within the protein-coding regions of the OLFR genes was further ana- lyzed to test if this pattern is unique to only the OLFR genes or also found in the ORFs of other genes. For this analysis, the entire set of the mouse mRNA database was scanned with the PWM-based Perl program to identify YY1 binding motifs. The number of the identified YY1 binding motifs within a given DNA sequence correspond- ing to the transcript was further divided by the size of the mRNA sequence, yielding a YY1 density score. The mouse mRNA sequences (total number = 20,191) were subse- quently binned based on their relative YY1 density scores (the X-axis on Figure 2a). The Y-axes of figure 2 represents the number of genes within a given range of the YY1 den- sity score. This analysis indicated that the majority of the non-olfactory mouse mRNA sequences (19,083 sequences) were distributed evenly and randomly throughout the varying ranges of the YY1 density score (0 to 0.114) (Figure 2a). In contrast, our detailed inspection revealed that about half of the OLFR gene set (1,108) show very high YY1 density scores. To better visualize this unusual pattern, we separated only the OLFR gene set from the rest of the mRNA set, and derived another histo- gram (Figure 2b). As shown in Figure 2b, about 45% (or 496 of 1,108) of the OLFR genes have YY1 density scores ranging from 0.032 to 0.094, which are equivalent to 4 to 8 (or more) YY1 binding sites per a 1-kb mRNA sequence. In contrast, a scan of the 1 kb upstream regions of 20,419 Refseq genes revealed that only 10% of these putative pro- Page 2 of 10 (page number not for citation purposes)