BMC Genomics 2009, 10:576 http://www.biomedcentral.com/1471-2164/10/576 Table 1: Dipeptide frequency in olfactory receptor proteins* Dipeptide All proteins Olfactory (Observed) Olfactory (Expected) Frequency Z-score *Only the 21/49 YY1 core forming dipeptides where observed count in olfactory receptor genes is greater than expected are shown. PL AI TI SI PM LH MA SH YG AM PF MV SM TM MG PI NH IH SW MD CH 77943 41587 35087 48732 15764 42086 30513 34455 25865 22983 29419 22565 26009 17689 19991 28078 14111 21733 17376 18778 10586 3222 2784 2444 2110 1971 1961 1817 1798 1130 1120 1065 939 926 786 746 689 661 609 478 475 330 contain the actual core motif of the YY1 binding consen- sus sequence. Therefore, the detection of 4 to 8 YY1 bind- ing motifs per one OLFR gene cannot be simply accounted for by the serendipitous overlap between the YY1 core motif and a subset of codon combinations encoding fre- quent peptide motifs of the OLFR genes. As a second measure, we carefully analyzed the positions of the identified YY1 binding motifs within the ORFs of the OLFR genes (Figure 5). Conservation in location of YY1 binding site would indicate conservation of the encoded dipeptide motif whereas a lack of conservation in motif location is consistent with selection for the presence of the motif at the DNA level. The full length YY1 binding motifs within the OLFR genes do not show any patterns in their relative positions to the protein sequences of the OLFR genes as would be detected by conserved dipeptide motifs. However, there is a bias in the orientation YY1 binding sites in OLFR genes. OLFR genes contain an aver- age of 3.4 forward sites for every reverse site per 1 kilo- base. Non-OLFR genes show only a slight bias of 1.2 reverse sites for every forward site per 1 kilobase. In con- trast, a similar analysis on the members of the histone 4 gene family (Hist1 h4) resulted in a different outcome. Two potential YY1 binding motifs were found within the protein-coding regions of this gene family, but the relative positions and orientations of these two motifs are identi- cal and fixed among all the members of this gene family in mouse. The two identified YY1 binding motifs also 1894 1010 852 1184 383 1022 741 837 628 558 715 548 632 430 486 682 343 528 422 456 257 1.70 2.76 2.87 1.78 5.15 1.92 2.45 2.15 1.80 2.01 1.49 1.71 1.47 1.83 1.54 1.01 1.93 1.15 1.13 1.04 1.28 3.72 3.01 2.46 1.92 1.69 1.68 1.44 1.41 0.33 0.31 0.23 0.02 0.00 -0.23 -0.29 -0.38 -0.43 -0.51 -0.73 -0.73 -0.97 coincide with two conserved peptide motifs (AM, and IA). Given the high levels of sequence conservation detected within the members of the histone 4 family (95% sequence identity), it is uncertain whether the identified YY1 binding motifs are genuine cis-regulatory elements or simply reflecting the serendipitous sequence match between the YY1 core motifs and some combinations of codons encoding conserved peptides. This pattern is in stark contrast to the random patterns associated with the position of YY1 binding motifs in the OLFR genes. This random pattern supports our initial idea that the clusters of the YY1 binding motifs within the OLFR genes most likely have been formed and maintained as cis-regulatory elements by purifying selection at the DNA sequence level. The bias favoring forward orientation may be an indication that forward sites enhance the efficiency of YY1 suppression of olfactory receptor genes. It is conceivable that multiple YY1 binding sites have been selected in OLFR genes with a preference for directionality as well as number. Alternatively, the non-random placement and orientation in histone 4 genes along with their extreme conservation prohibits a determination of whether selec- tion is occurring at the DNA level or the protein level. Discussion In the current study, we have shown an unusual enrich- ment of YY1 DNA-binding motifs in the OLFR gene fam- ily of mammals. About half of the members of the OLFR gene family have a range of 4-8 YY1 binding motifs within Page 5 of 10 (page number not for citation purposes)