BACKGROUND
segmental duplications  are dna sequences larger than 1 kb, which can be found at least twice with more than 90% sequence similarity in the genome.
they are a feature of various eukaryotic genomes, however, they have particularly accumulated during primate evolution .
thus the percentage of sds has increased from about 2% in the new world monkey marmoset  genome  <cit>  to approximately 5% in the human genome  <cit> .
it is not clear what has triggered this recent burst of sds, but the simultaneous decrease of point mutations and retrotransposition rate argues against that this is owed to a general increase of mutability  <cit> .
although sds pose a serious threat to genomic integrity by promoting non-allelic homologous recombination , this specific type of dna copy number variant has been fixed in the genome.
one reason for the manifestation of sds could be their preferential location in gene-rich genomic segments and their high gene content  <cit> .
several of the duplicated exons appear to be subject of accelerated evolution  <cit> , which has led to neofunctionalisation and subfunctionalisation of duplicated genes .
however, in most cases mutations have resulted in pseudogenisation of duplicated genes  <cit> , that nevertheless can show remarkably high transcriptional activity  <cit> .
yet, the large fraction of pericentromeric sds, which is less gene-rich  <cit> , points at alternative factors that could support positive selection of sds.
for example, sd insertion could also impact gene expression by demarcating euchromatin from transcriptional inactive heterochromatin  <cit> .
moreover, it has been discussed that sds, which frequently map to synteny breaks , may have mediated evolutionary rearrangements that have led to reproductive isolation of their carriers  <cit> .
however, the temporal order of events argues against the impact of sds on the generation of evolutionary rearrangements in many cases  <cit> .
on the contrary, a recent study supports the idea that the accumulation of sds may also be the consequence of evolutionary rearrangements rather than their cause  <cit> .
sds are not evenly distributed across the genome.
instead there are profound differences within and among chromosomes  <cit> .
apart from large sd clusters in the subtelomeric and pericentromeric regions of most chromosomes, sds can also accumulate in interstitial hubs  <cit> .
these hubs are characterised by an increased genomic instability, which manifests itself in a high probability of further sd insertion in their flanking regions, a phenomenon termed sd shadowing  <cit> .
furthermore, such hubs favour the presence of numerous structural variants with many of them having pathological relevance  <cit> .
yet, it is still uncertain what mechanisms have driven sd aggregation in the first place  <cit>  and whether the pro rata contribution of any such mechanism remained the same throughout evolution  <cit> .
a pivotal first step preceding formation of sd hubs may have been the insertion of core sds  <cit> .
recombination between repetitive elements may play a role too, as nearly 27% of all sds are flanked by alu repeats  <cit> .
in addition, the association of sds with g <dig> motifs and other sequence features promoting non-b dna conformations  <cit>  points at the possible relevance of chromatin conformation for sd insertion.
however, studies investigating sd distribution across the genome have so far based their analysis on the linear genome and have not taken into account its complex three dimensional organisation.
therefore, in this study we combined publicly available data on the three-dimensional organisation of the nucleus  <cit>  with own experimental data in order to explore the distribution of sds in relation to higher order chromatin organisation.
focusing on chromosome  <dig> with its particular high content of intrachromosomal and interstitial sds  <cit> , we demonstrate that paralogous sds, that have been separated in the course of evolution, are still in close spatial proximity.
proceeding on this observation we have explored a possible role of sds in sequence directed chromatin organisation and discuss how this may impact the emergence of genomic disorders such as the williams-beuren syndrome .
RESULTS
filtering and bundling of hi-c interaction bins
we have inferred spatial proximities of intrachromosomal sds from normalised hi-c data for chromosome  <dig>  <cit>  at a resolution of 20 kb.
hi-c is a derivative of the chromosome conformation capture protocol   <cit>  and facilitates the genome-wide analysis of chromatin interactions within the nucleus.
it is a proximity ligation based technology, where dna is cut, re-ligated and the products are analysed by paired-end sequencing.
the frequency of two dna sequences co-occurring in the same paired-end reads reflects their contact probability within the nucleus across a large population of heterogeneous cells in all phases of the cell cycle.
in order to concentrate on the most prevailing hi-c interactions and to minimise the influence of random noise, we have applied different criteria to filter hi-c data bins by changing 1) the normalised number of reads necessary to confirm the interaction of two given bins and 2) the minimal genomic distance of interacting bins.
for each of these data sets adjacent interaction bins were merged to regions of interaction bundles if their start and target sites locate within an interval of 500 kb, respectively, using circos tools  <cit> .
bundling all long distance interactions that have been confirmed by at least  <dig> interaction counts  with a minimum interaction span size of at least 25 mb using the bundling criteria “at least five interaction bins mapping within 500 kb at the start and the target site” to interaction bins, resulted in  <dig> bundles covering  <dig>  mb in total .
in line with the literature, these long distance interaction bundles preferentially connect regions with high transcriptional activity and open chromatin  <cit>  as demonstrated by our rna-seq and h4k8ac data .figure  <dig>
distribution of segmental duplications  and bundled long distance interactions in relation to acetylation of h4k <dig>  transcriptional activity and lamina associated domains on human chromosome  <dig> .
a) h4k <dig> acetylation profile, dark yellow: hyperacetylation of h4k8; blue: hypoacetylation of h4k <dig>  b) the red and blue curve represent rna-seq read counts/100 kb bin for coding and non-coding rna, respectively .
c) grey areas underlying the two histograms mark lamina associated domains .
d) idiogram of chromosome  <dig>  the williams-beuren syndrome region is highlighted in yellow beside the idiogram .
e) transparent blue shading of the idiogram illustrates the inversion-affected segments of chromosome  <dig> depicted in figure 2a-c. bundled long distance interactions  and segmental duplications  are depicted in the inner circle; green ribbons: long distance interactions between genomic regions; grey: sds with sequence similarity <98%; yellow: sds with sequence similarity 98-99%; orange: sds with sequence similarity >99%.
in accordance with the preferential insertion of sds into the gene-rich euchromatic portion of the genome, sd regions have a higher probability to be located within long distance interaction bundles .
in two out of  <dig> instances start and target site of long distance interaction bins directly coincide with the location of two sd paralogs .
although the initial sequence alignment of hi-c reads, as performed by dixon et al.
<cit> , employed a mapping quality score chosen to accept unique reads only, there is an apparent risk that some of these long distance interactions are owed to erroneous sequence alignment.
thus, we added a third filter for the hi-c data bins, namely 3) the exclusion of genomic bins overlapping with sds.
we tested the consequences on the bundling pattern after removing all interacting bins that connect two given sd paralogs , as well as ignoring all interaction bins that overlap with any sd at all .
these filter options are aimed at excluding all short distance interactions that have been misinterpreted as long distance interactions due to false alignment of one side of a paired-end read.
while this reduced the number of interaction bins by  <dig> % and  <dig> % , interactions of bins adjacent to the removed ones were sufficient to retain the basic triangular interaction pattern .
in addition to the filtering of sd overlapping interaction bins at the resolution of 20 kb, we performed a filtering also at the level of paired-end reads starting from the raw hi-c data  <cit> .
after exclusion of  <dig> intrachromosomal paired-end reads that ambiguously mapped to chromosome  <dig> , data were normalised and bundled .
in order to avoid threshold-induced interpretation bias we have tested in total  <dig> different combinations of cut-offs and filter criteria  with variations in interaction counts per bin, interaction distance and handling of genomic bins overlapping with known sds for the bundling of hi-c data.
the intersection of these  <dig> data sets revealed a core pattern of interactions independent of the threshold used .
therefore it is unlikely that the observed proximities of paralogous sds are solely result of ambiguous sequence alignments within segmental duplications.
however, we want to emphasise that given the paucity of reliable interaction counts within sds, this statement heavily depends on the interaction patterns of adjacent bins that lack any sds and is supported by shared regions of interactions as indicated by triangular interaction patterns.
chromosomal regions separated in the course of evolution retain spatial proximity
sds preferentially map to regions that are rich in long distance interactions.
at the same time they are known to accumulate at synteny breakpoints  <cit> .
this prompted us to search for particularities of long distance interaction patterns with respect to evolutionary breakpoints.
we have focused on two recent rearrangements of chromosome  <dig> that have occurred during hominoid evolution and are not present in the homologous chromosome of orang-utan, a pericentric inversion in the common ancestor of human/gorilla followed by a paracentric inversion in the human/chimpanzee ancestor.
as depicted in figure 2a-c, synteny breakpoints coincide with changes in the characteristics of interaction patterns.
to mimic the linear order of segments in gorilla and orang-utan we then recalculated the genomic coordinates of human chromosome  <dig> based on the fine-mapped evolutionary breakpoints .
figure 2a-c visualise the evolutionary split and relocation of a compact segment to three distant chromosomal regions in human and shows that these three - formerly adjacent - segments remain connected by long distance interactions.
these segments comprise almost all sequences of human chromosome  <dig> that are syntenic to a large block  of marmoset chromosome  <dig> .
genomic bins covering sequences of marmoset chromosome  <dig> were significantly overrepresented in regions rich in sds as indicated by low probability scores based on minimum hypergeometric statistics  <cit>  .
similarly a significant enrichment was detected in regions with a high frequency of alu repeats , as well as g <dig> dna motifs .figure  <dig>
long distance interactions of human chromosome  <dig> connect sequences syntenic to the most proximal  <dig>  mb of marmoset chromosome  <dig> and cluster in regions rich in sds, alu repeats and g <dig> motifs.
a-c) circos plots showing the patterns of long distance interactions  in relation to sds  within the three segments of human chromosome  <dig> affected by the pericentric and paracentric inversions ;  before and  after in silico reversion of the paracentric inversion and  after reverting the pericentric inversion.
the partial red and blue shading of the idiogram in a and b indicates the genomic interval inverted by the paracentric and pericentric inversion, respectively.
d) distribution of sds, long distance interactions , g <dig> motifs and alu repeats across human  chromosome  <dig>  and its relation to marmoset  chromosome  <dig> syntenic regions .
pink blocks highlight sequences syntenic to regions of marmoset chromosome  <dig>  e-g) enrichment of sds, alu repeats and g <dig> motifs within chromosome  <dig> segments homologous to sequences of marmoset chromosome  <dig> .
chromosome  <dig> segments  are displayed in ranked order according to feature count.
the red curve and red dot above each plot indicate the hypergeometric score and its minimum , respectively.
chromatin organisation of the williams-beuren region
one of the three segments affected by the evolutionary rearrangement described above – the most closest segment to the centromere - contains three sd clusters , two of which are involved in the aetiology of the williams-beuren syndrome .
together these three sd clusters are encompassed by a  <dig>  mb genomic interval at 7q <dig> -q <dig>   .
the most proximal sd cluster in the 7q <dig> segment starts at a transition of heterochromatin to euchromatin as demonstrated by our h4k8ac chip data and corroborated by numerous public data sets on posttranslational chromatin modifications .
this heterochromatin to euchromatin switch is accompanied by changed probabilities of dna attachment to the nuclear membrane  <cit>   and is also reflected by altered characteristics of replication timing and dna degradation during early phases of apoptosis.
in general, and in line with the literature, genome-wide analysis of apoptotic dna degradation revealed significant correlation with both lamina attachment  and replication timing  as defined by spearman’s rank correlation test .
the patterns of apoptotic dna degradation and its correlation to h4k <dig> acetylation were highly reproducible between two different cell lines .figure  <dig>
higher order chromatin organisation and sd localisation around the williams-beuren syndrome region.
all data are referring to genome release hg <dig> and are derived from imr <dig> unless indicated otherwise.
the proximal, central and distal sd clusters  of the 7q <dig> segment encompassing  <dig>  mb are highlighted in green within the chromosome banding track.
a-c) localisation of sds; colouring according to sequence similarity; grey: <98%, yellow: 98%-99%; orange: >99%; d) genomic interval commonly deleted in wbs and the distal 7q <dig>  deletion syndrome; e) topological domains as defined by dixon et al.
<cit> ; f) topological domains identified in the corresponding region in mouse  <cit>  after conversion to human hg <dig>  note that the murine topological domain homologous to sequences deleted in the distal 7q <dig>  syndrome is not fully represented due to a break of synteny within this genomic interval.
see figure  <dig> for details; g-h) heatmap and arc view of ctcf binding sites as detected by chia-pet in mcf7; i) number of g <dig> motifs/100 kb bins; j) average gc-content within 100 kb bins; k) number of alu repeats/100 kb bin; l) number of structural variants as annotated by database of genomic variance   <cit> , *maximum of  <dig> cnvs not shown; m) log <dig> ratio scores of the laminb <dig> damid map  as reported by guelen et al.
<cit> ; n) log <dig> ratio scores of dna regions prone to early apoptotic dna degradation in 20 kb windows, turquoise: degraded dna segments; o) log <dig> ratio scores of h4k <dig> acetylation profile in 20 kb windows, blue: hyperacetylation, grey: hypoacetylation; p) red curve representing the sum of all intrachromosomal interaction counts/bin divided by the median number of interactions for all bins of chromosome 7; q) percentage of interactions categorised according to their interaction span size; light grey: < <dig>  mb, grey:  <dig> -1 mb, light blue: 1–5 mb, light brown: 5–10 mb, dark grey: 10–25 mb, black: ≥25 mb.
gaps in this plot are due to alignment problems of hi-c data in regions harbouring sds with high sequence similarity.
given the reported association of gene density and chromatin organisation  <cit> , we compared gene distribution and intron size inside and outside of the 7q <dig> segment.
gene density in the genomic region of this segment is higher than in  <dig> randomly simulated intervals of chromosome  <dig> .
this difference in gene density was even more pronounced when focusing on the immediate genomic neighbourhood of the 7q <dig> segment; regions  <dig>  mb upstream and downstream contain an average of  <dig>  genes per mb  and  <dig>  genes per mb , respectively.
at the same time, intron size of the 7q <dig> segment is decreased when compared to the average of  <dig> simulations  and to the same number of genes  upstream and downstream of the segment .
gc-content is another aspect that is tightly linked to chromatin conformation.
gc-content within the 7q <dig> segment is  <dig> % on average with a standard deviation of  <dig> % based on 100 kb windows.
we observed a considerable drop of gc-content  within the most distal sd block and public data suggest that this interval of about 295 kb is located next to the nuclear membrane if mapped correctly.
g <dig> motifs show variable enrichment within the 7q <dig> segment, which is most prominent outside the sd blocks.
we also observed a relative depletion of g <dig> motifs within the central block of sds which is not reflected in a corresponding change of gc-content .
next we have asked whether this distinct dna conformation is also reflected in the hi-c data set.
the classification of hi-c interaction data referring to chromosome  <dig> into six categories based on their interaction span size  revealed that the change of chromatin state close to the wbs locus is also reflected by an increased proportion of interactions spanning less than  <dig>  mb , predominantly at the expense of interactions between  <dig> -5 mb and 10–25 mb.
this shift of span size characteristics is not accompanied by a general decrease of absolute interaction frequencies  and also lacks any symmetry around the gaps  within the hi-c data set, which would be expected if the observed changes in average span size are a consequence of mapping problems associated with the presence of sds .
furthermore, hi-c interaction patterns suggest that the recurrent deletion involved in the aetiology of wbs removes one topological domain, which is flanked by sds with highest sequence similarities.
in order to validate this assumption and to rule out that domain border definition at this site simply reflects sequence read depletion in large sd blocks, we performed an interspecies comparison of the human wbs locus and its homologous region in mouse.
topological domains were reported to have a high degree of evolutionary conservation.
indeed, the corresponding region in mice  comprises a distinct topological domain and the large sd blocks present in humans have inserted at sites that are homologous to murine topological domain borders .figure  <dig>
cross-species comparison showing that sds next to the wbs locus have inserted at topological domain borders.
hi-c interactions and topological domains in the human fetal fibroblast cell line imr <dig> are shown in dark green in the upper part as triangle view and bars, respectively.
sds with sequence similarity of 98%-99% and above 99%, respectively,  coincide with gaps within the hi-c data.
sd distribution and hi-c data of the corresponding region in mouse are given in the lower part of the image.
the position of fkbp <dig> and wbscr <dig>  the human orthologues of the two genes next to the murine topological domain borders are highlighted in green and red, respectively.
the intervals commonly affected in wbs and the distal 7q <dig>  syndrome are indicated by pale red bars.
note that the region distal to srrm <dig> including the distal sd block are homologous to a different mouse chromosome.
discussion
in this study we have investigated the relation between chromatin organisation of human chromosome  <dig> and the distribution of segmental duplications.
our study reveals that sds preferentially map to those regions of chromosome  <dig>  that are homologous to a  <dig>  mb large segment of marmoset chromosome  <dig>  in the course of evolution, this formerly compact chromosomal segment split up and relocated to human chromosome 7p <dig>  7q <dig> and 7q <dig> by a pericentric and paracentric inversion in the common ancestor of human/gorilla and human/chimpanzee, respectively  <cit> .
our analysis indicates that, despite these structural rearrangements, the three regions have retained their nuclear neighbourhood.
this observation corroborates findings of evolutionarily conserved principles of nuclear organisation at the resolution of interphase fish  <cit>  and is in line with a recent report on an increased hi-c interaction probability between murine syntenic breakpoint regions on human chromosomes, a phenomenon which has been termed spatial synteny  <cit> .
as a consequence of spatial synteny, sd paralogs that are separated by structural rearrangements and appear distant on the linear chromosome are still in close spatial proximity in the interphase nucleus.
a possible role for sds in spatial synteny
in light of the observed conservation of nuclear architecture, we have asked what factors could account for spatial synteny and whether the biased distribution of sds might play a role therein and in nuclear organisation in general  <cit> .
it is still unclear whether nuclear architecture is determined by a nuclear scaffold or represents the outcome of self-organisation choreographed by intrinsic properties of the chromatin itself , or a combination thereof.
although several dna-protein interactions and epigenetic marks clearly correlate with specific features of chromatin organisation, dna sequence by itself is likely to play a crucial role .
one dna sequence feature significantly enriched in those segments of chromosome  <dig> that are syntenic to a large block of marmoset chromosome  <dig> are g <dig> dna motifs   <cit>  .
these motifs can establish highly stable intramolecular and intermolecular connections via hoogsteen pairing between four guanines and have already been implicated in telomere organisation and in meiotic chromosome pairing .
the non-random distribution of g <dig> motifs along human chromosome  <dig>  as shown in this study, could point at a possible function of quadruplex structures in the retention of spatial proximities also in interphase nuclei.
high frequency of alu repeats is another, partly interrelated, sequence feature that we have found significantly enriched in these highly interacting regions .
alu repeat distribution is not the result of regional insertion preferences, but more likely the consequence of selective pressure on gc-content biased removal .
against this background, alu repeats have been implicated in higher order chromatin organisation  <cit> .
however, the overall presence of both alu repeats and g <dig> motifs throughout the genome raises the question how such a sequence-directed organisation of the nucleus might obtain its specificity in the first place.
the observed spatial proximity of sd paralogs , as well as their preferential insertion within alu repeat and g <dig> motif-rich areas  <cit>   makes sds ideal candidates to introduce sequence specificity into this process.
for example, temporal somatic pairing could influence polymer dynamics and in this way accelerate the establishment of higher order chromatin organisation.
allelic or ectopic somatic pairing of homologous sequences is a widespread phenomenon in eukaryotes that is known to impact gene regulation and nuclear architecture .
chromosomal structures enriched for interchromosomal sds such as the telomeres and centromeres have already been reported to colocalise in interphase nuclei .
notably, paralogous sds show a remarkably high rate of interlocus gene conversion  <cit> , which may indicate a high contact probability within the nucleus.
sd distribution at the heterochromatin to euchromatin boundary at 7q <dig>
previous studies have reported the occurrence of sds at the transition of heterochromatin to euchromatin .
this prompted us to re-evaluate the distribution of sds in the context of new models of chromatin organisation, particularily the concept of topological domains.
these megabase sized domains of highly interacting chromatin are remarkably stable between different cell types and highly conserved between mice and humans  <cit> .
we have focused on the three sd blocks localised at the border of 7q <dig>  to 7q <dig> .
these sds are of special interest to human geneticists as non-allelic homologous recombination between them underlies the development of williams-beuren syndrome , the 7q <dig>  duplication syndrome , the inversion that predisposes to the wbs deletion  <cit>  and the distal 7q <dig>  deletion syndrome .
several observations indicate that the 7q <dig> segment containing these three sd blocks has a particular dna conformation.
this segment meets all criteria that have been defined for ridges , i.e.
highly transcribed, gc-rich and gene-rich sequences with short introns and a high content of alu repeats.
ridges have a different degree of dna compaction as suggested by computational analysis  <cit> , an assumption, which is backed by the fact that the genomic characteristics of ridges largely overlap with those recently defined for dna domains in an underwound state  <cit> .
one factor for establishing and maintaining this specific chromatin conformation in this highly transcribed region may be g <dig> motifs, which are frequent in the 7q <dig> segment and have been reported to stabilise open chromatin  <cit> .
remarkably, sequences covered by the central and the distal sd cluster in the 7q <dig> segment show less g <dig> motif density and thus disrupt the continuity of g <dig> motif enrichment.
proceeding on the assumption that sequence reads were mapped unequivocally, the most distal sd block also has a high probability of being attached to the nuclear membrane .
evaluation of ctcf interaction characteristics and the re-analysis of hi-c data with focus on average interaction span sizes mirrors the particularities of chromatin conformation in the 7q <dig> segment .
moreover, hi-c data  <cit>  suggest that the genomic interval typically deleted in wbs patients comprises a distinct topological domain, which is flanked by sds at its borders.
clearly, the paucity of hi-c data mapping to sds with highest sequence similarities complicates the interpretation of sd-related interaction patterns and may have compromised the precise definition of topological domains.
in search of strategies which could enable us to discriminate sd-associated technical artefacts from biological relevant sd insertion at domain borders, we exploited the facts that topological domains are highly conserved between mice and humans  <cit>  and that the syntenic region in mice lack these large sd blocks  <cit> .
our cross-species comparison revealed that the single copy sequences deleted in wbs indeed compose a distinct topological domain in mice, and that the large sd blocks present in humans have inserted at sites homologous to the murine domain borders.
this insertion of dna sequences with different characteristics, for example in terms of g <dig> motif density or preference for attachment to the nuclear membrane , could emphasise the separation of topological domains.
thus sds may impact chromatin organisation at the level of topological domains in a way which is reminiscent of what has been proposed for pericentric sds at the chromosomal level, namely to facilitate differential gene regulation and to protect from the regulatory influence of adjacent sequences  <cit> .
the reciprocal event, a deletion of domain borders and linker region, has already been shown experimentally to provoke significant changes in the interaction pattern of two adjacent topological domains  <cit> .
further support for this assumption is provided by recent reports on the impact of wbs deletions on the interaction patterns of its adjacent topological domains  <cit> .
interestingly, although many sds show accelerated rates of sequence divergence  <cit> , sds involved in the aetiology of wbs and several other genomic disorders show a considerably high rate of gene conversion, which preserves their sequence similarity  and, as a consequence, the risk of recombination events that cause the genomic disorder  <cit> .
on one hand, recurrent recombinations of paralogous sds, which cause the high rate of intrachromosomal deletions and inversions in the wbs region, supports the assumption of a high contact probability between these paralogous sds within the nucleus.
on the other hand, it raises the question whether sequence similarity might serve a function that could compensate for the associated high susceptibility to structural rearrangements mediated by sds with high sequence similarity.
for example, sds could influence chromatin organisation by somatic pairing as discussed above or by rna-based mechanisms.
the latter option would be one explanation for the reported high transcriptional activity of pseudogenes mapping to sds  <cit> , with many of them regulated in a tissue-specific manner  <cit> .
notably, the frequent interaction of the prader-willi syndrome imprinting centre  with two adjacent sds has already inspired discussions on the functional impact of sds on chromatin organisation  <cit> .
CONCLUSIONS
our study suggests a link of nuclear architecture and the propagation of sds across chromosome  <dig>  higher contact probabilities could promote regional sd insertion, but also could be a factor of nuclear organisation themselves, which promotes their propagation and evolutionary fixation in the genome.

 segmental duplications are dna sequences larger than 1 kb, which can be found at least twice with more than 90% sequence similarity in the genome. they are a feature of various eukaryotic genomes, however, they have particularly accumulated during primate evolution. thus the percentage of sds has increased from about 2% in the new world monkey marmoset genome to approximately 5% in the human genome. it is not clear what has triggered this recent burst of sds, but the simultaneous decrease of point mutations and retrotransposition rate argues against that this is owed to a general increase of mutability. although sds pose a serious threat to genomic integrity by promoting non-allelic homologous recombination, this specific type of dna copy number variant has been fixed in the genome. one reason for the manifestation of sds could be their preferential location in gene-rich genomic segments and their high gene content. several of the duplicated exons appear to be subject of accelerated evolution, which has led to neofunctionalisation and subfunctionalisation of duplicated genes. however, in most cases mutations have resulted in pseudogenisation of duplicated genes, that nevertheless can show remarkably high transcriptional activity. yet, the large fraction of pericentromeric sds, which is less gene-rich, points at alternative factors that could support positive selection of sds. for example, sd insertion could also impact gene expression by demarcating euchromatin from transcriptional inactive heterochromatin. moreover, it has been discussed that sds, which frequently map to synteny breaks, may have mediated evolutionary rearrangements that have led to reproductive isolation of their carriers. however, the temporal order of events argues against the impact of sds on the generation of evolutionary rearrangements in many cases. on the contrary, a recent study supports the idea that the accumulation of sds may also be the consequence of evolutionary rearrangements rather than their cause. sds are not evenly distributed across the genome. instead there are profound differences within and among chromosomes. apart from large sd clusters in the subtelomeric and pericentromeric regions of most chromosomes, sds can also accumulate in interstitial hubs. these hubs are characterised by an increased genomic instability, which manifests itself in a high probability of further sd insertion in their flanking regions, a phenomenon termed sd shadowing. furthermore, such hubs favour the presence of numerous structural variants with many of them having pathological relevance. yet, it is still uncertain what mechanisms have driven sd aggregation in the first place and whether the pro rata contribution of any such mechanism remained the same throughout evolution. a pivotal first step preceding formation of sd hubs may have been the insertion of core sds. recombination between repetitive elements may play a role too, as nearly 27% of all sds are flanked by alu repeats. in addition, the association of sds with g motifs and other sequence features promoting non-b dna conformations points at the possible relevance of chromatin conformation for sd insertion. however, studies investigating sd distribution across the genome have so far based their analysis on the linear genome and have not taken into account its complex three dimensional organisation. therefore, in this study we combined publicly available data on the three-dimensional organisation of the nucleus with own experimental data in order to explore the distribution of sds in relation to higher order chromatin organisation. focusing on chromosome with its particular high content of intrachromosomal and interstitial sds, we demonstrate that paralogous sds, that have been separated in the course of evolution, are still in close spatial proximity. proceeding on this observation we have explored a possible role of sds in sequence directed chromatin organisation and discuss how this may impact the emergence of genomic disorders such as the williams-beuren syndrome.  filtering and bundling of hi-c interaction bins we have inferred spatial proximities of intrachromosomal sds from normalised hi-c data for chromosome at a resolution of 20 kb. hi-c is a derivative of the chromosome conformation capture protocol and facilitates the genome-wide analysis of chromatin interactions within the nucleus. it is a proximity ligation based technology, where dna is cut, re-ligated and the products are analysed by paired-end sequencing. the frequency of two dna sequences co-occurring in the same paired-end reads reflects their contact probability within the nucleus across a large population of heterogeneous cells in all phases of the cell cycle. in order to concentrate on the most prevailing hi-c interactions and to minimise the influence of random noise, we have applied different criteria to filter hi-c data bins by changing 1) the normalised number of reads necessary to confirm the interaction of two given bins and 2) the minimal genomic distance of interacting bins. for each of these data sets adjacent interaction bins were merged to regions of interaction bundles if their start and target sites locate within an interval of 500 kb, respectively, using circos tools. bundling all long distance interactions that have been confirmed by at least interaction counts with a minimum interaction span size of at least 25 mb using the bundling criteria “at least five interaction bins mapping within 500 kb at the start and the target site” to interaction bins, resulted in bundles covering  mb in total. in line with the literature, these long distance interaction bundles preferentially connect regions with high transcriptional activity and open chromatin as demonstrated by our rna-seq and h4k8ac data.figure distribution of segmental duplications and bundled long distance interactions in relation to acetylation of h4k transcriptional activity and lamina associated domains on human chromosome. a) h4k acetylation profile, dark yellow: hyperacetylation of h4k8; blue: hypoacetylation of h4k b) the red and blue curve represent rna-seq read counts/100 kb bin for coding and non-coding rna, respectively. c) grey areas underlying the two histograms mark lamina associated domains. d) idiogram of chromosome the williams-beuren syndrome region is highlighted in yellow beside the idiogram. e) transparent blue shading of the idiogram illustrates the inversion-affected segments of chromosome depicted in figure 2a-c. bundled long distance interactions and segmental duplications are depicted in the inner circle; green ribbons: long distance interactions between genomic regions; grey: sds with sequence similarity <98%; yellow: sds with sequence similarity 98-99%; orange: sds with sequence similarity >99%. in accordance with the preferential insertion of sds into the gene-rich euchromatic portion of the genome, sd regions have a higher probability to be located within long distance interaction bundles. in two out of instances start and target site of long distance interaction bins directly coincide with the location of two sd paralogs. although the initial sequence alignment of hi-c reads, as performed by dixon et al., employed a mapping quality score chosen to accept unique reads only, there is an apparent risk that some of these long distance interactions are owed to erroneous sequence alignment. thus, we added a third filter for the hi-c data bins, namely 3) the exclusion of genomic bins overlapping with sds. we tested the consequences on the bundling pattern after removing all interacting bins that connect two given sd paralogs, as well as ignoring all interaction bins that overlap with any sd at all. these filter options are aimed at excluding all short distance interactions that have been misinterpreted as long distance interactions due to false alignment of one side of a paired-end read. while this reduced the number of interaction bins by % and %, interactions of bins adjacent to the removed ones were sufficient to retain the basic triangular interaction pattern. in addition to the filtering of sd overlapping interaction bins at the resolution of 20 kb, we performed a filtering also at the level of paired-end reads starting from the raw hi-c data. after exclusion of intrachromosomal paired-end reads that ambiguously mapped to chromosome, data were normalised and bundled. in order to avoid threshold-induced interpretation bias we have tested in total different combinations of cut-offs and filter criteria with variations in interaction counts per bin, interaction distance and handling of genomic bins overlapping with known sds for the bundling of hi-c data. the intersection of these data sets revealed a core pattern of interactions independent of the threshold used. therefore it is unlikely that the observed proximities of paralogous sds are solely result of ambiguous sequence alignments within segmental duplications. however, we want to emphasise that given the paucity of reliable interaction counts within sds, this statement heavily depends on the interaction patterns of adjacent bins that lack any sds and is supported by shared regions of interactions as indicated by triangular interaction patterns. chromosomal regions separated in the course of evolution retain spatial proximity sds preferentially map to regions that are rich in long distance interactions. at the same time they are known to accumulate at synteny breakpoints. this prompted us to search for particularities of long distance interaction patterns with respect to evolutionary breakpoints. we have focused on two recent rearrangements of chromosome that have occurred during hominoid evolution and are not present in the homologous chromosome of orang-utan, a pericentric inversion in the common ancestor of human/gorilla followed by a paracentric inversion in the human/chimpanzee ancestor. as depicted in figure 2a-c, synteny breakpoints coincide with changes in the characteristics of interaction patterns. to mimic the linear order of segments in gorilla and orang-utan we then recalculated the genomic coordinates of human chromosome based on the fine-mapped evolutionary breakpoints. figure 2a-c visualise the evolutionary split and relocation of a compact segment to three distant chromosomal regions in human and shows that these three - formerly adjacent - segments remain connected by long distance interactions. these segments comprise almost all sequences of human chromosome that are syntenic to a large block of marmoset chromosome. genomic bins covering sequences of marmoset chromosome were significantly overrepresented in regions rich in sds as indicated by low probability scores based on minimum hypergeometric statistics. similarly a significant enrichment was detected in regions with a high frequency of alu repeats, as well as g dna motifs.figure long distance interactions of human chromosome connect sequences syntenic to the most proximal  mb of marmoset chromosome and cluster in regions rich in sds, alu repeats and g motifs. a-c) circos plots showing the patterns of long distance interactions in relation to sds within the three segments of human chromosome affected by the pericentric and paracentric inversions; before and after in silico reversion of the paracentric inversion and after reverting the pericentric inversion. the partial red and blue shading of the idiogram in a and b indicates the genomic interval inverted by the paracentric and pericentric inversion, respectively. d) distribution of sds, long distance interactions, g motifs and alu repeats across human chromosome and its relation to marmoset chromosome syntenic regions. pink blocks highlight sequences syntenic to regions of marmoset chromosome e-g) enrichment of sds, alu repeats and g motifs within chromosome segments homologous to sequences of marmoset chromosome. chromosome segments are displayed in ranked order according to feature count. the red curve and red dot above each plot indicate the hypergeometric score and its minimum, respectively. chromatin organisation of the williams-beuren region one of the three segments affected by the evolutionary rearrangement described above – the most closest segment to the centromere - contains three sd clusters, two of which are involved in the aetiology of the williams-beuren syndrome. together these three sd clusters are encompassed by a  mb genomic interval at 7q -q. the most proximal sd cluster in the 7q segment starts at a transition of heterochromatin to euchromatin as demonstrated by our h4k8ac chip data and corroborated by numerous public data sets on posttranslational chromatin modifications. this heterochromatin to euchromatin switch is accompanied by changed probabilities of dna attachment to the nuclear membrane and is also reflected by altered characteristics of replication timing and dna degradation during early phases of apoptosis. in general, and in line with the literature, genome-wide analysis of apoptotic dna degradation revealed significant correlation with both lamina attachment and replication timing as defined by spearman’s rank correlation test. the patterns of apoptotic dna degradation and its correlation to h4k acetylation were highly reproducible between two different cell lines.figure higher order chromatin organisation and sd localisation around the williams-beuren syndrome region. all data are referring to genome release hg and are derived from imr unless indicated otherwise. the proximal, central and distal sd clusters of the 7q segment encompassing  mb are highlighted in green within the chromosome banding track. a-c) localisation of sds; colouring according to sequence similarity; grey: <98%, yellow: 98%-99%; orange: >99%; d) genomic interval commonly deleted in wbs and the distal 7q deletion syndrome; e) topological domains as defined by dixon et al.; f) topological domains identified in the corresponding region in mouse after conversion to human hg note that the murine topological domain homologous to sequences deleted in the distal 7q syndrome is not fully represented due to a break of synteny within this genomic interval. see figure  for details; g-h) heatmap and arc view of ctcf binding sites as detected by chia-pet in mcf7; i) number of g motifs/100 kb bins; j) average gc-content within 100 kb bins; k) number of alu repeats/100 kb bin; l) number of structural variants as annotated by database of genomic variance, *maximum of cnvs not shown; m) log ratio scores of the laminb damid map as reported by guelen et al.; n) log ratio scores of dna regions prone to early apoptotic dna degradation in 20 kb windows, turquoise: degraded dna segments; o) log ratio scores of h4k acetylation profile in 20 kb windows, blue: hyperacetylation, grey: hypoacetylation; p) red curve representing the sum of all intrachromosomal interaction counts/bin divided by the median number of interactions for all bins of chromosome 7; q) percentage of interactions categorised according to their interaction span size; light grey: <  mb, grey: -1 mb, light blue: 1–5 mb, light brown: 5–10 mb, dark grey: 10–25 mb, black: ≥25 mb. gaps in this plot are due to alignment problems of hi-c data in regions harbouring sds with high sequence similarity. given the reported association of gene density and chromatin organisation, we compared gene distribution and intron size inside and outside of the 7q segment. gene density in the genomic region of this segment is higher than in randomly simulated intervals of chromosome. this difference in gene density was even more pronounced when focusing on the immediate genomic neighbourhood of the 7q segment; regions  mb upstream and downstream contain an average of genes per mb and genes per mb, respectively. at the same time, intron size of the 7q segment is decreased when compared to the average of simulations and to the same number of genes upstream and downstream of the segment. gc-content is another aspect that is tightly linked to chromatin conformation. gc-content within the 7q segment is % on average with a standard deviation of % based on 100 kb windows. we observed a considerable drop of gc-content within the most distal sd block and public data suggest that this interval of about 295 kb is located next to the nuclear membrane if mapped correctly. g motifs show variable enrichment within the 7q segment, which is most prominent outside the sd blocks. we also observed a relative depletion of g motifs within the central block of sds which is not reflected in a corresponding change of gc-content. next we have asked whether this distinct dna conformation is also reflected in the hi-c data set. the classification of hi-c interaction data referring to chromosome into six categories based on their interaction span size revealed that the change of chromatin state close to the wbs locus is also reflected by an increased proportion of interactions spanning less than  mb, predominantly at the expense of interactions between -5 mb and 10–25 mb. this shift of span size characteristics is not accompanied by a general decrease of absolute interaction frequencies and also lacks any symmetry around the gaps within the hi-c data set, which would be expected if the observed changes in average span size are a consequence of mapping problems associated with the presence of sds. furthermore, hi-c interaction patterns suggest that the recurrent deletion involved in the aetiology of wbs removes one topological domain, which is flanked by sds with highest sequence similarities. in order to validate this assumption and to rule out that domain border definition at this site simply reflects sequence read depletion in large sd blocks, we performed an interspecies comparison of the human wbs locus and its homologous region in mouse. topological domains were reported to have a high degree of evolutionary conservation. indeed, the corresponding region in mice comprises a distinct topological domain and the large sd blocks present in humans have inserted at sites that are homologous to murine topological domain borders.figure cross-species comparison showing that sds next to the wbs locus have inserted at topological domain borders. hi-c interactions and topological domains in the human fetal fibroblast cell line imr are shown in dark green in the upper part as triangle view and bars, respectively. sds with sequence similarity of 98%-99% and above 99%, respectively, coincide with gaps within the hi-c data. sd distribution and hi-c data of the corresponding region in mouse are given in the lower part of the image. the position of fkbp and wbscr the human orthologues of the two genes next to the murine topological domain borders are highlighted in green and red, respectively. the intervals commonly affected in wbs and the distal 7q syndrome are indicated by pale red bars. note that the region distal to srrm including the distal sd block are homologous to a different mouse chromosome. discussion in this study we have investigated the relation between chromatin organisation of human chromosome and the distribution of segmental duplications. our study reveals that sds preferentially map to those regions of chromosome that are homologous to a  mb large segment of marmoset chromosome in the course of evolution, this formerly compact chromosomal segment split up and relocated to human chromosome 7p 7q and 7q by a pericentric and paracentric inversion in the common ancestor of human/gorilla and human/chimpanzee, respectively. our analysis indicates that, despite these structural rearrangements, the three regions have retained their nuclear neighbourhood. this observation corroborates findings of evolutionarily conserved principles of nuclear organisation at the resolution of interphase fish and is in line with a recent report on an increased hi-c interaction probability between murine syntenic breakpoint regions on human chromosomes, a phenomenon which has been termed spatial synteny. as a consequence of spatial synteny, sd paralogs that are separated by structural rearrangements and appear distant on the linear chromosome are still in close spatial proximity in the interphase nucleus. a possible role for sds in spatial synteny in light of the observed conservation of nuclear architecture, we have asked what factors could account for spatial synteny and whether the biased distribution of sds might play a role therein and in nuclear organisation in general. it is still unclear whether nuclear architecture is determined by a nuclear scaffold or represents the outcome of self-organisation choreographed by intrinsic properties of the chromatin itself, or a combination thereof. although several dna-protein interactions and epigenetic marks clearly correlate with specific features of chromatin organisation, dna sequence by itself is likely to play a crucial role. one dna sequence feature significantly enriched in those segments of chromosome that are syntenic to a large block of marmoset chromosome are g dna motifs. these motifs can establish highly stable intramolecular and intermolecular connections via hoogsteen pairing between four guanines and have already been implicated in telomere organisation and in meiotic chromosome pairing. the non-random distribution of g motifs along human chromosome as shown in this study, could point at a possible function of quadruplex structures in the retention of spatial proximities also in interphase nuclei. high frequency of alu repeats is another, partly interrelated, sequence feature that we have found significantly enriched in these highly interacting regions. alu repeat distribution is not the result of regional insertion preferences, but more likely the consequence of selective pressure on gc-content biased removal. against this background, alu repeats have been implicated in higher order chromatin organisation. however, the overall presence of both alu repeats and g motifs throughout the genome raises the question how such a sequence-directed organisation of the nucleus might obtain its specificity in the first place. the observed spatial proximity of sd paralogs, as well as their preferential insertion within alu repeat and g motif-rich areas makes sds ideal candidates to introduce sequence specificity into this process. for example, temporal somatic pairing could influence polymer dynamics and in this way accelerate the establishment of higher order chromatin organisation. allelic or ectopic somatic pairing of homologous sequences is a widespread phenomenon in eukaryotes that is known to impact gene regulation and nuclear architecture. chromosomal structures enriched for interchromosomal sds such as the telomeres and centromeres have already been reported to colocalise in interphase nuclei. notably, paralogous sds show a remarkably high rate of interlocus gene conversion, which may indicate a high contact probability within the nucleus. sd distribution at the heterochromatin to euchromatin boundary at 7q previous studies have reported the occurrence of sds at the transition of heterochromatin to euchromatin. this prompted us to re-evaluate the distribution of sds in the context of new models of chromatin organisation, particularily the concept of topological domains. these megabase sized domains of highly interacting chromatin are remarkably stable between different cell types and highly conserved between mice and humans. we have focused on the three sd blocks localised at the border of 7q to 7q. these sds are of special interest to human geneticists as non-allelic homologous recombination between them underlies the development of williams-beuren syndrome, the 7q duplication syndrome, the inversion that predisposes to the wbs deletion and the distal 7q deletion syndrome. several observations indicate that the 7q segment containing these three sd blocks has a particular dna conformation. this segment meets all criteria that have been defined for ridges, i.e. highly transcribed, gc-rich and gene-rich sequences with short introns and a high content of alu repeats. ridges have a different degree of dna compaction as suggested by computational analysis, an assumption, which is backed by the fact that the genomic characteristics of ridges largely overlap with those recently defined for dna domains in an underwound state. one factor for establishing and maintaining this specific chromatin conformation in this highly transcribed region may be g motifs, which are frequent in the 7q segment and have been reported to stabilise open chromatin. remarkably, sequences covered by the central and the distal sd cluster in the 7q segment show less g motif density and thus disrupt the continuity of g motif enrichment. proceeding on the assumption that sequence reads were mapped unequivocally, the most distal sd block also has a high probability of being attached to the nuclear membrane. evaluation of ctcf interaction characteristics and the re-analysis of hi-c data with focus on average interaction span sizes mirrors the particularities of chromatin conformation in the 7q segment. moreover, hi-c data suggest that the genomic interval typically deleted in wbs patients comprises a distinct topological domain, which is flanked by sds at its borders. clearly, the paucity of hi-c data mapping to sds with highest sequence similarities complicates the interpretation of sd-related interaction patterns and may have compromised the precise definition of topological domains. in search of strategies which could enable us to discriminate sd-associated technical artefacts from biological relevant sd insertion at domain borders, we exploited the facts that topological domains are highly conserved between mice and humans and that the syntenic region in mice lack these large sd blocks. our cross-species comparison revealed that the single copy sequences deleted in wbs indeed compose a distinct topological domain in mice, and that the large sd blocks present in humans have inserted at sites homologous to the murine domain borders. this insertion of dna sequences with different characteristics, for example in terms of g motif density or preference for attachment to the nuclear membrane, could emphasise the separation of topological domains. thus sds may impact chromatin organisation at the level of topological domains in a way which is reminiscent of what has been proposed for pericentric sds at the chromosomal level, namely to facilitate differential gene regulation and to protect from the regulatory influence of adjacent sequences. the reciprocal event, a deletion of domain borders and linker region, has already been shown experimentally to provoke significant changes in the interaction pattern of two adjacent topological domains. further support for this assumption is provided by recent reports on the impact of wbs deletions on the interaction patterns of its adjacent topological domains. interestingly, although many sds show accelerated rates of sequence divergence, sds involved in the aetiology of wbs and several other genomic disorders show a considerably high rate of gene conversion, which preserves their sequence similarity and, as a consequence, the risk of recombination events that cause the genomic disorder. on one hand, recurrent recombinations of paralogous sds, which cause the high rate of intrachromosomal deletions and inversions in the wbs region, supports the assumption of a high contact probability between these paralogous sds within the nucleus. on the other hand, it raises the question whether sequence similarity might serve a function that could compensate for the associated high susceptibility to structural rearrangements mediated by sds with high sequence similarity. for example, sds could influence chromatin organisation by somatic pairing as discussed above or by rna-based mechanisms. the latter option would be one explanation for the reported high transcriptional activity of pseudogenes mapping to sds, with many of them regulated in a tissue-specific manner. notably, the frequent interaction of the prader-willi syndrome imprinting centre with two adjacent sds has already inspired discussions on the functional impact of sds on chromatin organisation.  our study suggests a link of nuclear architecture and the propagation of sds across chromosome higher contact probabilities could promote regional sd insertion, but also could be a factor of nuclear organisation themselves, which promotes their propagation and evolutionary fixation in the genome. 
focusing on sd clusters implicated in the aetiology of the williams-beuren syndrome locus we demonstrate by cross-species comparison that these sds have inserted at the borders of a topological domain and that they flank regions with distinct dna conformation. focusing on chromosome which is particularly rich in interstitial sds, we have investigated the distribution of sds in the context of evolution and the three dimensional organisation of the chromosome in order to gain insights into the mutual relationship of sds and chromatin topology. the latter could compensate for the high risk of structural rearrangements and thus may have contributed to their evolutionary fixation in the human genome. accumulation of sds is associated with increased genomic instability, which can lead to structural variants and genomic disorders such as the williams-beuren syndrome. our study suggests a link of nuclear architecture and the propagation of sds across chromosome either by promoting regional sd insertion or by contributing to the establishment of higher order chromatin organisation themselves. intrachromosomal sds preferentially accumulate in those segments of chromosome that are homologous to marmoset chromosome although this formerly compact segment has been re-distributed to three different sites during primate evolution, we can show by means of public data on long distance chromatin interactions that these three intervals, and consequently the paralogous sds mapping to them, have retained their spatial proximity in the nucleus. the reasons for this biased susceptibility to sd insertion are poorly understood. segmental duplications are not evenly distributed along chromosomes. higher order chromatin organisationsegmental duplicationwilliams-beuren syndromechromosome evolutionhi-cissue-copyright-statement© the author 2014
 segmental duplications are not evenly distributed along chromosomes. the reasons for this biased susceptibility to sd insertion are poorly understood. accumulation of sds is associated with increased genomic instability, which can lead to structural variants and genomic disorders such as the williams-beuren syndrome. despite these adverse effects, sds have become fixed in the human genome. focusing on chromosome which is particularly rich in interstitial sds, we have investigated the distribution of sds in the context of evolution and the three dimensional organisation of the chromosome in order to gain insights into the mutual relationship of sds and chromatin topology.  intrachromosomal sds preferentially accumulate in those segments of chromosome that are homologous to marmoset chromosome although this formerly compact segment has been re-distributed to three different sites during primate evolution, we can show by means of public data on long distance chromatin interactions that these three intervals, and consequently the paralogous sds mapping to them, have retained their spatial proximity in the nucleus. focusing on sd clusters implicated in the aetiology of the williams-beuren syndrome locus we demonstrate by cross-species comparison that these sds have inserted at the borders of a topological domain and that they flank regions with distinct dna conformation.  our study suggests a link of nuclear architecture and the propagation of sds across chromosome either by promoting regional sd insertion or by contributing to the establishment of higher order chromatin organisation themselves. the latter could compensate for the high risk of structural rearrangements and thus may have contributed to their evolutionary fixation in the human genome. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords higher order chromatin organisationsegmental duplicationwilliams-beuren syndromechromosome evolutionhi-cissue-copyright-statement© the author 2014 