BMC Genomics 2006, 7:133 http://www.biomedcentral.com/1471-2164/7/133 Alu. The situation is different for Gfi-1; there the PSSM [30] is based on 21 target sites, only five of which are part of Alu (Table 3). As to PITX2, it's PSSM [31] was based on target sites on the promoter of PLOD1, all of which reside on Alu subsequences [40]. Finally, the heart development marker Nkx2.5 is the only one whose PSSM was calculated using only off-Alu target sites – nevertheless, the target site found by us, that resides on Alu, is the one with the high- est possible score. Evidence (in live cells) for regulation by TFs bound to Alu The regulatory role of single – gene BSs on Alu was dem- onstrated for several TFs by a transfection assay for hor- mone ligands [16,22], for YY1 [20], and for Estrogen Receptors (ER) [18]. We found experimental evidence indicating that Gfi-1, PITX2 and Nkx2.5 regulate a reporter gene's expression [40-42] via BSs that reside on Alu; where the PITX2 function was validated in two differ- ent experiments [40,42]. In addition, Alu was essential for regulation of PLOD1 by PITX2, shown by using two con- structs of PLOD1 promoter: one with several Alu elements and one without any Alu. These researchers [40] found a 3-fold change in the induction of a reporter gene with constructs that contained several Alu elements with respect to the expression level of the construct from which Alu elements were excluded. The same procedure of com- paring promoters with and without Alu was done again on PLOD1 [42], but this time they checked PITX2 together with Nkx2.5 in order to prove the synergic effect on PLOD1 expression, and again a big difference between the expression levels of the reporter gene was found. ChIP analysis was done on 21 genes' promoters that con- tain Gfi-1 putative target sites; five of these promoters con- tained suspected BSs on Alu (table 3). However, only one of these five genes (JAK3) had its Gfi-1 BS exclusively on Alu [41]. Hence the positive result of the ChIP experiment on this gene shows that Gfi-1 actually binds in vivo to Alu. Developmental and stress response TFs constitute a majority of the BSs on Alu Most of the stress response TFs that bind to Alu are nuclear factors and hormone ligands that were validated experi- mentally, such as ER, RAR, LXRE and SP1 (Tables 2, 4). As was mentioned above, a subset of them are common to all Alu subfamilies while some appear only on the young Alu sequences, or on Alu elements that were mutated [20]. Another interesting group of TFs is developmental (usu- ally referred to either as early markers or regulators of development); more than 30 members of this group passed the T5 threshold on at least one Alu consensuses sequence. The largest set of developmental TF binding sites that appear on Alu belong to hemateopoietic tran- scription factors [43] such as Gfi-1, LYF-1, GATA3, GATA2 and ONECUT2 (Table 4). Another set of BMs belong to TFs that are related to muscle and heart development [44], such as PITX2, MEF2, NKX2.5 and LUN1 (Tables 2, 4), and some to brain development: PAX4, NRL, OTX2 (Tables 2, 4). Note that even though the PAX6.01 PSSM did pass our thresholds (and hence appears in Table 4), it has been demonstrated [45] that PAX6 actually binds to very specific Alu sequences, that constitute only 0.2% of the genome-wide Alu elements. The PSSM that was used in our analysis (taken from MatInspector) was con- structed from BMs that were identified in different experi- ments and its dominant binding sequences do not coincide with those that [45] identified as the Alu medi- ated PAX6 BMs. Table 3: List of human target genes of (murine) Gfi-1 which contain Alu in their 5' upstream region. In these genes some of the Gfi-1 target sites are on Alu sequences; such sequences are denoted by '+' in the third column. The first column contains the target gene name; the second column is a potential binding sequence in the promoter region of the gene. The fourth column is a result of EMSA binding experiment for the sequence in column 2 [41]. part of Alu Binding in EMSA *These sites were tested in transient transfection assays. Gene AZU AAT ACT Jak3 P21 Potential Gfi-1 BSs 1, 5'-CTCCCAAAGTGCTGAGATTACAGGTGTGAG-3'† 2, 5'-CGGCCTGGAGACCAGATTTGAGCCCTGGGA-3' 3, 5'-ACTTCCTGCCTCTGCAGATTTGCTTGTTCT-3' 4, 5'-TTTCACACCTGTAATCCCAGCATTTTGGGA-3' 5, 5'-GGTGGACAACTGTAATCCTAGCTACTCAGG-3" 1, 5'-GCACACGCCTGTAATCCCAGCTATTTGGGA-3' 2, 5'-CCCAGGAGGTTTAATCACAGCCCCTCCATG-3'* 3, 5'-ATAGCTGACCTAATCTCTCTGGCTTTGGTT-3'* 1, 5'-GCTCATGCCTGTAATCCCAGCACTTTGGGA-3' 2, 5'-TTCTTGGCTGCCACTGATTCCCTGTGCCTT-3' 3, 5'-GCCAAAATAAAATCCCAGTCCAAGTCTGGG-3'* 1, 5'-CTCACGTGTGTAATCACAGCACTTTGGG-3' 1, 5'-GCATGCCTGTAATCCCAGCTACTCGGG-3' 2, 5'-ATGTCTGGGCAGAGATTTCCAGACTCT-3' + - - + - + - - + - - + + - + + - + - + + - + + + + + + Page 5 of 15 (page number not for citation purposes)