BMC Genomics 2009, 10:576 http://www.biomedcentral.com/1471-2164/10/576 Mouse chromosome 7 100 kb YFYig1ubreind1ing sites in genomic context YY1 binding sites in genomic context. YY1 binding sites are mapped onto the Custom Track of the UCSC Genome Browser http://genome.ucsc.edu/. YY1 binding sites are marked by blue and red bars indicating forward and reverse directions respectively. Height on the Y-axis represents the score of each YY1 binding site in relation to the consensus sequence. This 100 kb segment of the genome contains five olfactory receptor genes and shows association of several YY1 binding sites corre- lated with the exons of the genes. Mammalian conservation is shown in the plot at the bottom of the map. This map was cre- ated through scanning the RepeatMasked sequence with a scoring matrix representing the YY1 binding site consensus. Mammalian conservation correlates with exons as expected, but also with many YY1 binding sites in this region. moter containing regions had a density greater than 4 YY1 binding sites. that the unusual clustering of YY1 binding motifs within the OLFR genes is a feature of the mammalian lineage. We also repeated the above analyses using the mRNA data sets derived from human, cow, and zebrafish transcrip- tomes to test the evolutionary conservation of this unu- sual clustering of YY1 binding motifs within OLFR genes (Figure 2c &2d). The results from human and cow (data not shown) mRNA data sets also showed a pattern con- sistent with the mouse data set: an unusual clustering of YY1 binding motifs within many OLFR genes. In humans we found 57% (or 215 of 380) of OLFRs contain more than 4 YY1 binding sites while percentage in cow rose to 82% (or 740 of 900). It is important to note that the total number of the human OLFR genes (380) in the figure is smaller than those of the other mammals since a large fraction of the human OLFR genes are known to have become pseudogenes in recent evolutionary times and we removed all genes annotated as hypothetical. In contrast, the OLFR genes of zebrafish do not show a similar pattern to mammals. The total 25 OLFR genes of zebrafish show as wide a range of the YY1 density scores as seen in the other non-OLFR genes (Figure 2d). These results confirm Statistical tests for the clustering of YY1 binding motifs within OLFR genes We performed two different series of analyses to test the functional significance of the observed clustering of YY1 binding motifs within the mammalian OLFR genes. First, we tested whether some peptide motifs enriched within the protein sequences of the OLFR genes are responsible for a spurious display of the YY1 binding motifs in the nucleotide sequences of the OLFR genes. If some peptide motifs are enriched then we expect a significant difference in the number of those motifs found in OLFR genes versus the rest of the proteome. For this test, we identified 49 individual dipeptides that can be encoded by the core motifs (CCAT or ATGG) of the YY1 binding consensus sequence, and determined if any of these dipeptides were over-represented in the protein sequences of the OLFR genes (Figure 3). We first calculated the frequencies of all 400 possible combinations of dipeptides using all other protein sequences except OLFR genes of the mouse to derive a reference set of the expected frequencies for the Page 3 of 10 (page number not for citation purposes)