BACKGROUND
over 90% of the human and other vertebrate genomes don't have any known functional role  <cit> , and as such were considered until recently as "junk dna" or genomic "dark matter".
a large fraction of the "non-functional" dna originated from mobile elements  <cit>  such as alu, which comprise about 10% of the nucleotides of the human genome  <cit>  with over one million inserted copies.
this abundance is somewhat of a surprise, since alu is a non-autonomous retroelement, i.e.
it doesn't encode proteins that assist its mobilization, and therefore it needs to rely on the cell's machinery for its duplication in the genome  <cit> .
currently alu is believed to be a parasite of the mobilization machinery of long interspersed elements   <cit> .
a typical alu is a dimer, comprised of a central a-rich region which is flanked by two similar sequence elements of about  <dig> bp .
during evolution the alu elements have spread in the human genome in several bursts at different times, which allow their classification into several families and subfamilies, on the basis of their insertions, deletions and mutations.
the major families are: old aluj, whose members are alujb and alujo ; the middle, alus family, which includes alusx, alusg, alusp alusc and alusq , and the youngest alu family, aluy   <cit> .
one of the biggest mysteries associated with alu concerns the non-random manner in which these elements are distributed throughout the human genome  <cit> .
the distribution of alu was found to be highly correlated with local gc content and with the density of genes and with intron density  <cit> .
furthermore, the density of alu is higher in intragenic  than in intergenic regions   <cit> , which is surprising if indeed alu elements are non-functional bits of dna.
in addition, alu elements are negatively selected in imprinted regions of primate genomes  <cit> .
transposable elements are underrepresented in the region surrounding the tss of genes  <cit> .
another analysis revealed that almost 20% of the genes contain transposable elements in the 3' utr  <cit> .
significant differences were observed also when alu density was compared among full length gene sequences of different biological process classes .
genes that were related to metabolism, transport and signalling were mostly alu rich, whereas genes that had poor alu content belonged mostly to structural proteins and to information processing and storage pathways  <cit> .
it was also shown recently that housekeeping genes contain more alu than tissue specific genes  <cit> .
this non-random of distribution of alu, in particular the high concentration near and within genes, might be the result of positive feedback; selective pressure drives the organism to use these elements for some new functions, and the functions acquired by alu generate advantage for phenotypes that have high alu concentration near genes.
indeed, the possible functional roles of alu in genomic organization and gene expression has received increasing attention during the last two decades .
one of the more fascinating possible consequences of integration of mobile elements near genes is acquisition of a transcriptional regulatory role by the alu element, as a carrier of different tf binding sites.
the alu element harbours bss of several transcription factors that were shown to bind to alu; most of these tfs were nuclear factors, hormones, calcium nuclear factors, as well as other tfs  <cit> .
the idea of alu acting as a carrier of cis regulatory elements was suggested by britten  <cit>  and by oei at el.
<cit>  who found yy <dig> and sp <dig> bss on young alu family members.
these experimental results imply that alu elements have influence on the regulation of expression levels of many genes that have alu in their promoter regions; however, such a wide ranged regulatory impact has not yet been tested experimentally.
the main aim of our study was to find new putative bss that reside on alu.
very recently, several groups have performed similar analysis for all transposable element classes; however, the set of pssms studied  <cit>  contained about half of what is presented here.
in addition, our analysis is complemented by an extensive literature search of past experimental work on the alu-associated binding sites that we found, as well as the biological functions of the corresponding tfs and target genes.
we started by searching the alu consensus sequences and found new, previously unnoticed bss, many of which turned out to be related to developmental processes.
the regulatory role played by alu for genes that are related to differentiation or development was already established for at least six genes  <cit> .
in these cases alu was shown to act as enhancer or silencer involved in regulating the expression of six developmental genes.
for one of these, the t-cell marker gene cd8α, the silencing mechanism was shown to act via binding of lyf- <dig>  bhlh and gata <dig> to the alu situated in the last intron of cd8α  <cit> .
these experimental results demonstrated the regulatory impact of alu on developmental genes and during developmental stages.
to explore further and enhance the possible regulatory roles of alu we focused on the genomic region most likely to have a regulatory function – the upstream regions of genes.
we scanned these regions and searched for bss on the particular alu sequences that were identified.
our findings show for the first time that tfs that are known to regulate the developmental processes may bind to alu elements that are incorporated in the promoter regions of genes that need to be activated or suppressed during development .
RESULTS
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  <cit> , whose sequences were downloaded from repbase  <cit> .
we refer to the ensemble of sequences that bind a tf as it's binding motif .
in order to find bms of a tf, we used its position specific score matrix ;  <dig> pssms were downloaded from two databases  <cit> .
for each pssm we set two threshold scores, t <dig> and t <dig>  selected so that the probability to find a target bm with score higher than t <dig> in a suitably chosen random sequence  is less than  <dig>  .
furthermore, for each bm we identified tmax, the maximal score observed on all the above listed alu subfamilies' consensus sequences.
for  <dig> bms tmax was higher than t <dig> and for  <dig> tfs – higher than t <dig> .
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  <dig> bms with scores higher than their respective t <dig>  the probability to find this many bms in a random model  is  <dig>  .
in order to control the problem of multiple testing we performed a false discovery rate  analysis at 20% fdr <cit> .
the  <dig> bms that passed the fdr test with q =  <dig>  in alusx are listed in table  <dig>  where we also compare the iupac consensus sequences of these bms  <cit>  with the target sequence that had the highest score in all the major alu subfamilies.
most sequences with scores higher than t <dig> differ at not more than two nucleotides from the iupac consensus , and there are several pssms, such as pitx2_q <dig>  lyf- <dig> , pax <dig> , six <dig> , deltaef.
<dig> and nkx <dig>  , whose iupac consensus sequences agree with the highest-scoring target sites on at least one of the alu consensus sequences.
bms tend to cluster together on specific parts of alu consensus sequences, that also tend to be more conserved during evolution
we checked the bm's locations along the consensus sequences on both strands of the eight alu subfamilies.
in general, most of the putative bss were found on the sense strand.
it is interesting to note that alu elements show a 55% to 45% bias towards antisense orientation  <cit>  relative to nearby genes.
it would be of considerable interest to check experimentally whether this orientational difference is reflected in functional roles of the tfs.
the target sites that passed t <dig> cover, on average, 50% of the consensus sequence of each subfamily.
on the other hand, the total length of all the binding motifs found on one alu consensus sequence exceeds, on the average,  <dig> nucleotides .
this redundancy in putative target sites is caused by the fact that several pssms have very similar structure, giving top scores to the same dna sequence.
the short sequences ggtaatccc on the 5' to 3' strand and its complementary sequence gggattacc serve as a putative bs of five tfs, four of which are homeodomain proteins .
this sequence is part of the consensus of all eight alu subfamilies, and in the five middle alus subfamilies the bs appears on both the left and right monomers.
moreover, this is precisely the sequence identified by britten  <cit>  as one of the most conserved subsequences on alu, which was seen as a surprise since it did not have any role in retrotransportation or for any other function.
later on, it was found that in the germ line the specific alu binding protein  can bind to this sequence and prevent methylation  <cit> , but since the importance of this function of is still not understood, and hence the high level of conservation is still a mystery.
another region is a putative harbour for several bms is the middle poly a- stretch .
we found this sequence to be a putative target site for many binding motifs such as all mef <dig> family members, hnf <dig> , oc.
<dig> and pax <dig> .
these putative target sites were also found to accumulate less mutations than the expected rate  <cit> .
more recently both the central poly a stretch and the subsequence mentioned above  <cit>  were found to be associated with a-to-i editing.
existing evidence for tf binding to alu
having identified bss of various tfs on alu, we searched for existing experimental evidence for binding and regulation associated with these motifs.
indeed, several tfs that are known to bind to motifs that passed our thresholds have already been shown to bind to dna, and the fact that the binding sequences reside on alu has been noticed .
we show in table  <dig> four additional bms that we found and report here for the first time; their corresponding tfs were shown to bind to the target sites identified by us, but the fact that they reside on alu has not been previously noted.
these tfs  are pitx <dig>  nkx <dig>  gfi- <dig> and lun <dig>  the most striking example was lun-1; chu et al.
<cit>  observed  that it binds to  <dig> dna sequences,  <dig> of which are palindromic; we discovered that all of these  <dig> reside on alu.
the situation is different for gfi-1; there the pssm  <cit>  is based on  <dig> target sites, only five of which are part of alu .
as to pitx <dig>  it's pssm  <cit>  was based on target sites on the promoter of plod <dig>  all of which reside on alu subsequences  <cit> .
finally, the heart development marker nkx <dig>  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 highest possible score.
evidence  for regulation by tfs bound to alu
the regulatory role of single – gene bss on alu was demonstrated for several tfs by a transfection assay for hormone ligands  <cit> , for yy <dig>  <cit> , and for estrogen receptors   <cit> .
we found experimental evidence indicating that gfi- <dig>  pitx <dig> and nkx <dig>  regulate a reporter gene's expression  <cit>  via bss that reside on alu; where the pitx <dig> function was validated in two different experiments  <cit> .
in addition, alu was essential for regulation of plod <dig> by pitx <dig>  shown by using two constructs of plod <dig> promoter: one with several alu elements and one without any alu.
these researchers  <cit>  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 comparing promoters with and without alu was done again on plod <dig>  <cit> , but this time they checked pitx <dig> together with nkx <dig>  in order to prove the synergic effect on plod <dig> expression, and again a big difference between the expression levels of the reporter gene was found.
chip analysis was done on  <dig> genes' promoters that contain gfi- <dig> putative target sites; five of these promoters contained suspected bss on alu .
however, only one of these five genes  had its gfi- <dig> bs exclusively on alu  <cit> .
hence the positive result of the chip experiment on this gene shows that gfi- <dig> 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 experimentally, such as er, rar, lxre and sp <dig> .
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  <cit> .
another interesting group of tfs is developmental ; more than  <dig> members of this group passed the t <dig> threshold on at least one alu consensuses sequence.
the largest set of developmental tf binding sites that appear on alu belong to hemateopoietic transcription factors  <cit>  such as gfi- <dig>  lyf- <dig>  gata <dig>  gata <dig> and onecut <dig> .
another set of bms belong to tfs that are related to muscle and heart development  <cit> , such as pitx <dig>  mef <dig>  nkx <dig>  and lun <dig> , and some to brain development: pax <dig>  nrl, otx <dig> .
note that even though the pax <dig>  pssm did pass our thresholds , it has been demonstrated  <cit>  that pax <dig> actually binds to very specific alu sequences, that constitute only  <dig> % of the genome-wide alu elements.
the pssm that was used in our analysis  was constructed from bms that were identified in different experiments and its dominant binding sequences do not coincide with those that  <cit>  identified as the alu mediated pax <dig> bms.
the distribution of transposable elements in the 5' upstream regions of human genes
the distribution of transposable elements in the 5' upstream regions of human genesis not uniform.
we computed the density of different transposable elements on  <dig> bp long upstream sequences of about  <dig> genes.
in the region between  <dig> bp and  <dig> bp from the transcription start site , 45% of the nucleotides belong to transposable elements.
this fraction is the same as the overall density in the entire genome – see fig  <dig>  in contrast, in the region closer to the tss, between  <dig> and  <dig> bp – the density of transposable elements is lower.
the alu density exhibits a more interesting behaviour; for short distances – between  <dig> and  <dig> bp – it is lower than the genomic average of about 10%.
this is not surprising since the immediate proximal region to the tss is rich with important binding sites for initiating factors and insertion of alu and other transposable elements in this region is most probably selected against.
the density increases steadily till about  <dig> bp, where it reaches a level of 18% of the base pairs , after which it maintains a steady level .
the alu content in the deep intergenic spacer regions is less than 10%; hence the density of on-alu bss in these regions is also lower than in the upstream  <dig> bp regions of genes.
the presences of less bss in these regions might imply less functional relevance.
finally, 85% of the genes in our database contained at least one alu insertion in the  <dig> kbp upstream region.
non random distribution of alu elements on promoter regions of different gene groups
we analyzed the distribution of alu elements in the  <dig> bp upstream regions of genes, treating separately groups of genes that belong to different biological processes.
protein biosynthesis genes are enriched with alu compared to the genome-wide average value of  <dig>  copies in the  <dig> bp long upstream regions .
in contrast, genes that are related to various kinds of developmental processes, tissue formation  contain a smaller number of alu elements in their  <dig> bp upstream region than the average value .
many of these differences between the alu content of genes of a particular biological process and the alu content of the entire genome  were found  to be highly significant .
as mentioned above, similar differences in alu content were reported  <cit>  when the full length gene sequences on chromosomes  <dig> and  <dig> were analyzed.
studying bs content in specific  alu elements in the promoter regions
hundreds of thousands of putative bss reside on alu elements in the upstream regions of genes.
for several tfs one observes that nearly all the putative bss  are located on alu.
the search for bms presented in the previous sections was done on the consensus sequences of various alu subfamilies.
since, however, alu elements have mutated after insertion, there is considerable deviation from the consensus in the specific alu sequences that are found across the genome.
this variation may change the bs content of specific individual alu elements versus the consensus.
hence we repeated our search of bss in the actual  <dig> bp upstream regions of about  <dig>  genes, and if a bs passed a significance filter, we checked whether it resides on a specific alu.
this search was limited to those  <dig> bms that passed our filter based on the consensus sequences, i.e.
their largest score, tmax, exceeded t <dig> for the consensus of at least one of the eight alu subfamilies studied.
for each of these  <dig> bms we searched the  <dig>  upstream regions for putative bms whose score exceeds  tmax.
we found that for  <dig> bms out of the  <dig> the majority of putative bss that pass tmax reside on alu sequences; the relative on and off alu bm content is presented, for  <dig> out of these  <dig>  in fig  <dig>  for example, for  <dig> out the  <dig> nuclear factors  whose bms appear among the  <dig>  between 66% and 99% of the bms  are on alu .
the highest over-representations in alu were observed for the bs of pitx2; over 99% of the  <dig> bss that pass the threshold were on alu, and for lxre's bss over 99% of it's  <dig> bss that pass the threshold were on alu, .
in general, the distributions of putative bss of tfs that bind to tgtaatccc  exhibit remarkable differences between the number of putative bs on and off alu; over 90% of the total number of putative bss of these tfs are located within alu sequences.
more moderate differences were found for developmental tfs such as six <dig> , pax <dig> , nrl.
<dig>  mef <dig>  and nkx <dig>  .
in general, for every bm that passed fdr of q =  <dig>   over 80% of the bss on the  <dig> kbp upstream region are located on alu.
on the other hand, for the  <dig>  remaining pssms the majority of bss were not located on alu, like irf <dig>   and hnf <dig>  .
still, even for these tfs, with mainly off-alu bms, the alu repeats provide between hundreds to thousands of putative bss in the  <dig> kbp upstream regions .
as mentioned above, in the  <dig> bp upstream region, which constitutes the proximal promoter  <cit>  and hence is believed to be most important for regulation, the alu density is below the genome average .
to study this region in more detail, we repeated the analysis that was done for the  <dig> bp region, and compared the distributions of bss on and off alu in the first  <dig> bp upstream to the tss .
only  <dig> out of the  <dig> bms had more on alu than off alu bms.
these bms include almost all homeodomain proteins, lxre.
<dig>  lyf- <dig> and pax <dig>  .
in spite of the relatively low alu density, on-alu bms do constitute a sizeable fraction of the putative bms that are found in the  <dig> bp long upstream region .
genes of different biological processes contain different amounts of bss as a result of differences in their alu content
we used the results of the search for on and off alu bms to look for differences between genes that belong to different biological processes.
as expected, for those biological processes whose genes were previously found to be enriched with alu , we also found that a high percentage of their regulatory signals were on-alu.
the alu rich groups in this category include metabolic and bio-synthesis associated genes.
since for several tfs a large majority of bms reside on alu, we find that alu-rich functional families have a large number of these alu-associated bms, whereas alu-poor biological processes  will have less of these bms .
for example, the average number of putative bss of pitx <dig> is almost  <dig> times greater in protein biosynthetic genes than in cns developmental genes .
interestingly, as a result, developmental tfs such as lyf- <dig>  pax <dig>  mef2d and pitx <dig>  that have on-alu bms, appear less in the  upstream region of developmental genes than in the alu-rich non-developmental group of protein biosynthesis related genes.
one possible explanation of this may be that the developmental tfs main role is to suppress pathways that are in competition with or exclusive of the developmental process .
one should emphasize that the statements made above are based mainly on in-silico evidence, which must be supplemented by firm experimental observations of binding and functional roles of the bound tfs.
inter-species comparisons: b <dig>  the murine 'alu like' elements, contain several bss that are similar to the primate alu bss
we scanned the b <dig> consensus sequences that were downloaded from repbase  <cit> .
these sequences were less enriched with bss that pass the t <dig> threshold; this might be due to the fact that the length of the b <dig> sequences is only half that of alu.
twenty three binding motifs passed t <dig> on b <dig>  and we found that  <dig> of these passed t <dig> also in alu, as expected from the similarity between b <dig> and alu  <cit>  .
one of these shared bss was ttaatccc where g appears in all alu families and t appears in the consensus of b1s.
this bs is the target of otx <dig> , a homeodomain protein in both human and mouse.
an additional homeodomain protein, crx <dig> , is also bound to this bs in mouse; it did not pass t <dig> in alu.
the members of the mef <dig> family of bms appear in mouse  with a weaker score than in alu, .
the pssm with the most significant score on b <dig> was pax <dig> , which is not surprising since this pssm is built from those bss of pax <dig> that were known to reside on b <dig>  <cit> .
in addition, a group of bms that are related to nuclear factors and response proteins passed t <dig> on b <dig>  but not on alu.
this is surprising since there are experiments that validated that the tfs that correspond to these bms bind to primate alu  <cit> .
the distributions of b <dig> in mouse promoters and alu in human promoters are positively correlated
when the mouse genome was first sequenced, regions orthologous to human dna were identified.
the densities of b <dig> and b <dig> in mouse and alu in human were measured in windows of  <dig>  bp and the alu density in humans was found to be correlated with b <dig> and b <dig> density in mouse  <cit> .
a high correlation was quite unexpected, since alu and the mouse sine elements b <dig> and b <dig> spread in the rodent and human genomes after the divergence of rodents and primates.
we checked whether this trend is present also in the regulatory regions of genes, of size  <dig> kbp, and we found a positive correlation between b <dig> and alu densities even in this relatively short region.
in more than  <dig> pairs of orthologous genes, that had available upstream sequences in both human and mouse, we found positive correlation  of  <dig>  .
in addition we ordered the genes by increasing alu content in the  <dig> upstream bp region, and divided the genes on this basis into  <dig> sets, with each set containing  <dig> genes.
we then computed the average alu and b <dig> associated nucleotide densities for each of the  <dig> sets, and plotted the two average alu/b <dig> contents .
each point in fig  <dig> represents a group of  <dig> genes, with the x  coordinates representing the alu  densities in human .
the monotonic behaviour evident in fig  <dig> indicates that the alu and b <dig> contents of orthologous genes are largely similar in human and mouse.
this similarity is far from being obvious, since spreading of alu and b <dig> across the genome is believed to have happened after the divergence of primates and rodents.
CONCLUSIONS
summary
this research suggests that evolution used transposable elements to insert modules of transcription factor binding motifs into promoters and, by means of their presence, assemble higher level regulatory networks.
in order to explore this question we focused on alu elements, which are good potential candidates to be part of the building blocks of regulatory networks for two reasons.
first, alu elements are abundant in the upstream region of the tss of genes, and second, alu elements contain dozens of putative bss for tfs.
some of these bss were found before and their association with alu was also reported, whereas in some cases although the bss were found, the fact that they reside on alu went unnoticed.
finally, we list here also bss on alu that were not identified previously.
our findings imply that the biological pathway on which alu-mediated regulation appears to have the most significant impact is the development process.
many of the tfs that have binding motifs on alu are associated with development; moreover, some of these bss were previously demonstrated to be functional in vivo and essential to regulation of some target genes.
we have also shown that a few subsequences on alu, that were known to be highly conserved, provide many of the binding sites for the transcription factors.
we studied the alu distribution in the upstream region of genes and found that while alu are "repelled" from the  <dig> bp long region immediately preceding the transcription starts site, the  <dig> bp long sequence further upstream is enriched .
the alu content in the promoters varies between functional families of genes.
comparison of alu in human and b <dig> in mice shows a significant correlation between their distributions , and high similarity between the binding motifs that reside on the two mobile elements.
several particular cases  were discussed in detail.
future directions
most importantly, more experiments are needed to verify that the bms that were found by us  indeed bind their corresponding tfs and, furthermore, to verify their regulatory roles.
of particular interest is the role of alu in developmental processes.
we found that promoters of genes involved in biosynthesis and metabolism are enriched  in bms of several developmental tfs .
it is our belief that since development and cell proliferation  are opposing processes  <cit> , these bms play an inhibitory role, suppressing proliferation during differentiation.
this hypothesis is supported by the known suppressory role of gfi <dig>  but the presently known activities on pitx <dig> and nkx <dig>  do not fit this picture.
further experiments that address this issue by measuring the regulatory effects of other alu-associated developmental tfs are needed.
clearly, many of the genes and their functions date earlier than the rodent/primate divergence, whereas the alu insertions happened later.
this poses an open and debated  <cit>  question about the mechanism by which pre-existing regulatory circuits must have been replaced, modified or supplemented by alu insertions.

 over 90% of the human and other vertebrate genomes don't have any known functional role, and as such were considered until recently as "junk dna" or genomic "dark matter". a large fraction of the "non-functional" dna originated from mobile elements such as alu, which comprise about 10% of the nucleotides of the human genome with over one million inserted copies. this abundance is somewhat of a surprise, since alu is a non-autonomous retroelement, i.e. it doesn't encode proteins that assist its mobilization, and therefore it needs to rely on the cell's machinery for its duplication in the genome. currently alu is believed to be a parasite of the mobilization machinery of long interspersed elements. a typical alu is a dimer, comprised of a central a-rich region which is flanked by two similar sequence elements of about bp. during evolution the alu elements have spread in the human genome in several bursts at different times, which allow their classification into several families and subfamilies, on the basis of their insertions, deletions and mutations. the major families are: old aluj, whose members are alujb and alujo; the middle, alus family, which includes alusx, alusg, alusp alusc and alusq, and the youngest alu family, aluy. one of the biggest mysteries associated with alu concerns the non-random manner in which these elements are distributed throughout the human genome. the distribution of alu was found to be highly correlated with local gc content and with the density of genes and with intron density. furthermore, the density of alu is higher in intragenic than in intergenic regions, which is surprising if indeed alu elements are non-functional bits of dna. in addition, alu elements are negatively selected in imprinted regions of primate genomes. transposable elements are underrepresented in the region surrounding the tss of genes. another analysis revealed that almost 20% of the genes contain transposable elements in the 3' utr. significant differences were observed also when alu density was compared among full length gene sequences of different biological process classes. genes that were related to metabolism, transport and signalling were mostly alu rich, whereas genes that had poor alu content belonged mostly to structural proteins and to information processing and storage pathways. it was also shown recently that housekeeping genes contain more alu than tissue specific genes. this non-random of distribution of alu, in particular the high concentration near and within genes, might be the result of positive feedback; selective pressure drives the organism to use these elements for some new functions, and the functions acquired by alu generate advantage for phenotypes that have high alu concentration near genes. indeed, the possible functional roles of alu in genomic organization and gene expression has received increasing attention during the last two decades. one of the more fascinating possible consequences of integration of mobile elements near genes is acquisition of a transcriptional regulatory role by the alu element, as a carrier of different tf binding sites. the alu element harbours bss of several transcription factors that were shown to bind to alu; most of these tfs were nuclear factors, hormones, calcium nuclear factors, as well as other tfs. the idea of alu acting as a carrier of cis regulatory elements was suggested by britten and by oei at el. who found yy and sp bss on young alu family members. these experimental results imply that alu elements have influence on the regulation of expression levels of many genes that have alu in their promoter regions; however, such a wide ranged regulatory impact has not yet been tested experimentally. the main aim of our study was to find new putative bss that reside on alu. very recently, several groups have performed similar analysis for all transposable element classes; however, the set of pssms studied contained about half of what is presented here. in addition, our analysis is complemented by an extensive literature search of past experimental work on the alu-associated binding sites that we found, as well as the biological functions of the corresponding tfs and target genes. we started by searching the alu consensus sequences and found new, previously unnoticed bss, many of which turned out to be related to developmental processes. the regulatory role played by alu for genes that are related to differentiation or development was already established for at least six genes. in these cases alu was shown to act as enhancer or silencer involved in regulating the expression of six developmental genes. for one of these, the t-cell marker gene cd8α, the silencing mechanism was shown to act via binding of lyf- bhlh and gata to the alu situated in the last intron of cd8α. these experimental results demonstrated the regulatory impact of alu on developmental genes and during developmental stages. to explore further and enhance the possible regulatory roles of alu we focused on the genomic region most likely to have a regulatory function – the upstream regions of genes. we scanned these regions and searched for bss on the particular alu sequences that were identified. our findings show for the first time that tfs that are known to regulate the developmental processes may bind to alu elements that are incorporated in the promoter regions of genes that need to be activated or suppressed during development.  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, whose sequences were downloaded from repbase. we refer to the ensemble of sequences that bind a tf as it's binding motif. in order to find bms of a tf, we used its position specific score matrix; pssms were downloaded from two databases. for each pssm we set two threshold scores, t and t selected so that the probability to find a target bm with score higher than t in a suitably chosen random sequence is less than. furthermore, for each bm we identified tmax, the maximal score observed on all the above listed alu subfamilies' consensus sequences. for bms tmax was higher than t and for tfs – higher than t. 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 bms with scores higher than their respective t the probability to find this many bms in a random model is. in order to control the problem of multiple testing we performed a false discovery rate analysis at 20% fdr. the bms that passed the fdr test with q = in alusx are listed in table where we also compare the iupac consensus sequences of these bms with the target sequence that had the highest score in all the major alu subfamilies. most sequences with scores higher than t differ at not more than two nucleotides from the iupac consensus, and there are several pssms, such as pitx2_q lyf-, pax, six, deltaef. and nkx, whose iupac consensus sequences agree with the highest-scoring target sites on at least one of the alu consensus sequences. bms tend to cluster together on specific parts of alu consensus sequences, that also tend to be more conserved during evolution we checked the bm's locations along the consensus sequences on both strands of the eight alu subfamilies. in general, most of the putative bss were found on the sense strand. it is interesting to note that alu elements show a 55% to 45% bias towards antisense orientation relative to nearby genes. it would be of considerable interest to check experimentally whether this orientational difference is reflected in functional roles of the tfs. the target sites that passed t cover, on average, 50% of the consensus sequence of each subfamily. on the other hand, the total length of all the binding motifs found on one alu consensus sequence exceeds, on the average, nucleotides. this redundancy in putative target sites is caused by the fact that several pssms have very similar structure, giving top scores to the same dna sequence. the short sequences ggtaatccc on the 5' to 3' strand and its complementary sequence gggattacc serve as a putative bs of five tfs, four of which are homeodomain proteins. this sequence is part of the consensus of all eight alu subfamilies, and in the five middle alus subfamilies the bs appears on both the left and right monomers. moreover, this is precisely the sequence identified by britten as one of the most conserved subsequences on alu, which was seen as a surprise since it did not have any role in retrotransportation or for any other function. later on, it was found that in the germ line the specific alu binding protein can bind to this sequence and prevent methylation, but since the importance of this function of is still not understood, and hence the high level of conservation is still a mystery. another region is a putative harbour for several bms is the middle poly a- stretch. we found this sequence to be a putative target site for many binding motifs such as all mef family members, hnf, oc. and pax. these putative target sites were also found to accumulate less mutations than the expected rate. more recently both the central poly a stretch and the subsequence mentioned above were found to be associated with a-to-i editing. existing evidence for tf binding to alu having identified bss of various tfs on alu, we searched for existing experimental evidence for binding and regulation associated with these motifs. indeed, several tfs that are known to bind to motifs that passed our thresholds have already been shown to bind to dna, and the fact that the binding sequences reside on alu has been noticed. we show in table four additional bms that we found and report here for the first time; their corresponding tfs were shown to bind to the target sites identified by us, but the fact that they reside on alu has not been previously noted. these tfs are pitx nkx gfi- and lun the most striking example was lun-1; chu et al. observed that it binds to dna sequences, of which are palindromic; we discovered that all of these reside on alu. the situation is different for gfi-1; there the pssm is based on target sites, only five of which are part of alu. as to pitx it's pssm was based on target sites on the promoter of plod all of which reside on alu subsequences. finally, the heart development marker nkx 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 highest possible score. evidence for regulation by tfs bound to alu the regulatory role of single – gene bss on alu was demonstrated for several tfs by a transfection assay for hormone ligands, for yy, and for estrogen receptors. we found experimental evidence indicating that gfi- pitx and nkx regulate a reporter gene's expression via bss that reside on alu; where the pitx function was validated in two different experiments. in addition, alu was essential for regulation of plod by pitx shown by using two constructs of plod promoter: one with several alu elements and one without any alu. these researchers 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 comparing promoters with and without alu was done again on plod, but this time they checked pitx together with nkx in order to prove the synergic effect on plod expression, and again a big difference between the expression levels of the reporter gene was found. chip analysis was done on genes' promoters that contain gfi- putative target sites; five of these promoters contained suspected bss on alu. however, only one of these five genes had its gfi- bs exclusively on alu. hence the positive result of the chip experiment on this gene shows that gfi- 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 experimentally, such as er, rar, lxre and sp. 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. another interesting group of tfs is developmental; more than members of this group passed the t threshold on at least one alu consensuses sequence. the largest set of developmental tf binding sites that appear on alu belong to hemateopoietic transcription factors such as gfi- lyf- gata gata and onecut. another set of bms belong to tfs that are related to muscle and heart development, such as pitx mef nkx and lun, and some to brain development: pax nrl, otx. note that even though the pax pssm did pass our thresholds, it has been demonstrated that pax actually binds to very specific alu sequences, that constitute only % of the genome-wide alu elements. the pssm that was used in our analysis was constructed from bms that were identified in different experiments and its dominant binding sequences do not coincide with those that identified as the alu mediated pax bms. the distribution of transposable elements in the 5' upstream regions of human genes the distribution of transposable elements in the 5' upstream regions of human genesis not uniform. we computed the density of different transposable elements on bp long upstream sequences of about genes. in the region between bp and bp from the transcription start site, 45% of the nucleotides belong to transposable elements. this fraction is the same as the overall density in the entire genome – see fig in contrast, in the region closer to the tss, between and bp – the density of transposable elements is lower. the alu density exhibits a more interesting behaviour; for short distances – between and bp – it is lower than the genomic average of about 10%. this is not surprising since the immediate proximal region to the tss is rich with important binding sites for initiating factors and insertion of alu and other transposable elements in this region is most probably selected against. the density increases steadily till about bp, where it reaches a level of 18% of the base pairs, after which it maintains a steady level. the alu content in the deep intergenic spacer regions is less than 10%; hence the density of on-alu bss in these regions is also lower than in the upstream bp regions of genes. the presences of less bss in these regions might imply less functional relevance. finally, 85% of the genes in our database contained at least one alu insertion in the kbp upstream region. non random distribution of alu elements on promoter regions of different gene groups we analyzed the distribution of alu elements in the bp upstream regions of genes, treating separately groups of genes that belong to different biological processes. protein biosynthesis genes are enriched with alu compared to the genome-wide average value of copies in the bp long upstream regions. in contrast, genes that are related to various kinds of developmental processes, tissue formation contain a smaller number of alu elements in their bp upstream region than the average value. many of these differences between the alu content of genes of a particular biological process and the alu content of the entire genome were found to be highly significant. as mentioned above, similar differences in alu content were reported when the full length gene sequences on chromosomes and were analyzed. studying bs content in specific alu elements in the promoter regions hundreds of thousands of putative bss reside on alu elements in the upstream regions of genes. for several tfs one observes that nearly all the putative bss are located on alu. the search for bms presented in the previous sections was done on the consensus sequences of various alu subfamilies. since, however, alu elements have mutated after insertion, there is considerable deviation from the consensus in the specific alu sequences that are found across the genome. this variation may change the bs content of specific individual alu elements versus the consensus. hence we repeated our search of bss in the actual bp upstream regions of about genes, and if a bs passed a significance filter, we checked whether it resides on a specific alu. this search was limited to those bms that passed our filter based on the consensus sequences, i.e. their largest score, tmax, exceeded t for the consensus of at least one of the eight alu subfamilies studied. for each of these bms we searched the upstream regions for putative bms whose score exceeds tmax. we found that for bms out of the the majority of putative bss that pass tmax reside on alu sequences; the relative on and off alu bm content is presented, for out of these in fig for example, for out the nuclear factors whose bms appear among the between 66% and 99% of the bms are on alu. the highest over-representations in alu were observed for the bs of pitx2; over 99% of the bss that pass the threshold were on alu, and for lxre's bss over 99% of it's bss that pass the threshold were on alu,. in general, the distributions of putative bss of tfs that bind to tgtaatccc exhibit remarkable differences between the number of putative bs on and off alu; over 90% of the total number of putative bss of these tfs are located within alu sequences. more moderate differences were found for developmental tfs such as six, pax, nrl. mef and nkx. in general, for every bm that passed fdr of q = over 80% of the bss on the kbp upstream region are located on alu. on the other hand, for the remaining pssms the majority of bss were not located on alu, like irf and hnf. still, even for these tfs, with mainly off-alu bms, the alu repeats provide between hundreds to thousands of putative bss in the kbp upstream regions. as mentioned above, in the bp upstream region, which constitutes the proximal promoter and hence is believed to be most important for regulation, the alu density is below the genome average. to study this region in more detail, we repeated the analysis that was done for the bp region, and compared the distributions of bss on and off alu in the first bp upstream to the tss. only out of the bms had more on alu than off alu bms. these bms include almost all homeodomain proteins, lxre. lyf- and pax. in spite of the relatively low alu density, on-alu bms do constitute a sizeable fraction of the putative bms that are found in the bp long upstream region. genes of different biological processes contain different amounts of bss as a result of differences in their alu content we used the results of the search for on and off alu bms to look for differences between genes that belong to different biological processes. as expected, for those biological processes whose genes were previously found to be enriched with alu, we also found that a high percentage of their regulatory signals were on-alu. the alu rich groups in this category include metabolic and bio-synthesis associated genes. since for several tfs a large majority of bms reside on alu, we find that alu-rich functional families have a large number of these alu-associated bms, whereas alu-poor biological processes will have less of these bms. for example, the average number of putative bss of pitx is almost times greater in protein biosynthetic genes than in cns developmental genes. interestingly, as a result, developmental tfs such as lyf- pax mef2d and pitx that have on-alu bms, appear less in the upstream region of developmental genes than in the alu-rich non-developmental group of protein biosynthesis related genes. one possible explanation of this may be that the developmental tfs main role is to suppress pathways that are in competition with or exclusive of the developmental process. one should emphasize that the statements made above are based mainly on in-silico evidence, which must be supplemented by firm experimental observations of binding and functional roles of the bound tfs. inter-species comparisons: b the murine'alu like' elements, contain several bss that are similar to the primate alu bss we scanned the b consensus sequences that were downloaded from repbase. these sequences were less enriched with bss that pass the t threshold; this might be due to the fact that the length of the b sequences is only half that of alu. twenty three binding motifs passed t on b and we found that of these passed t also in alu, as expected from the similarity between b and alu. one of these shared bss was ttaatccc where g appears in all alu families and t appears in the consensus of b1s. this bs is the target of otx, a homeodomain protein in both human and mouse. an additional homeodomain protein, crx, is also bound to this bs in mouse; it did not pass t in alu. the members of the mef family of bms appear in mouse with a weaker score than in alu,. the pssm with the most significant score on b was pax, which is not surprising since this pssm is built from those bss of pax that were known to reside on b. in addition, a group of bms that are related to nuclear factors and response proteins passed t on b but not on alu. this is surprising since there are experiments that validated that the tfs that correspond to these bms bind to primate alu. the distributions of b in mouse promoters and alu in human promoters are positively correlated when the mouse genome was first sequenced, regions orthologous to human dna were identified. the densities of b and b in mouse and alu in human were measured in windows of bp and the alu density in humans was found to be correlated with b and b density in mouse. a high correlation was quite unexpected, since alu and the mouse sine elements b and b spread in the rodent and human genomes after the divergence of rodents and primates. we checked whether this trend is present also in the regulatory regions of genes, of size kbp, and we found a positive correlation between b and alu densities even in this relatively short region. in more than pairs of orthologous genes, that had available upstream sequences in both human and mouse, we found positive correlation of. in addition we ordered the genes by increasing alu content in the upstream bp region, and divided the genes on this basis into sets, with each set containing genes. we then computed the average alu and b associated nucleotide densities for each of the sets, and plotted the two average alu/b contents. each point in fig represents a group of genes, with the x coordinates representing the alu densities in human. the monotonic behaviour evident in fig indicates that the alu and b contents of orthologous genes are largely similar in human and mouse. this similarity is far from being obvious, since spreading of alu and b across the genome is believed to have happened after the divergence of primates and rodents.  summary this research suggests that evolution used transposable elements to insert modules of transcription factor binding motifs into promoters and, by means of their presence, assemble higher level regulatory networks. in order to explore this question we focused on alu elements, which are good potential candidates to be part of the building blocks of regulatory networks for two reasons. first, alu elements are abundant in the upstream region of the tss of genes, and second, alu elements contain dozens of putative bss for tfs. some of these bss were found before and their association with alu was also reported, whereas in some cases although the bss were found, the fact that they reside on alu went unnoticed. finally, we list here also bss on alu that were not identified previously. our findings imply that the biological pathway on which alu-mediated regulation appears to have the most significant impact is the development process. many of the tfs that have binding motifs on alu are associated with development; moreover, some of these bss were previously demonstrated to be functional in vivo and essential to regulation of some target genes. we have also shown that a few subsequences on alu, that were known to be highly conserved, provide many of the binding sites for the transcription factors. we studied the alu distribution in the upstream region of genes and found that while alu are "repelled" from the bp long region immediately preceding the transcription starts site, the bp long sequence further upstream is enriched. the alu content in the promoters varies between functional families of genes. comparison of alu in human and b in mice shows a significant correlation between their distributions, and high similarity between the binding motifs that reside on the two mobile elements. several particular cases were discussed in detail. future directions most importantly, more experiments are needed to verify that the bms that were found by us indeed bind their corresponding tfs and, furthermore, to verify their regulatory roles. of particular interest is the role of alu in developmental processes. we found that promoters of genes involved in biosynthesis and metabolism are enriched in bms of several developmental tfs. it is our belief that since development and cell proliferation are opposing processes, these bms play an inhibitory role, suppressing proliferation during differentiation. this hypothesis is supported by the known suppressory role of gfi but the presently known activities on pitx and nkx do not fit this picture. further experiments that address this issue by measuring the regulatory effects of other alu-associated developmental tfs are needed. clearly, many of the genes and their functions date earlier than the rodent/primate divergence, whereas the alu insertions happened later. this poses an open and debated question about the mechanism by which pre-existing regulatory circuits must have been replaced, modified or supplemented by alu insertions. 
we performed a search for new putative bss on alu elements, using a database of position specific score matrices. we found dozens of new putative bss. we propose that evolution used transposable elements to insert tf binding motifs into promoter regions. since development and cell proliferation were proposed to be opposing processes, these tfs possibly play inhibitory roles, suppressing proliferation during differentiation. we found that the upstream regions of the tss are enriched with alu elements, and the alu consensus sequences contain dozens of putative bss for tfs. for several tfs most of the putative bss reside on alu; a few of these were previously found and their association with alu was also reported. this hypothesis is gaining strength due to findings that indicate involvement of alu elements in a variety of functions; for example, alu sequences were found to contain several functional transcription factor binding sites. we searched consensus alu sequences as well as specific alu elements that appear on the kbp regions upstream to the transcription start site of about genes. we observed enrichment of biosynthesis genes with alu-associated bss of developmental tfs. while metabolism-related genes were shown to be enriched with alu, in structural genes alu elements are under-represented. such observations led researchers to suggest that alu elements were involved in gene regulation and were selected to be present in some genes and absent from others. finally, we found a correlation between the mouse b and human alu densities within the corresponding upstream regions of orthologous genes. interestingly, many of the corresponding tfs are associated with early markers of development, even though the upstream regions of development-related genes are alu-poor, compared with translational and protein biosynthesis related genes, which are alu-rich.
 the human genome contains over one million alu repeat elements whose distribution is not uniform. while metabolism-related genes were shown to be enriched with alu, in structural genes alu elements are under-represented. such observations led researchers to suggest that alu elements were involved in gene regulation and were selected to be present in some genes and absent from others. this hypothesis is gaining strength due to findings that indicate involvement of alu elements in a variety of functions; for example, alu sequences were found to contain several functional transcription factor binding sites. we performed a search for new putative bss on alu elements, using a database of position specific score matrices. we searched consensus alu sequences as well as specific alu elements that appear on the kbp regions upstream to the transcription start site of about genes.  we found that the upstream regions of the tss are enriched with alu elements, and the alu consensus sequences contain dozens of putative bss for tfs. hence several tfs have alu-associated bss upstream of the tss of many genes. for several tfs most of the putative bss reside on alu; a few of these were previously found and their association with alu was also reported. in four cases the fact that the identified bss resided on alu went unnoticed, and we report this association for the first time. we found dozens of new putative bss. interestingly, many of the corresponding tfs are associated with early markers of development, even though the upstream regions of development-related genes are alu-poor, compared with translational and protein biosynthesis related genes, which are alu-rich. finally, we found a correlation between the mouse b and human alu densities within the corresponding upstream regions of orthologous genes.  we propose that evolution used transposable elements to insert tf binding motifs into promoter regions. we observed enrichment of biosynthesis genes with alu-associated bss of developmental tfs. since development and cell proliferation were proposed to be opposing processes, these tfs possibly play inhibitory roles, suppressing proliferation during differentiation. 