BMC Genomics 2006, 7:133 http://www.biomedcentral.com/1471-2164/7/133 Results and discussion The Alu consensus sequences are enriched with Binding Motifs We focused on the eight major Alu subfamilies AluJo, Jb, Sx, Sz, Sc, Sq, Sp and Y [3], whose sequences were down- loaded from RepBase [29]. We refer to the ensemble of sequences that bind a TF as it's Binding Motif (BM). In order to find BMs of a TF, we used its Position Specific Score Matrix (PSSM); 410 PSSMs were downloaded from two databases [30,31]. For each PSSM we set two thresh- old scores, T5 and T1, selected so that the probability to find a target BM with score higher than T5 in a suitably chosen random sequence (see Methods) is less than 0.05 (0.01 for T1). Furthermore, for each BM we identified Tmax, the maximal score observed on all the above listed Alu subfamilies' consensus sequences. For 66 BMs Tmax was higher than T5 and for 25 TFs – higher than T1 (see Table 1). A good measure for the statistical significance of enrichment of Alu with BMs is the number of BMs whose score exceeds T5; for example, we found on AluSx 35 BMs with scores higher than their respective T5. The probabil- ity to find this many BMs in a random model (i.e. p-value) is 0.023 (see Methods). In order to control the problem of multiple testing we per- formed a false discovery rate (FDR) analysis at 20% FDR[32]. The 14 BMs that passed the FDR test with Q = 0.20 in AluSx are listed in Table 1, where we also compare the IUPAC consensus sequences of these BMs [33] with the target sequence that had the highest score in all the major Alu subfamilies. Most sequences with scores higher than T1 differ at not more than two nucleotides from the IUPAC consensus (see Table 1 and Additional data file 1), and there are several PSSMs, such as PITX2_Q2, LYF-1.01, PAX4.01, SIX3.01, DELTAEF.01 and NKX25.01 (Table 1, and Additional data file 1), whose IUPAC consensus sequences agree with the highest-scoring target sites on at least one of the Alu consensus sequences. Table 1: Twenty five binding motifs whose scores pass T1. Those that pass FDR of Q = 0.20 on at least one major Alu subfamily sequence are in bold. The consensus sites of the PSSMs (from the IUPAC convention [33]) is in the second column and the target sequences with the highest scores among all the major Alu consensus sequences – in the third, with nucleotides that agree with the consensus denoted by capital letters. The fourth column contains the locations of the putative target sites on the Alu sequences of the subfamily with the highest score (in case the same sequence appears on several Alu elements, we choose the one with the highest number of copies in the 5 kb upstream regions). Two Alu subsequences serve as putative target sites of several TFs (designated by*, and **). The fifth column contains the p-values (see text and methods) and the number of subfamilies on which the BSs of the third column resides is listed in column 6. BM name Consensus V$OTX2.01 NNGGATTAANNN V$GSC.01 ANGRGATTASMN V$GFI1.01 NNAAATCNNAGNN V$PITX2_Q2 WNTAATCCCAR V$LUN1_01 TCCCAGCTACTTTGGGA V$LYF1.01 TTTGGGARR V$LXRE.01 NGNTYACTNNMGKTCA V$SREBP.03 NATCACGTGAN V$RORA1.01 NNWWNNAGGTCAN V$NKX25.01 TYAAGTG V$HNF4.02 NNGGNCNAAAGNTCN V$ER.02 NNAGGTCANNGTGACCT V$CHREBP _MLX_01 CAYGNGNNANNSNNGTG V$AARE.01 RTTKCATCA V$MEF2.04 TGTTACTAAAAATAGAAM V$MEF2.03 NNKWKCTAWAAATAGMNN V$MEF2.02 NNNCTAWAAATAGMNN V$RSRFC4.01 NWKCTAWAAATAGMNN V$RSRFC4.02 ANKCTAWAAATAGMWNN V$OC2.01 NNANANAATCMANANNT V$BRN2.03 NNMTWNATTWNMWTN V$HNF1.01 GGTTAATNWTTAMM V$HNF1.03 GGTTAATNWTTRNC V$PAX4.01 NAMWAATTASS V$IRF1.01 SNAAAGYGAAACY Binding site TGGGATTAcAGG cTGGGATTACAG TGtAATCCCAGCA TGTAATCCCAG TCCCAGCactTTgGGa TTTGGGAGG GGATCACcTGAGGTCA GATCACcTGAG ATcACGAGGTCAA TCAAGTG GAGGTCAAgAGATCG GTtGGTCAGGcTGgtCT CACGGtGAAACCCCGTc GTTTCAcCA ctcTACTAAAAATAcAAA CTcTaCTAAAAATAcAAA CTACTAAAAATAcAAA CTaCTAAAAATAcAAA cTaCTAAAAATAcAAAA ACAAAAAATaCAAAAAT ACAaAAATTAGCTgG GGcTAATTTTTgtA GGcTAATTTTTGTa AAAAAATTAGC CAAgAGCGAAACT location AluSx (-22:-33)* AluSx (-23:-34)* AluSx (24:36)* AluSx (24:34)* AluSx (29:45) AluSx (38:46) AluSx (58:73) AluSx (59:69) AluSc (60:72) AluJb (-62:-68) AluSc (65:79) AluSp (-82:-98) AluY (96:112) AluSx (-100:-108) AluSx (114:131)** AluSx (114:131)** AluSx (116:131)** AluSx (116:131)** AluSx (116:132)** AluJo (118:134)** AluSc (125:139)** AluSx (-126:-139)** AluSx (-126:-139)** AluY (127:137)** AluSp (264:276) p-val 4.00E-03 2.60E-03 2.00E-03 1.00E-05 1.00E-05 1.70E-03 1.00E-04 1.60E-03 8.50E-03 1.00E-05 6.60E-03 4.90E-03 6.50E-03 3.60E-03 4.00E-04 3.50E-03 2.60E-03 3.30E-03 3.00E-03 4.00E-04 8.30E-03 8.80E-03 6.60E-03 4.00E-04 3.60E-03 n. fam 8 8 8 8 8 8 2 3 1 1 1 1 1 5 6 6 6 6 6 2 1 6 6 1 2 Page 3 of 15 (page number not for citation purposes)