BACKGROUND
transposable elements  are abundant inhabitants of eukaryotic genomes, representing e.g.
about 50% of the human genome and up to 90% in some plant species.
long terminal repeat  retrotransposons are most common in plant genomes while animal genomes, including the human genome, are often flooded by non-ltr retrotransposons.
most of the human genome is transcribed and tes therefore greatly contribute to cellular transcriptome and proteome
<cit> .
recent insertions of tes underlie the variability of human populations and can cause several human diseases
<cit> .
somatic retrotranspositions occur during neuronal development
<cit>  and tumorigenesis
<cit> .
during the last two decades, it became widely accepted that tes, as an inherently dynamic genome component, have an important role in both cell functioning
<cit>  and genome evolution
<cit> .
human ltr retrotransposons are represented by endogenous retroviruses  but their activity is currently very limited: most hervs were inserted into the genomes of our ancestors earlier that  <dig> mya
<cit> .
ltr retrotransposons have ltr sequences at both ends, carry gag and pol genes and several regulatory regions like promoter located inside ltr, primer binding site  and polypurine  sites where reverse transcription of the first and second strand of dna starts, respectively.
the majority of human tes result from the present and past activity of non-ltr retrotransposons, including the line- <dig>  alu and sva elements
<cit> .
line- <dig>  have two orfs coding for rna binding protein  and endonuclease and reverse transcriptase .
orfs are flanked with 5’-utr and 3’-utr regions.
there are at least  <dig>  l <dig> copies in the human genome
<cit> .
alu elements are about  <dig> bp long and have dimeric structure formed by the fusion of two monomers derived from 7sl rna gene.
alus were active over the past  <dig> mya and the human genome contains more than  <dig> million copies.
sva elements are about  <dig> kb long and are composed of a hexamer repeat region, vntr region, an alu-like region, a herv-k10-like region and polyadenylation signal ending with oligo-rich tail.
svas were active throughout the last  <dig> mya of hominoid evolution and have about  <dig>  copies
<cit> .
both alu and sva are trans-mobilized by the l <dig> machinery
<cit> .
molecular processes participating in the retrotransposon life cycle are regulated both by enzymes encoded by these elements themselves and by several host factors.
it is probable that the activity of retrotransposons can also be affected by the changes of dna conformation that are known to influence many molecular processes .
formation of multi-stranded dna structures, namely quadruplex dna, is probably involved in dimerization of the hiv- <dig> genomic rna molecules found in virus particles
<cit> .
similarly, long polypurine tract  located in 3’-utr of l <dig> retrotransposons, where reverse transcription of the second cdna strand starts, can form intrastrand quadruplex
<cit> .
relationship between quadruplexes and transposons can be seen in the cleavage of quadruplexes by rag <dig> protein during translocations in human lymphomas
<cit>  because rag <dig> protein evolved from transposase of the transib family of dna transposons
<cit> .
recently, we found
<cit>  that potential quadruplex sequences  are often located inside ltrs of plant ltr retrotransposons at specific distances from their promoter indicating a possible effect of quadruplexes on transcription.
quadruplexes were better preserved in evolutionary young elements which supports their functional role
<cit> .
similar observation was made by savage et al.
<cit>  who found that younger human sva elements contain more pqs sequences than older sva elements but the ability of candidate sequences to adopt quadruplex conformation was not experimentally confirmed.
although quadruplexes were found in many regions of human genome, especially in promoters
, systematic analysis of quadruplexes in all main types of human retrotransposons was lacking.
in this study, we searched for pqs sequences in human line- <dig>  herv, sva and alu elements.
we analyzed the prominent regions of their location as well as the effect of element age and localization on chromosomes.
the ability of candidate motifs to adopt quadruplex was verified by circular dichroism and gel electrophoresis.
RESULTS
potential quadruplex-forming sequences are located in specific regions of human transposable elements
we analyzed the localization of pqss inside main groups of human transposable elements , namely in line- <dig>  alu elements, herv retrotransposons and sva elements.
we searched for the  motif representing potential g-quadruplex cluster inside  <dig>  line- <dig> elements,  <dig> , <dig> alu elements,  <dig>  herv and  <dig>  sva elements or their fragments.
altogether, we found  <dig>  pqs in all annotated repeats or their  <dig> bp flanking sequence .
of those,  <dig>  were associated with the four studied classes .
the overall highest abundance of pqss was observed in sva  followed by line- <dig>  and herv elements .
the occurrence of pqss was lowest in alu elements  , showing pqs distribution and frequency in repeatmasker subfamilies of alu, ervl-malr, ervl, erv <dig>  hat-charlie, l <dig>  l <dig>  mir and sva elements).
in line- <dig> elements, pqss were located almost exclusively in the 3’-utr region.
only very low numbers of pqss were found outside this region .
herv ltr retrotransposons contained pqss along the whole element with accumulation in ltr regions .
in sva elements, pqss were specifically located in hex region in minus strand and along the larger vntr region in plus strand .
the occurrence of pqss inside alu elements was low throughout most of element length .
there was only one peak of pqs in the left part of left monomer .
all mentioned pqs peaks were above the markov model random background threshold, with sva-vntr region being much closer to it than the other pqss, as expected for a long g-rich tandem repeat.
we analyzed the abundance of pqss in line- <dig>  herv, sva and alu elements separately on the y and x chromosomes and on autosomes.
in four main types of elements, the number of quadruplexes was measured on both plus and minus strands for respective chromosomes.
the distribution of pqss along all elements slightly differed between chromosomes being more similar in autosomes and x chromosome and different in the y chromosome where peaks of pqss were located in different parts of elements than in autosomes and the x chromosome .
the most striking difference between pqs distribution or frequency was observed in the sva family of transposable elements, where those on the y chromosome had a reduced pqs content .
intriguingly, we noticed an increased occurrence of pqss in the orf <dig> region of line- <dig> elements from chromosome x and y compared to their autosome counterparts .figure  <dig>
occurrence of pqss along human line- <dig> , herv , sva  and alu .
the density of pqs clusters containing a minimum of four adequately spaced ggg groups in the sense strand  and antisense strand  visualized along line- <dig> , herv , sva  and alu elements .
sliding window covered between 40- <dig> bp of the element length.
frequency represents the number of pqss in such window in the entire family.
green boxes show annotation with main structural components from typical full-length elements .
chromosomes are visualized separately where autosomes are in yellow, x chromosomes in red and y chromosomes in blue.
the dashed red line shows pqs frequency in randomized control sequences generated by an equivalent 2nd-order markov chain model.
the black triangles on the right show reference densities of pqs sites in the entire human genome recalculated into the coordinates of the given family.
the abundance of pqss inside line- <dig>  sva and alu elements related to the element length, activity or age.
the abundance of pqss inside all line- <dig>  full-length lines and active lines  located on autosomes and x and y chromosomes.
the abundance of pqss inside sva of different age  located on autosomes and the x and y chromosomes.
the abundance of pqss upstream inside and downstream of alu elements of different age  located on autosomes and the x and y chromosomes.
we clustered pqss from individual families to determine the most common patterns of guanines.
we have chosen  <dig> specific motifs for each te type among the most common pqs motifs and used them for dna conformational studies .
the selected sequences originated from the 5’-utr and 3’-utr regions of line- <dig>  ltr and gag-pol gene of herv, hex and vntr regions of sva and left part of the left monomer of alu .table  <dig>
oligonucleotides used in this study
names and the sequences of oligonucleotides are shown.
clusters of three or more guanines are shown as bold.
the abundance of pqss in the neighborhood of transposable elements
we compared the abundance of pqss inside and in the vicinity of line- <dig>  herv, sva and alu elements.
in full-length line- <dig> elements, the density of pqss was markedly higher inside elements than in element vicinity.
the greater abundance of pqss inside line- <dig> elements compared to the element vicinity was observed only in plus strand while the neighborhood contained more pqss than element when minus strand was analyzed .
elements with the 3’-utr pqs were much less likely to have the pqss in the 3’ downstream flanking region .
in herv elements, many more pqss were present inside elements than in their neighborhood, especially when full-length elements were taken into account .
high enrichment of elements compared to their neighborhood was also observed in sva elements .
this trend was stronger in plus than in minus strand.
in minus strand, sva contained more pqss upstream than downstream of elements.
alu elements differed from line- <dig>  herv and sva.
the density of pqss inside alu elements was lower than in regions located upstream and downstream .figure  <dig>
the density of pqss inside tes and in tes vicinity.
the number of pqss per  <dig> bp of zone located inside, upstream and downstream of line- <dig> , herv , sva  and alu elements .
for each element type pqs in plus strand  and minus strand  are shown.
we analyzed either all elements  or only full-length elements .
the abundance of pqss within transposable elements of different age and activity
we compared the pqs abundance in all line- <dig> elements, full-length line- <dig> and transcriptionally active line- <dig> families
<cit> .
we found that full-length lines contained much more pqss than truncated line elements .
among full-length elements, the transcriptionally active l1hs and l1pa <dig> families contained more pqss than was the average abundance of pqss inside full-length lines.
truncated l1hs and l1pa <dig> homologues contained much less pqss.
these trends were observed both on autosomes and on x and y sex chromosomes.
we analyzed the abundance of pqss inside sva elements of different age - sva-a  to sva-f .
we found that the abundance of pqss was higher in younger elements  than in older elements  and this trend was same both in autosomes and sex chromosomes .
the abundance of pqss was highest in middle-aged sva elements .
the pqss were common in the central part of elements in plus strand.
detailed analysis revealed that in older elements, the pqs abundance in the central part of plus strand decreased and predominated in the left part of sva in the minus strand .
the peak of pqss in sva-e present on the y chromosome was caused by the low number of elements and the sva-f elements even absented on the y chromosome.
we made similar analysis of alu elements where alu-j are oldest, alu-s are middle-aged and alu-y are youngest elements.
we found that in contrast to line- <dig> and sva, the age did not markedly affect the abundance of pqss inside alu elements.
there is a slight pqs-increasing trend with age in the main families, however the youngest subfamilies
<cit>  are also depleted of pqss .
because alu elements have more pqss in their vicinity than inside elements  we also analyzed the upstream and downstream regions.
we found that older alu elements contained more pqss than younger elements in their downstream regions .
the pqs abundance did not differ markedly between autosomes and sex chromosomes, a small decrease in pqss on the y chromosome was registered .
the most active families of alu  had lower abundance of pqss than average alu-y elements.
pqss can form quadruplexes as revealed by circular dichroism
we probed dna conformational properties of  <dig> oligonucleotides  representing pqss obtained from sva, herv, line- <dig> and alu elements by circular dichroism .
we tested their ability to form quadruplex structures upon increasing concentration of potassium ions.
first, we measured cd spectra of pqss originating from sva elements because they contain pqss more often than any other human tes.
we divided sva elements into three families with different age - oldest sva-a family, middle-aged sva-c family and youngest sva-f family - and for each family we analyzed the ability of one hex region and one vntr region to adopt a quadruplex structure.
vntr consensus sequences in older families were always present in younger families as well, therefore we used consensus sequences common for multiple families - sva-bf for families b to f and sva-cf for families c to f. only sva-f vntr oligonucleotide was specific for the youngest sva family.
as shown in figure
4a, the positive cd band at about  <dig> nm, which is characteristic of the presence of a parallel quadruplex
<cit> , increased steeply and at lower potassium concentrations with sva-f  than with sva-c .
much less increase in this band and only at the highest k+ concentrations used was observed with the older sva-a family and the common consensus oligonucleotides sva-bf and sva-cf.
similarly, the thermal stability of quadruplexes was highest in sva-f and lowest in sva-a .
native gel electrophoresis at  <dig> mm k+ showed that hex region of three groups of sva adopted bimolecular quadruplexes .
vntr region provided cd spectra of the b-dna type at low k+ concentrations marked out by low amplitudes and a slightly predominating  <dig> nm band, which is characteristic of duplexes of g-rich and c-rich dna strands
<cit> .
these monomolecular structures  may thus correspond to hairpins containing rather accidental, namely g.c, base pairs.
the increase in the  <dig> nm band with increasing k+ concentration indicating quadruplex formation was again most obvious with sva-f and less so with sva-bf and sva-cf.
the quadruplexes were formed non-cooperatively and not much willingly.
pqss originating from the line- <dig> elements were selected from the 5’-utr and 3’-utr regions .
the pqss from the 5’-utr  provided cd spectrum corresponding to antiparallel quadruplex , while the cd spectrum of the pqs from 3’-utr  corresponded to that of the parallel-stranded quadruplex .
native page revealed that both the anti-parallel l1_ <dig> and the parallel l1_ <dig> quadruplexes were monomolecular at low as well as at room temperature .
antiparallel folding of the l1_ <dig> quadruplex was enabled by the sufficiently long  loops between all four g blocks.
two pqss were selected from herv elements.
the first pqs corresponded to a minor pqs peak in the ltr in the minus strand  and the second pqs originated from the gag-pol region of the minus strand .
cd measurements indicated gradual formation of parallel-stranded quadruplexes with both pqss .
native page revealed that a monomolecular quadruplex structure dominated in both, herv_ <dig> and herv_ <dig> at room temperature, while a bimolecular quadruplex, in addition to two types of monomolecular ones, were formed by herv_ <dig> at low temperatures .
in alu elements, two pqss  were selected for cd measurements, both from the left part of left monomer located in plus strand .
both pqss corresponded to the middle-aged alu elements .
although cd spectra of both oligonucleotides indicated the formation of parallel-stranded quadruplex, the spectral changes induced by the increasing potassium concentration were gradual and limited in the case of alu-s_ <dig>  this along with the shoulder on the long wavelength part of the positive 260nm cd band  indicates that a substantial part of alu-s_ <dig> sequence formed a hairpin.
alu-s_ <dig> formed the quadruplex at much lower potassium concentration  and the transition was highly cooperative.
the quadruplex was parallel and intramolecular in the same way as was the quadruplex of alu-s_ <dig> .
note that the mobility of the studied quadruplexes is slower than would correspond to their length markers.
this is usually the case with the heavy g-rich strands.
moreover, the mobilities of the intramolecular qudruplexes differ , which may be partly a consequence of their distinct compactness, and mainly, by distinct hindering effects of the overlapping nucleotides not involved in the quadruplex structure.
in addition, we measured pqss selected from older and younger alu families  but we found no correlation between susceptibility to form quadruplex and the age of alu elements.figure  <dig>
circular dichroism measurements and gel electrophoreses of pqss from sva elements of different age.
cd spectra of sva-a, sva-c and sva-f  obtained at various concentrations of potassium ions and at various temperatures are marked by different colors.
the peak at  <dig> nm corresponds to a parallel-stranded quadruplex.
sketches correspond to the most probable folding of the dominating quadruplex structure according to cd and electrophoretic results.
native gel electrophoreses of sva pqs in the presence of  <dig> mm kcl at 20°c or 1°c.
circular dichroism measurements and native gel electrophoreses of pqss from line- <dig>  herv and alu elements.
cd spectra of the oligonucleotides  obtained at various concentrations of potassium and at various temperatures are marked by different colors.
the peak at  <dig> nm indicates formation of the parallel-stranded quadruplex while maximum at  <dig> nm corresponds to an antiparallel-stranded quadruplex.
sketches correspond to the most probable folding of the dominating quadruplex structure according to cd and electrophoretic results.
native gel electrophoreses of line- <dig>  herv and alu pqs in the presence of  <dig> mm kcl at 20°c or 1°c.
discussion
we found that potential quadruplex-forming sequences are located in specific regions of human transposable elements and experimentally verified the ability of such sequences to adopt quadruplex dna conformation.
full-length and active l <dig> elements and younger sva elements had a larger number of pqss.
the propensity of these sequences to form quadruplex and quadruplex stability  were higher than in older elements.
alu elements contained pqss not inside but in their neighborhood where more pqss were present in downstream regions of older elements.
two available counts of g4-quadruplexes in the entire human genome found about  <dig>  pqss
<cit> .
this allows us to express our numbers as proportions of mobile element pqss to whole-genome pqs content with a value of 71%.
the four main classes of elements studied here carry 49% of total predicted pqss.
these numbers reflect the current human genome sequencing and annotation status and are very likely to miss potential pqss in centromeres, telomeres or other difficult-to-map regions of the human genome.
our results are in agreement with savage et al.
<cit>  who also found that the youngest sva  contained more quadruplexes than older elements.
such age-dependent distribution of pqss  can be explained by the action of constraints leading to fixation of quadruplexes in recent and active elements while non-active older elements accumulate mutations that hinder quadruplex formation.
moreover, we found that quadruplexes are present in the central part of sva elements in plus strand and in the left part of minus strand.
if the localization of quadruplexes in plus strand has negative effect on transcription and their presence in minus strand has a positive effect
<cit> , then the potential evolutionary balancing of quadruplexes abundance  in complementary strands could regulate element activity over time.
the greater abundance of pqss  in the neighborhood of older alu elements is probably related to generally high gc-content of isochores containing older alus
<cit> .
surprisingly, despite the age-dependent increase of gc-content of alu neighborhood, the abundance of pqss inside alu elements was very low  and did not increase with the element age .
we have shown that pqss are strongly accumulated in 3’-utr of line- <dig> elements.
quadruplexes located in 3’-utr can have an effect on target-primed reverse transcription  that starts at the 3’ end.
quadruplexes formed either by rna template or by the growing first dna strand can represent a barrier for reverse transcription.
however, quadruplex dna can regulate not only the transposable element itself but can also influence neighboring genes as was proposed recently by kejnovsky and lexa
<cit> .
because sva elements are preferentially located inside genes or in their neighborhood
<cit>  we suggest that recent sva elements could spread quadruplex motifs close to genes or into genes and in this way they regulate expression of these genes.
the regulatory potential of quadruplexes inside tes decreases as the element gets older and is eroded by mutations and rearrangements.
in this way, quadruplexes can enlarge the potential of transposable elements to respond to environmental challenges as was suggested by mcclintock
<cit>  long time ago.
quadruplexes carried by tes can also affect other cellular processes like replication or epigenetic regulation.
it is remarkable that quadruplexes are located close to the line- <dig> poly tail that represents the labile region of duplex dna.
other labile  regions are represented by replication origins and, surprisingly, also here quadruplexes are located
<cit> .
because quadruplexes also represent barriers for replication, or at least can slow it down, the spreading of pqss by retrotransposons can also contribute to the regulation of replication speed.
in addition, the quadruplexes can represent epigenetic marks in large introns that contain repetitive dna and are also at-rich
<cit> .
moreover, if non-b dna conformations are nucleosome-free
<cit>  and some transposable elements are preferentially inserted into naked dna
<cit> , then one would expect that such regions could represent sites for nested insertions, at least in some te families.
several proteins were shown to bind quadruplex dna
<cit> .
for example, p <dig> protein, that has binding sites inside human alu and l <dig> elements
<cit> , can strongly bind quadruplex dna
<cit> .
another example is the recombination and repair protein ku <dig> that was shown to bind cdna of ty <dig> yeast retrotransposons
<cit>  and has high affinity to quadruplex dna
<cit> .
in this context, it is interesting that human lines have many ku70/ <dig> binding sites
<cit> .
taken together, the remarkable ability of some proteins to bind both tes and quadruplex dna underlining the relationship of these unusual dna conformations with transposable elements as well as the higher abundance of pqss inside younger, full-length and active elements indicates the role of quadruplexes in te spreading.
such a role can consist in negative or positive regulation of te activity, e.g.
in response to current intracellular ionic conditions influencing the stability of quadruplexes.
in the long-term perspective, quadruplexes can represent an evolutionary feedback suppressing non-controlled amplification of active elements.
CONCLUSIONS
the results suggest that activity of transposable elements, especially line- <dig> and sva elements, contributes towards genome-wide quadruplex distribution in human.
conservation of quadruplexes at specific positions implies their function either in the life cycle of transposable elements or host genome maintenance, or both.
all tested pqss were able to form quadruplex structure in vitro, albeit with differing willingness, strand orientation and molecularity.
line- <dig> and sva families displayed an age-dependent pattern with younger elements containing a higher number of more stable quadruplexes.
further studies should be done to determine how the conserved elements are selected for during evolution.

 transposable elements are abundant inhabitants of eukaryotic genomes, representing e.g. about 50% of the human genome and up to 90% in some plant species. long terminal repeat retrotransposons are most common in plant genomes while animal genomes, including the human genome, are often flooded by non-ltr retrotransposons. most of the human genome is transcribed and tes therefore greatly contribute to cellular transcriptome and proteome. recent insertions of tes underlie the variability of human populations and can cause several human diseases. somatic retrotranspositions occur during neuronal development and tumorigenesis. during the last two decades, it became widely accepted that tes, as an inherently dynamic genome component, have an important role in both cell functioning and genome evolution. human ltr retrotransposons are represented by endogenous retroviruses but their activity is currently very limited: most hervs were inserted into the genomes of our ancestors earlier that mya. ltr retrotransposons have ltr sequences at both ends, carry gag and pol genes and several regulatory regions like promoter located inside ltr, primer binding site and polypurine sites where reverse transcription of the first and second strand of dna starts, respectively. the majority of human tes result from the present and past activity of non-ltr retrotransposons, including the line- alu and sva elements. line- have two orfs coding for rna binding protein and endonuclease and reverse transcriptase. orfs are flanked with 5’-utr and 3’-utr regions. there are at least l copies in the human genome. alu elements are about bp long and have dimeric structure formed by the fusion of two monomers derived from 7sl rna gene. alus were active over the past mya and the human genome contains more than million copies. sva elements are about kb long and are composed of a hexamer repeat region, vntr region, an alu-like region, a herv-k10-like region and polyadenylation signal ending with oligo-rich tail. svas were active throughout the last mya of hominoid evolution and have about copies. both alu and sva are trans-mobilized by the l machinery. molecular processes participating in the retrotransposon life cycle are regulated both by enzymes encoded by these elements themselves and by several host factors. it is probable that the activity of retrotransposons can also be affected by the changes of dna conformation that are known to influence many molecular processes. formation of multi-stranded dna structures, namely quadruplex dna, is probably involved in dimerization of the hiv- genomic rna molecules found in virus particles. similarly, long polypurine tract located in 3’-utr of l retrotransposons, where reverse transcription of the second cdna strand starts, can form intrastrand quadruplex. relationship between quadruplexes and transposons can be seen in the cleavage of quadruplexes by rag protein during translocations in human lymphomas because rag protein evolved from transposase of the transib family of dna transposons. recently, we found that potential quadruplex sequences are often located inside ltrs of plant ltr retrotransposons at specific distances from their promoter indicating a possible effect of quadruplexes on transcription. quadruplexes were better preserved in evolutionary young elements which supports their functional role. similar observation was made by savage et al. who found that younger human sva elements contain more pqs sequences than older sva elements but the ability of candidate sequences to adopt quadruplex conformation was not experimentally confirmed. although quadruplexes were found in many regions of human genome, especially in promoters, systematic analysis of quadruplexes in all main types of human retrotransposons was lacking. in this study, we searched for pqs sequences in human line- herv, sva and alu elements. we analyzed the prominent regions of their location as well as the effect of element age and localization on chromosomes. the ability of candidate motifs to adopt quadruplex was verified by circular dichroism and gel electrophoresis.  potential quadruplex-forming sequences are located in specific regions of human transposable elements we analyzed the localization of pqss inside main groups of human transposable elements, namely in line- alu elements, herv retrotransposons and sva elements. we searched for the motif representing potential g-quadruplex cluster inside line- elements,, alu elements, herv and sva elements or their fragments. altogether, we found pqs in all annotated repeats or their bp flanking sequence. of those, were associated with the four studied classes. the overall highest abundance of pqss was observed in sva followed by line- and herv elements. the occurrence of pqss was lowest in alu elements, showing pqs distribution and frequency in repeatmasker subfamilies of alu, ervl-malr, ervl, erv hat-charlie, l l mir and sva elements). in line- elements, pqss were located almost exclusively in the 3’-utr region. only very low numbers of pqss were found outside this region. herv ltr retrotransposons contained pqss along the whole element with accumulation in ltr regions. in sva elements, pqss were specifically located in hex region in minus strand and along the larger vntr region in plus strand. the occurrence of pqss inside alu elements was low throughout most of element length. there was only one peak of pqs in the left part of left monomer. all mentioned pqs peaks were above the markov model random background threshold, with sva-vntr region being much closer to it than the other pqss, as expected for a long g-rich tandem repeat. we analyzed the abundance of pqss in line- herv, sva and alu elements separately on the y and x chromosomes and on autosomes. in four main types of elements, the number of quadruplexes was measured on both plus and minus strands for respective chromosomes. the distribution of pqss along all elements slightly differed between chromosomes being more similar in autosomes and x chromosome and different in the y chromosome where peaks of pqss were located in different parts of elements than in autosomes and the x chromosome. the most striking difference between pqs distribution or frequency was observed in the sva family of transposable elements, where those on the y chromosome had a reduced pqs content. intriguingly, we noticed an increased occurrence of pqss in the orf region of line- elements from chromosome x and y compared to their autosome counterparts.figure occurrence of pqss along human line-, herv, sva and alu. the density of pqs clusters containing a minimum of four adequately spaced ggg groups in the sense strand and antisense strand visualized along line-, herv, sva and alu elements. sliding window covered between 40- bp of the element length. frequency represents the number of pqss in such window in the entire family. green boxes show annotation with main structural components from typical full-length elements. chromosomes are visualized separately where autosomes are in yellow, x chromosomes in red and y chromosomes in blue. the dashed red line shows pqs frequency in randomized control sequences generated by an equivalent 2nd-order markov chain model. the black triangles on the right show reference densities of pqs sites in the entire human genome recalculated into the coordinates of the given family. the abundance of pqss inside line- sva and alu elements related to the element length, activity or age. the abundance of pqss inside all line- full-length lines and active lines located on autosomes and x and y chromosomes. the abundance of pqss inside sva of different age located on autosomes and the x and y chromosomes. the abundance of pqss upstream inside and downstream of alu elements of different age located on autosomes and the x and y chromosomes. we clustered pqss from individual families to determine the most common patterns of guanines. we have chosen specific motifs for each te type among the most common pqs motifs and used them for dna conformational studies. the selected sequences originated from the 5’-utr and 3’-utr regions of line- ltr and gag-pol gene of herv, hex and vntr regions of sva and left part of the left monomer of alu.table oligonucleotides used in this study names and the sequences of oligonucleotides are shown. clusters of three or more guanines are shown as bold. the abundance of pqss in the neighborhood of transposable elements we compared the abundance of pqss inside and in the vicinity of line- herv, sva and alu elements. in full-length line- elements, the density of pqss was markedly higher inside elements than in element vicinity. the greater abundance of pqss inside line- elements compared to the element vicinity was observed only in plus strand while the neighborhood contained more pqss than element when minus strand was analyzed. elements with the 3’-utr pqs were much less likely to have the pqss in the 3’ downstream flanking region. in herv elements, many more pqss were present inside elements than in their neighborhood, especially when full-length elements were taken into account. high enrichment of elements compared to their neighborhood was also observed in sva elements. this trend was stronger in plus than in minus strand. in minus strand, sva contained more pqss upstream than downstream of elements. alu elements differed from line- herv and sva. the density of pqss inside alu elements was lower than in regions located upstream and downstream.figure the density of pqss inside tes and in tes vicinity. the number of pqss per bp of zone located inside, upstream and downstream of line-, herv, sva and alu elements. for each element type pqs in plus strand and minus strand are shown. we analyzed either all elements or only full-length elements. the abundance of pqss within transposable elements of different age and activity we compared the pqs abundance in all line- elements, full-length line- and transcriptionally active line- families. we found that full-length lines contained much more pqss than truncated line elements. among full-length elements, the transcriptionally active l1hs and l1pa families contained more pqss than was the average abundance of pqss inside full-length lines. truncated l1hs and l1pa homologues contained much less pqss. these trends were observed both on autosomes and on x and y sex chromosomes. we analyzed the abundance of pqss inside sva elements of different age - sva-a to sva-f. we found that the abundance of pqss was higher in younger elements than in older elements and this trend was same both in autosomes and sex chromosomes. the abundance of pqss was highest in middle-aged sva elements. the pqss were common in the central part of elements in plus strand. detailed analysis revealed that in older elements, the pqs abundance in the central part of plus strand decreased and predominated in the left part of sva in the minus strand. the peak of pqss in sva-e present on the y chromosome was caused by the low number of elements and the sva-f elements even absented on the y chromosome. we made similar analysis of alu elements where alu-j are oldest, alu-s are middle-aged and alu-y are youngest elements. we found that in contrast to line- and sva, the age did not markedly affect the abundance of pqss inside alu elements. there is a slight pqs-increasing trend with age in the main families, however the youngest subfamilies are also depleted of pqss. because alu elements have more pqss in their vicinity than inside elements we also analyzed the upstream and downstream regions. we found that older alu elements contained more pqss than younger elements in their downstream regions. the pqs abundance did not differ markedly between autosomes and sex chromosomes, a small decrease in pqss on the y chromosome was registered. the most active families of alu had lower abundance of pqss than average alu-y elements. pqss can form quadruplexes as revealed by circular dichroism we probed dna conformational properties of oligonucleotides representing pqss obtained from sva, herv, line- and alu elements by circular dichroism. we tested their ability to form quadruplex structures upon increasing concentration of potassium ions. first, we measured cd spectra of pqss originating from sva elements because they contain pqss more often than any other human tes. we divided sva elements into three families with different age - oldest sva-a family, middle-aged sva-c family and youngest sva-f family - and for each family we analyzed the ability of one hex region and one vntr region to adopt a quadruplex structure. vntr consensus sequences in older families were always present in younger families as well, therefore we used consensus sequences common for multiple families - sva-bf for families b to f and sva-cf for families c to f. only sva-f vntr oligonucleotide was specific for the youngest sva family. as shown in figure 4a, the positive cd band at about nm, which is characteristic of the presence of a parallel quadruplex, increased steeply and at lower potassium concentrations with sva-f than with sva-c. much less increase in this band and only at the highest k+ concentrations used was observed with the older sva-a family and the common consensus oligonucleotides sva-bf and sva-cf. similarly, the thermal stability of quadruplexes was highest in sva-f and lowest in sva-a. native gel electrophoresis at mm k+ showed that hex region of three groups of sva adopted bimolecular quadruplexes. vntr region provided cd spectra of the b-dna type at low k+ concentrations marked out by low amplitudes and a slightly predominating nm band, which is characteristic of duplexes of g-rich and c-rich dna strands. these monomolecular structures may thus correspond to hairpins containing rather accidental, namely g.c, base pairs. the increase in the nm band with increasing k+ concentration indicating quadruplex formation was again most obvious with sva-f and less so with sva-bf and sva-cf. the quadruplexes were formed non-cooperatively and not much willingly. pqss originating from the line- elements were selected from the 5’-utr and 3’-utr regions. the pqss from the 5’-utr provided cd spectrum corresponding to antiparallel quadruplex, while the cd spectrum of the pqs from 3’-utr corresponded to that of the parallel-stranded quadruplex. native page revealed that both the anti-parallel l1_ and the parallel l1_ quadruplexes were monomolecular at low as well as at room temperature. antiparallel folding of the l1_ quadruplex was enabled by the sufficiently long loops between all four g blocks. two pqss were selected from herv elements. the first pqs corresponded to a minor pqs peak in the ltr in the minus strand and the second pqs originated from the gag-pol region of the minus strand. cd measurements indicated gradual formation of parallel-stranded quadruplexes with both pqss. native page revealed that a monomolecular quadruplex structure dominated in both, herv_ and herv_ at room temperature, while a bimolecular quadruplex, in addition to two types of monomolecular ones, were formed by herv_ at low temperatures. in alu elements, two pqss were selected for cd measurements, both from the left part of left monomer located in plus strand. both pqss corresponded to the middle-aged alu elements. although cd spectra of both oligonucleotides indicated the formation of parallel-stranded quadruplex, the spectral changes induced by the increasing potassium concentration were gradual and limited in the case of alu-s_ this along with the shoulder on the long wavelength part of the positive 260nm cd band indicates that a substantial part of alu-s_ sequence formed a hairpin. alu-s_ formed the quadruplex at much lower potassium concentration and the transition was highly cooperative. the quadruplex was parallel and intramolecular in the same way as was the quadruplex of alu-s_. note that the mobility of the studied quadruplexes is slower than would correspond to their length markers. this is usually the case with the heavy g-rich strands. moreover, the mobilities of the intramolecular qudruplexes differ, which may be partly a consequence of their distinct compactness, and mainly, by distinct hindering effects of the overlapping nucleotides not involved in the quadruplex structure. in addition, we measured pqss selected from older and younger alu families but we found no correlation between susceptibility to form quadruplex and the age of alu elements.figure circular dichroism measurements and gel electrophoreses of pqss from sva elements of different age. cd spectra of sva-a, sva-c and sva-f obtained at various concentrations of potassium ions and at various temperatures are marked by different colors. the peak at nm corresponds to a parallel-stranded quadruplex. sketches correspond to the most probable folding of the dominating quadruplex structure according to cd and electrophoretic results. native gel electrophoreses of sva pqs in the presence of mm kcl at 20°c or 1°c. circular dichroism measurements and native gel electrophoreses of pqss from line- herv and alu elements. cd spectra of the oligonucleotides obtained at various concentrations of potassium and at various temperatures are marked by different colors. the peak at nm indicates formation of the parallel-stranded quadruplex while maximum at nm corresponds to an antiparallel-stranded quadruplex. sketches correspond to the most probable folding of the dominating quadruplex structure according to cd and electrophoretic results. native gel electrophoreses of line- herv and alu pqs in the presence of mm kcl at 20°c or 1°c. discussion we found that potential quadruplex-forming sequences are located in specific regions of human transposable elements and experimentally verified the ability of such sequences to adopt quadruplex dna conformation. full-length and active l elements and younger sva elements had a larger number of pqss. the propensity of these sequences to form quadruplex and quadruplex stability were higher than in older elements. alu elements contained pqss not inside but in their neighborhood where more pqss were present in downstream regions of older elements. two available counts of g4-quadruplexes in the entire human genome found about pqss. this allows us to express our numbers as proportions of mobile element pqss to whole-genome pqs content with a value of 71%. the four main classes of elements studied here carry 49% of total predicted pqss. these numbers reflect the current human genome sequencing and annotation status and are very likely to miss potential pqss in centromeres, telomeres or other difficult-to-map regions of the human genome. our results are in agreement with savage et al. who also found that the youngest sva contained more quadruplexes than older elements. such age-dependent distribution of pqss can be explained by the action of constraints leading to fixation of quadruplexes in recent and active elements while non-active older elements accumulate mutations that hinder quadruplex formation. moreover, we found that quadruplexes are present in the central part of sva elements in plus strand and in the left part of minus strand. if the localization of quadruplexes in plus strand has negative effect on transcription and their presence in minus strand has a positive effect, then the potential evolutionary balancing of quadruplexes abundance in complementary strands could regulate element activity over time. the greater abundance of pqss in the neighborhood of older alu elements is probably related to generally high gc-content of isochores containing older alus. surprisingly, despite the age-dependent increase of gc-content of alu neighborhood, the abundance of pqss inside alu elements was very low and did not increase with the element age. we have shown that pqss are strongly accumulated in 3’-utr of line- elements. quadruplexes located in 3’-utr can have an effect on target-primed reverse transcription that starts at the 3’ end. quadruplexes formed either by rna template or by the growing first dna strand can represent a barrier for reverse transcription. however, quadruplex dna can regulate not only the transposable element itself but can also influence neighboring genes as was proposed recently by kejnovsky and lexa. because sva elements are preferentially located inside genes or in their neighborhood we suggest that recent sva elements could spread quadruplex motifs close to genes or into genes and in this way they regulate expression of these genes. the regulatory potential of quadruplexes inside tes decreases as the element gets older and is eroded by mutations and rearrangements. in this way, quadruplexes can enlarge the potential of transposable elements to respond to environmental challenges as was suggested by mcclintock long time ago. quadruplexes carried by tes can also affect other cellular processes like replication or epigenetic regulation. it is remarkable that quadruplexes are located close to the line- poly tail that represents the labile region of duplex dna. other labile regions are represented by replication origins and, surprisingly, also here quadruplexes are located. because quadruplexes also represent barriers for replication, or at least can slow it down, the spreading of pqss by retrotransposons can also contribute to the regulation of replication speed. in addition, the quadruplexes can represent epigenetic marks in large introns that contain repetitive dna and are also at-rich. moreover, if non-b dna conformations are nucleosome-free and some transposable elements are preferentially inserted into naked dna, then one would expect that such regions could represent sites for nested insertions, at least in some te families. several proteins were shown to bind quadruplex dna. for example, p protein, that has binding sites inside human alu and l elements, can strongly bind quadruplex dna. another example is the recombination and repair protein ku that was shown to bind cdna of ty yeast retrotransposons and has high affinity to quadruplex dna. in this context, it is interesting that human lines have many ku70/ binding sites. taken together, the remarkable ability of some proteins to bind both tes and quadruplex dna underlining the relationship of these unusual dna conformations with transposable elements as well as the higher abundance of pqss inside younger, full-length and active elements indicates the role of quadruplexes in te spreading. such a role can consist in negative or positive regulation of te activity, e.g. in response to current intracellular ionic conditions influencing the stability of quadruplexes. in the long-term perspective, quadruplexes can represent an evolutionary feedback suppressing non-controlled amplification of active elements.  the results suggest that activity of transposable elements, especially line- and sva elements, contributes towards genome-wide quadruplex distribution in human. conservation of quadruplexes at specific positions implies their function either in the life cycle of transposable elements or host genome maintenance, or both. all tested pqss were able to form quadruplex structure in vitro, albeit with differing willingness, strand orientation and molecularity. line- and sva families displayed an age-dependent pattern with younger elements containing a higher number of more stable quadruplexes. further studies should be done to determine how the conserved elements are selected for during evolution. 
transposable elements form a significant proportion of eukaryotic genomes. conservation of quadruplexes at specific positions of transposable elements implies their importance in their life cycle. the increasing quadruplex presence in evolutionarily young line- and sva families makes these elements important contributors toward present genome-wide quadruplex distribution. here, we searched for pqss in human retrotransposons and found that pqss are specifically localized in the 3’-utr of line- elements, in ltrs of herv elements and are strongly accumulated in specific regions of sva elements. reported that plant long terminal repeat retrotransposons often contain potential quadruplex sequences in their ltrs and experimentally confirmed their ability to adopt four-stranded dna conformations. evolutionarily young sva elements contained more pqss than older elements and their propensity to form quadruplex dna was higher.
 transposable elements form a significant proportion of eukaryotic genomes. recently, lexa et al. reported that plant long terminal repeat retrotransposons often contain potential quadruplex sequences in their ltrs and experimentally confirmed their ability to adopt four-stranded dna conformations.  here, we searched for pqss in human retrotransposons and found that pqss are specifically localized in the 3’-utr of line- elements, in ltrs of herv elements and are strongly accumulated in specific regions of sva elements. circular dichroism spectroscopy confirmed that most pqss had adopted monomolecular or bimolecular guanine quadruplex structures. evolutionarily young sva elements contained more pqss than older elements and their propensity to form quadruplex dna was higher. full-length l elements contained more pqss than truncated elements; the highest proportion of pqss was found inside transpositionally active l elements.  conservation of quadruplexes at specific positions of transposable elements implies their importance in their life cycle. the increasing quadruplex presence in evolutionarily young line- and sva families makes these elements important contributors toward present genome-wide quadruplex distribution. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords g quadruplexretrotransposonsgenomeissue-copyright-statement© the author 2014 