BACKGROUND
eukaryotic nucleus is a complex organelle, where dna is highly compacted but still is accessible for nuclear processes in a precisely controlled manner.
the structural basis for such compact but orderly organization is provided by a proteinaceous meshwork known as nuclear matrix ,  <cit>  that has been visualized by electron microscopy  <cit> .
although existence of numat in vivo is debated in the context of possible self assembly, this alone is not sufficient to explain all the features of nuclear organization  <cit> .
non-diffusible fraction of lamin in the interior of the nucleus of living cells indicates the existance of a nucleoskeleton involving such components  <cit> .
the presence of actin, myosin and several cytoskeletal proteins in the nucleus and numat further strengthens this view  <cit> .
whole proteome analysis of numat from arabidopsis, drosophila and human have shown that the major categories of proteins these preparations remain the same across species and cytoskeletal proteins are a conserved category .
numat as a framework offers potential basis for compartmentalization of the nucleus and explains why several markers of different nuclear substructures have been found in the numat proteome  <cit> .
numat is also envisaged as a scaffold on which higher order organization of chromatin loops takes place.
such topologically independent loops define the unit of compartmentalized chromatin that along with differential epigenetic marks bring about proper regulation of gene expression  <cit> .
biochemically, numat consists of dna, rna and protein and, while the underlying nuclear structure is not fully characterised, the dna component belongs to the sequences that help attach the base of chromatin loops to the numat  <cit>  and are defined as matrix/scaffold associated regions, m/sar <cit> .
although not much sequence similarity is noticed in different mars, their biochemical properties have been shown to be conserved  <cit>  presumably due to the secondary structure features and other physical properties of underlying sequences .
they are nuclease protected regions of chromatin that functionally associate with varitey of cis-regulatory elements including origin of replication, chromatin domain boundaries and locus control regions .
considering the importance of mars in genome organization and regulation, a genome–wide map of these sequences is needed to understand the relationship between mar and known regulatory sequences at genome scale.
efforts have been made in past to experimentally identify number of mars and to create computational tools to predict them  <cit> .
here we report, for the first time, mapping and characterization of mars across the entire 120 mb of drosophila euchromatic genome.
we identify  <dig> mars accounting for ~ <dig> % of this genome and gives insight into their role in chromatin organization and regulation in the context of genomic packaging.
RESULTS
identification of mar elements in drosophila melanogastergenome
d. melanogaster embryos  were collected and used for numat preparation using serial extraction steps including dnasei to digest accessible chromatin dna   <cit> .
quality of the numat preparation was checked using several parameters: retention of 1-2% dna, ~10% proteins and 30% rna compared to the nucleus.
this preparation was used as source to purify associated genomic fragments as mar dna of ~100 bp size that were dnasei sensitive and rnasea resistant .
this uniformity in the size of mar dna reflects the region that remains in-accessible to the nuclease, while some flanking relevant features may not be retained here.
this mar preparation was further checked for the enrichment of known mars   <cit> .
the mar dna preparation was directly sequenced without any further fragmentation.
two biological replicates of mar preparations were used for direct sequencing that gave a total of  <dig>  million mappable reads.
the sequenced reads were mapped to the d. melanogaster genome  using bowtie short read mapping algorithm and enriched regions were identified using macs software   <cit> .
the two replicates were processed independently and gave  <dig>  and  <dig>  peaks.
as shown in additional file 1: figure s <dig>  we observed very close similarity in pattern of peaks between the biological replicates.
overall, we found  <dig> peaks common between the two replicates that amounts to ~70% of overlap.
a windowed  comparison of tag counts between the replicates indicates a pearson’s correlation coefficient of  <dig> .
this implies that ~97% of the reads from the biological replicates agree with each other.
of the  <dig> peaks common to both the biological replicates, we selected  <dig> peaks that mapped to euchromatic regions for further analysis, while peaks that mapped to heterochromatic or un-annotated stretches of sequences were left out .
figure  <dig> shows a ~100 kb region from chromosome 2 l encompassing acx and vha <dig> gene clusters.
correlation of mars in the region with at richness, repeats and paused polymerase ii sites is evident.figure  <dig>
isolation and validation of
drosophila
embryo mars.
overview of mar dna isolation procedure.
size distribution of mar dna after electrophoresis on  <dig> % agarose gel.
m-molecular weight marker; lane 1-genomic dna; lane 2-mar dna; lane 3-mar dna + dnasei; lane 4-mar dna + rnasea.
slot-blot hybridization to show enrichment of known mars in the mar dna preparation.
equal quantity of plasmids  carrying sequences of beaf protein exon as –ve control , mar at histone gene locus  and scs’ mar at hsp <dig> locus  were loaded in each slot.
genomic dna and mar dna were 32p labelled by random primer labelling method and used for hybridization of the upper and the lower panel respectively.
southern validation for  <dig> mars and  <dig> non-mar regions chosen on the basis of sequencing data.
the upper panel shows the etbr stained gel profile of the pcr amplified fragments and lower panel shows the blot probed with 32p labelled mar dna.
distribution of mars on the acx and vha <dig> gene clusters.
a 98 kb region of chromosome 2 l that encompasses four closely related genes  and three genes of vha <dig> cluster is drawn to scale.
each boxed sub part shows genes, transcripts, paused pol ii, mar peaks, actual sequence tag counts , at rich regions and repeat regions.
the tag counts box shows ‘xy’ plot using the total tag count values of two replicates .
a / at rich and repeat region tracks are reproduced from the ucsc genome browser.
we randomly picked a few of the identified mars for validation, before carrying out further analysis.
to ascertain that the peaks obtained by sequencing represent true mars, we performed an in vivo mar-assay with some modification in the original protocol  <cit> .
we used the mar preparation to make probe and analyzed pcr amplified potential mars.
this modification in the protocol makes it possible to test many candidate sequences on a single southern blot.
we chose  <dig> mar and  <dig> non-mar sequences with similar at content for this purpose and, as shown in figure 1d, almost all of the candidate mars tested were found to be in vivo mars.
sequences corresponding to negative controls were absent in numat preparations.
mar prediction tools detect only a fraction of mars identified in this study
mar finder and smartest are the two commonly used online tools for prediction of mars in a given sequence using set of rules related to dna sequence motifs and conformational features  <cit> .
large scale, genome or chromosome level, validity of these tools, however, remains to be experimentally established.
we find that only 10-15% of the mars identified in our study are predictable by these in silico tools.
vast majority of in vivo mars identified in our study, therefore, are not detectable by existing computational tools.
there is also considerable lack of overlap between the output of these two tools.
for example, while mar finder predicted  <dig> mars on chromosome 3r, smartest predicted  <dig> mars, of which only  <dig> % were common with the mar finder hits.
our analysis gives  <dig> mars in this part of the genome of which only  <dig> % were picked by at least one of these tools.
significant improvement is needed to make mar analysis and prediction tools more effective.
mar content and its possible influence on topological feature of genome
mars identified in this study fall in size range of  <dig> -3 kb with a median size of 400 bp.
about 90% of the mar sequences were less than 600 bp long .
total genomic contribution by the  <dig> mars add up to  <dig>  mb which represents  <dig> % of the 120 mb of euchromatic genome of drosophila.
as mars are associated with higher order organization of chromatin and are expected to hold the base of chromatin loops, we looked into the inter-mar distance across the genome that may reflect the average chromatin loop-size.
we find this distance ranged from <1 kb to 150 kb with an average distance of 16 kb.
overall, 75% of the mars were less than 20 kb apart .
the data agrees with the reported loop size of 5–200 kb  <cit> .
our analysis, however, is restricted to the euchromatic region of the genome.
the heterochromatic part that contributes up to 30% of the genome, may have different topological features.figure  <dig>
mar characteristics.
size of mars – in base pairs plotted against their frequency of occurrence in the genome.
distribution of inter-mar distances measured as distance between the midpoints of two contiguous mars, plotted against their frequency of occurrence in the genome.
genomic location of mars, percentage of mars localising in various genomic elements.
mars occurring from -1 kb to +1 kb of tss were plotted as a line graph.
the graph shows that mars associate preferentially with paused pol ii site at +100 bp.
genomic context of mars
to find out whether mars are distributed randomly across the euchromatic regions or have a preferential locations, we looked at the distribution of mars on different chromosomal arms .
we observed a distinct increase in mar density on x chromosome as compared to the autosomes.
while, on autosomes we find one mar for ~20 kb of genomic dna, the x chromosome has one mar for ~9 kb dna.
we also noticed gene frequency per mar on the x chromosome to be ~ <dig> fold less than that on autosome, x chromosome has ~ <dig> gene/mar while autosomes have ~ <dig> genes/mar.
since half of the embryo are likely to be male where x chromosome is hyperactive for dosage compensation and active genes associate with mar  <cit> , this might be a reflection of the higher activity of genes on the x chromosome in male embryos for dosage compensation.table  <dig>
distribution of mars on chromosomes
while 90% of all the mars are in non-coding regions  we do find mars associated with exons, mostly from highly expressed genes like rplp <dig>  ef1alpha 48d, hsc70- <dig>  lamc, mhc and cg <dig> .
75% of the exonic mars correlate with moderate to highly expressed genes  <cit> .
this further substantiates the view that mars have a link to transcriptional activity of the locus associated with them.
it remains to be established, however, if structural basis provided by mar association helps in coupling of transcription and related activity, viz., splicing, or differential epigenetic status and pol ii occupancy of exons/introns  <cit>  results in preferential retention of exons in numat.
we also noticed that non-genic mars are smaller compared to exonic ones, average size of 386 bp and 584 bp, respectively, which may reflect different nature or roles of these mars.
analysis of mar sequences with the known genomic features
association to known mar features
we looked for the enrichment of dna sequence features that are reported to be associated with mars  <cit> .
as shown in additional file 2: table s <dig>  ~94% of the mars follow the atc rule of one strand having at least a  <dig> nucleotide stretch of a, t, or c without intervening g, ~95% have origin of replication  sequence motifs  and significant proportion of them have features such as curved dna, at-rich stretch, etc.
base un-pairing sequences and dtopo ii binding sites, however, are missing in most of the mar sequences identified in this study.
the dtopoii binding site used in mar finder as well as in our study has been derived from in vitro conditions  <cit> .
later studies show that dtopoii sequence derived in vitro was not operative in vivo <cit>  which may explain this apparent inconsistency.
however, whether drosophila has different sequence motif for these features and that is why they appear to be missing remains to be ruled out.
as satb <dig> does not have a homologue in flies, this motif is likely to have got included in mars later at the time of emergence of vertebrates.
paused polymerase ii regions are associated with mars
we plotted the occurrence of mars with relation to transcription start sites .
as shown in figure 3d, most of the mars around tss, localize approximately at 100 bp after the tss.
it has been shown that in genes that respond to environmental and developmental cues, pol ii is engaged in early elongation and remains poised at ~ <dig> nucleotides downstream of tss.
its release into elongation is the rate-limiting step, and stalling pol ii is a way to regulate stimulus response.
we find that ~16% of the stalled pol ii regions listed by a study that used 2–4 hrs drosophila embryo, are mars  <cit>  .
it is probable that stalled pol ii regions are dynamic and many may not remain stalled at later stages of embryonic development and thus do not show up as mars in our study where 0–16 hrs old embryos were used.
this also indicates that the mars corresponding to paused pol ii regions are dynamic.
as this study provides only a snapshot of mars present in developing embryo, it remains to be seen if these sequences are numat associated to cause polymerase stalling or a mere consequence of the process.
enrichment of repeats in mars
simple sequence repeats  have been proposed to have role in genome packaging and mediation of long-range interactions  <cit> .
we looked for mar association with ssrs ≥12 bp repeats and present > <dig> locations in the genome.
a subset of ssrs show significant association with mars .
29% of a≥ <dig> and 27% of c≥ <dig> repeats present in euchromatic region are associated with mars.
among the dimeric repeats,  <dig> and  <dig> were enriched significantly in mars.
about 6% of the trimeric repeats,  <dig>   <dig>   <dig> and  <dig> are associated with mars.
among the hexameric repeats,  <dig>   <dig> and  <dig> that closely resemble poly-a stretches with a single mis-match, and  <dig> are enriched in mars.
interestingly when we looked for novel sequence motifs present in the mars identified in this study using mumod, that uses a bayesian approach to detect enriched novel dna motifs without relying on any motifs database,  <cit>  the four most abundant sequence motifs turned out to be ssr related,  <dig>   <dig>   <dig> and  <dig> .
finally, among transposable elements  the 5′-untranslated region of the drosophila gypsy retrotransposon has been shown to contain an ‘insulator’ that acts as transcriptional activator of gypsy as well as a mar/sar  <cit> .
thus we looked for enrichment of sequences from tes in the mars.
we find that sequences from a few retrotransposons are enriched in numat .
from the  <dig> ltr and  <dig> line-like families of retrotransposons, only  <dig> show significant enrichment of more than  <dig> copies being present in the mars.
lines show enrichment in the mars and in most of the cases, the at-rich 3′-utr of the te is a mar.figure  <dig>
mar sequence motifs.
abundant ssrs present among the mars are represented as percent of total genomic occurrence and fold enrichment.
sequence motifs found to be enriched in mars based on mumod search.
transposable elements that associate with numat
*
*based on blast  <dig> sequence comparison of mar sequence hit region with euchromatic genome sequence  carried out on flybase release  <dig>  annotation including partial tes.
mars as chromatin domain boundaries
as mars are proposed to be responsible for compartmentalization of genome into functional domains, we looked if these sequences overlap with chromatin domain boundaries.
based on modencode chip data, two types of boundaries - class i  and class ii  were identified earlier  <cit> .
of the  <dig> mars, only  <dig>  overlap with class i and class ii boundaries.
about 90% of the experimentally determined sites bound with beaf, cp <dig>  ctcf or su do not map to the mars identified in this study.
along with this, we also looked into a list of predicted boundaries obtained by an in silico prediction tool cdbest, that uses clusters of binding sites of boundary interacting proteins for prediction  <cit> .
of the cdbest predicted boundaries,  <dig>  overlap with mars.
we, therefore, conclude that only a small subset of mars function as chromatin domain boundaries.
for a closer look, we analysed the drosophila bithorax complex for overlap of boundaries and mars .
the region has four identified and  <dig> predicted boundaries.
out of these  <dig> boundaries only one coincides with one of the thirteen mars present in this region.
discussion
sequencing of the genome of higher eukaryotes promises insight into the complexity of how genetic information is contained and regulated.
to achieve that, however, annotation of various functional elements of the genome needs to be accomplished.
other than protein coding genes, some rna coding genes and few regulatory elements, much of the genomic content remains to be explored.
in the present study, we sequenced mars from drosophila embryo and mapped to annotate ~ <dig> % of the euchromatic genome that opens up ways to understand structural basis of genomic packaging and its functional implications.
sequence analysis of the mars from our study shows abundance of several known mar motifs including at-rich stretches and ori site.
in addition to the known mar features, our analysis shows enrichment of certain ssrs.
ability of several repeats to influence nucleosome positioning and chromatin level regulatory functions gives structural context of numat to these functional repeats  <cit> .
we also found retrotransposon sequences associated with mars.
transposons are implicated in shaping up of genome architecture, occupy ~5% of euchromatic genome and many occurrences are restraint by transcriptional inactivation  <cit> .
while functional link between mar and retrotransposons remains be investigated, our findings and earlier report that gypsy retrotransposon, which functions as chromatin insulator element, is also associated with nuclear matrix  <cit> , provide possible structural basis to it.
mars link the genomic loci to the nuclear architecture and by that are proposed to define topologically independent domains.
numat proteins that bind to dna may have a role in the process.
for example, lamin b, an abundant structural protein in the numat has been shown to bind to s/mars  <cit> .
we earlier identified  <dig> numat proteins from drosophila embryo, where ~13% were related to dna binding or chromatin remodelling and, possibly, bridge numat to chromatin in vivo <cit> .
we find an average inter-mar distance and, thereby, chromatin loop size to be 16 kb, which supports the earlier reports showing the loops in active chromatin regions to range from  <dig> to 13 kb  <cit> .
this also raises the possibility of a cohabitation of chromatin domain boundary and mar in agreement with the mechanism where boundaries interact with nuclear lamina, nucleolus, and numat as well  <cit> .
however, we found only ~5% overlap between boundaries and mars.
this shows that most of the boundaries do not associate with numat and insulator bodies may be a numat independent feature.
it remains, however, to be formally ruled out if only a subset of boundaries are functional in a given cell type and partial overlap with mar reflects that.
we find several mars in coding regions that are actively transcribed or are poised for transcription.
it has been shown that association of chromosomes with numat determines its transcriptional activity  <cit> .
we expect transcription related mars to be dynamic and dependent on the transcription profile of the cell in question, although it remains to be firmly established.
interestingly, we find that x chromosome, that is known to be hyper-activated in drosophila males for dosage compensation, has double the mar density compared to the rest of the genome.
a direct link between mar and dosage compensation remains to be explored.
another striking feature, in the context of transcription and mar emerged as the association of stalled pol ii promoters with mar  <cit> .
this indicates that transcriptionally engaged pol ii accumulates just downstream of the promoters and structural basis for this state is provided by the association with numat.
in the actively transcribed genes, we also noticed that only exons were associated with numat.
it has become increasingly clear that transcription and splicing are coupled events as both the processes are executed in numat in a co-ordinated manner.
it has also been shown that pol ii accumulates mostly at exons during transcription and only exons are tethered to pol ii transcription machinery excluding introns by looping out  <cit> .
such an arrangement helps in accurate splice site selection especially across large introns that may have many alternative splice sites  <cit> .
as pol ii transcription machinery is closely associated with numat, enrichment of exonic sequences in mars provides the structural link between the two.
we also noticed that the average mar size measures to 2– <dig> nucleosomal space.
this incidentally is the number of nucleosomes that get displaced during many nuclear processes like transcription, and double stranded break repair  <cit> .
as these processes are executed in close proximity of numat, mars of this size may be the reflection of functional dna in action at a given instance.
mars in coding regions were, however, relatively longer than those in non-coding regions.
while the relevance of this remains to be investigated, this observation is in agreement with earlier report of a similar difference in size of mars from coding and non-coding regions that was observed in hela cell  <cit> .
we observed very little overlap between the output of mar prediction tools as well as the mars identified in this study.
one possibility is that various ‘rules’ may not have been given appropriate weightage or these rules apply less to drosophila genome.
while further studies will be required to settle this issue, mar prediction tools will need to incorporate functional/epigenetic state of the region and its dynamic nature among the parameters.
several observations in our study indicate functional links of mar, viz., association with exons of highly expressed genes, longer mars from exons and association with sites of paused rna pol ii which adds conditional mar situation in addition to generally considered facultative mars.
CONCLUSIONS
in conclusion, mapping of nuclear matrix attachment regions across 120 mb of euchromatic drosophila genome reveals strong link between mar and transcriptional status of the locus.
we also find mars cohabiting with regulatory functions, viz., ori sequences, paused pol ii sites, domain boundaries, etc.
these observations reflect the structural perspective of nuclear architecture in context of functioning of these elements.
it is also possible that there are two kinds of mars, one that function as structural elements – attach genome to the nuclear matrix, for example, to define chromatin loops and the other that associate with components of nuclear architecture as a consequence being part of a regulatory mechanism, for example, pausing of transcription.
while the first kind is expected to be constant or static mar, the second one is likely to be transient or dynamic which will depend on cell type or functional state of the associated loci.
our study leads to a better understanding of the elements that define the chromatin landscape and co-ordinate packaging and regulation of genome in nuclear space.
mapping of mars at genome scale offers a structural look at the high throughput epigenetic modifications now available in public domain and an understanding of the structural basis of epigenetic regulation of gene expression.
further studies will be needed to examine such regulatory elements in the context of specific cell types.

 eukaryotic nucleus is a complex organelle, where dna is highly compacted but still is accessible for nuclear processes in a precisely controlled manner. the structural basis for such compact but orderly organization is provided by a proteinaceous meshwork known as nuclear matrix, that has been visualized by electron microscopy. although existence of numat in vivo is debated in the context of possible self assembly, this alone is not sufficient to explain all the features of nuclear organization. non-diffusible fraction of lamin in the interior of the nucleus of living cells indicates the existance of a nucleoskeleton involving such components. the presence of actin, myosin and several cytoskeletal proteins in the nucleus and numat further strengthens this view. whole proteome analysis of numat from arabidopsis, drosophila and human have shown that the major categories of proteins these preparations remain the same across species and cytoskeletal proteins are a conserved category. numat as a framework offers potential basis for compartmentalization of the nucleus and explains why several markers of different nuclear substructures have been found in the numat proteome. numat is also envisaged as a scaffold on which higher order organization of chromatin loops takes place. such topologically independent loops define the unit of compartmentalized chromatin that along with differential epigenetic marks bring about proper regulation of gene expression. biochemically, numat consists of dna, rna and protein and, while the underlying nuclear structure is not fully characterised, the dna component belongs to the sequences that help attach the base of chromatin loops to the numat and are defined as matrix/scaffold associated regions, m/sar. although not much sequence similarity is noticed in different mars, their biochemical properties have been shown to be conserved presumably due to the secondary structure features and other physical properties of underlying sequences. they are nuclease protected regions of chromatin that functionally associate with varitey of cis-regulatory elements including origin of replication, chromatin domain boundaries and locus control regions. considering the importance of mars in genome organization and regulation, a genome–wide map of these sequences is needed to understand the relationship between mar and known regulatory sequences at genome scale. efforts have been made in past to experimentally identify number of mars and to create computational tools to predict them. here we report, for the first time, mapping and characterization of mars across the entire 120 mb of drosophila euchromatic genome. we identify mars accounting for ~ % of this genome and gives insight into their role in chromatin organization and regulation in the context of genomic packaging.  identification of mar elements in drosophila melanogastergenome d. melanogaster embryos were collected and used for numat preparation using serial extraction steps including dnasei to digest accessible chromatin dna. quality of the numat preparation was checked using several parameters: retention of 1-2% dna, ~10% proteins and 30% rna compared to the nucleus. this preparation was used as source to purify associated genomic fragments as mar dna of ~100 bp size that were dnasei sensitive and rnasea resistant. this uniformity in the size of mar dna reflects the region that remains in-accessible to the nuclease, while some flanking relevant features may not be retained here. this mar preparation was further checked for the enrichment of known mars. the mar dna preparation was directly sequenced without any further fragmentation. two biological replicates of mar preparations were used for direct sequencing that gave a total of million mappable reads. the sequenced reads were mapped to the d. melanogaster genome using bowtie short read mapping algorithm and enriched regions were identified using macs software. the two replicates were processed independently and gave and peaks. as shown in additional file 1: figure s we observed very close similarity in pattern of peaks between the biological replicates. overall, we found peaks common between the two replicates that amounts to ~70% of overlap. a windowed comparison of tag counts between the replicates indicates a pearson’s correlation coefficient of. this implies that ~97% of the reads from the biological replicates agree with each other. of the peaks common to both the biological replicates, we selected peaks that mapped to euchromatic regions for further analysis, while peaks that mapped to heterochromatic or un-annotated stretches of sequences were left out. figure  shows a ~100 kb region from chromosome 2 l encompassing acx and vha gene clusters. correlation of mars in the region with at richness, repeats and paused polymerase ii sites is evident.figure isolation and validation of drosophila embryo mars. overview of mar dna isolation procedure. size distribution of mar dna after electrophoresis on % agarose gel. m-molecular weight marker; lane 1-genomic dna; lane 2-mar dna; lane 3-mar dna + dnasei; lane 4-mar dna + rnasea. slot-blot hybridization to show enrichment of known mars in the mar dna preparation. equal quantity of plasmids carrying sequences of beaf protein exon as –ve control, mar at histone gene locus and scs’ mar at hsp locus were loaded in each slot. genomic dna and mar dna were 32p labelled by random primer labelling method and used for hybridization of the upper and the lower panel respectively. southern validation for mars and non-mar regions chosen on the basis of sequencing data. the upper panel shows the etbr stained gel profile of the pcr amplified fragments and lower panel shows the blot probed with 32p labelled mar dna. distribution of mars on the acx and vha gene clusters. a 98 kb region of chromosome 2 l that encompasses four closely related genes and three genes of vha cluster is drawn to scale. each boxed sub part shows genes, transcripts, paused pol ii, mar peaks, actual sequence tag counts, at rich regions and repeat regions. the tag counts box shows ‘xy’ plot using the total tag count values of two replicates. a / at rich and repeat region tracks are reproduced from the ucsc genome browser. we randomly picked a few of the identified mars for validation, before carrying out further analysis. to ascertain that the peaks obtained by sequencing represent true mars, we performed an in vivo mar-assay with some modification in the original protocol. we used the mar preparation to make probe and analyzed pcr amplified potential mars. this modification in the protocol makes it possible to test many candidate sequences on a single southern blot. we chose mar and non-mar sequences with similar at content for this purpose and, as shown in figure 1d, almost all of the candidate mars tested were found to be in vivo mars. sequences corresponding to negative controls were absent in numat preparations. mar prediction tools detect only a fraction of mars identified in this study mar finder and smartest are the two commonly used online tools for prediction of mars in a given sequence using set of rules related to dna sequence motifs and conformational features. large scale, genome or chromosome level, validity of these tools, however, remains to be experimentally established. we find that only 10-15% of the mars identified in our study are predictable by these in silico tools. vast majority of in vivo mars identified in our study, therefore, are not detectable by existing computational tools. there is also considerable lack of overlap between the output of these two tools. for example, while mar finder predicted mars on chromosome 3r, smartest predicted mars, of which only % were common with the mar finder hits. our analysis gives mars in this part of the genome of which only % were picked by at least one of these tools. significant improvement is needed to make mar analysis and prediction tools more effective. mar content and its possible influence on topological feature of genome mars identified in this study fall in size range of -3 kb with a median size of 400 bp. about 90% of the mar sequences were less than 600 bp long. total genomic contribution by the mars add up to  mb which represents % of the 120 mb of euchromatic genome of drosophila. as mars are associated with higher order organization of chromatin and are expected to hold the base of chromatin loops, we looked into the inter-mar distance across the genome that may reflect the average chromatin loop-size. we find this distance ranged from <1 kb to 150 kb with an average distance of 16 kb. overall, 75% of the mars were less than 20 kb apart. the data agrees with the reported loop size of 5–200 kb. our analysis, however, is restricted to the euchromatic region of the genome. the heterochromatic part that contributes up to 30% of the genome, may have different topological features.figure mar characteristics. size of mars – in base pairs plotted against their frequency of occurrence in the genome. distribution of inter-mar distances measured as distance between the midpoints of two contiguous mars, plotted against their frequency of occurrence in the genome. genomic location of mars, percentage of mars localising in various genomic elements. mars occurring from -1 kb to +1 kb of tss were plotted as a line graph. the graph shows that mars associate preferentially with paused pol ii site at +100 bp. genomic context of mars to find out whether mars are distributed randomly across the euchromatic regions or have a preferential locations, we looked at the distribution of mars on different chromosomal arms. we observed a distinct increase in mar density on x chromosome as compared to the autosomes. while, on autosomes we find one mar for ~20 kb of genomic dna, the x chromosome has one mar for ~9 kb dna. we also noticed gene frequency per mar on the x chromosome to be ~ fold less than that on autosome, x chromosome has ~ gene/mar while autosomes have ~ genes/mar. since half of the embryo are likely to be male where x chromosome is hyperactive for dosage compensation and active genes associate with mar, this might be a reflection of the higher activity of genes on the x chromosome in male embryos for dosage compensation.table distribution of mars on chromosomes while 90% of all the mars are in non-coding regions we do find mars associated with exons, mostly from highly expressed genes like rplp ef1alpha 48d, hsc70- lamc, mhc and cg. 75% of the exonic mars correlate with moderate to highly expressed genes. this further substantiates the view that mars have a link to transcriptional activity of the locus associated with them. it remains to be established, however, if structural basis provided by mar association helps in coupling of transcription and related activity, viz., splicing, or differential epigenetic status and pol ii occupancy of exons/introns results in preferential retention of exons in numat. we also noticed that non-genic mars are smaller compared to exonic ones, average size of 386 bp and 584 bp, respectively, which may reflect different nature or roles of these mars. analysis of mar sequences with the known genomic features association to known mar features we looked for the enrichment of dna sequence features that are reported to be associated with mars. as shown in additional file 2: table s ~94% of the mars follow the atc rule of one strand having at least a nucleotide stretch of a, t, or c without intervening g, ~95% have origin of replication sequence motifs and significant proportion of them have features such as curved dna, at-rich stretch, etc. base un-pairing sequences and dtopo ii binding sites, however, are missing in most of the mar sequences identified in this study. the dtopoii binding site used in mar finder as well as in our study has been derived from in vitro conditions. later studies show that dtopoii sequence derived in vitro was not operative in vivo which may explain this apparent inconsistency. however, whether drosophila has different sequence motif for these features and that is why they appear to be missing remains to be ruled out. as satb does not have a homologue in flies, this motif is likely to have got included in mars later at the time of emergence of vertebrates. paused polymerase ii regions are associated with mars we plotted the occurrence of mars with relation to transcription start sites. as shown in figure 3d, most of the mars around tss, localize approximately at 100 bp after the tss. it has been shown that in genes that respond to environmental and developmental cues, pol ii is engaged in early elongation and remains poised at ~ nucleotides downstream of tss. its release into elongation is the rate-limiting step, and stalling pol ii is a way to regulate stimulus response. we find that ~16% of the stalled pol ii regions listed by a study that used 2–4 hrs drosophila embryo, are mars. it is probable that stalled pol ii regions are dynamic and many may not remain stalled at later stages of embryonic development and thus do not show up as mars in our study where 0–16 hrs old embryos were used. this also indicates that the mars corresponding to paused pol ii regions are dynamic. as this study provides only a snapshot of mars present in developing embryo, it remains to be seen if these sequences are numat associated to cause polymerase stalling or a mere consequence of the process. enrichment of repeats in mars simple sequence repeats have been proposed to have role in genome packaging and mediation of long-range interactions. we looked for mar association with ssrs ≥12 bp repeats and present > locations in the genome. a subset of ssrs show significant association with mars. 29% of a≥ and 27% of c≥ repeats present in euchromatic region are associated with mars. among the dimeric repeats, and were enriched significantly in mars. about 6% of the trimeric repeats, and are associated with mars. among the hexameric repeats, and that closely resemble poly-a stretches with a single mis-match, and are enriched in mars. interestingly when we looked for novel sequence motifs present in the mars identified in this study using mumod, that uses a bayesian approach to detect enriched novel dna motifs without relying on any motifs database, the four most abundant sequence motifs turned out to be ssr related, and. finally, among transposable elements the 5′-untranslated region of the drosophila gypsy retrotransposon has been shown to contain an ‘insulator’ that acts as transcriptional activator of gypsy as well as a mar/sar. thus we looked for enrichment of sequences from tes in the mars. we find that sequences from a few retrotransposons are enriched in numat. from the ltr and line-like families of retrotransposons, only show significant enrichment of more than copies being present in the mars. lines show enrichment in the mars and in most of the cases, the at-rich 3′-utr of the te is a mar.figure mar sequence motifs. abundant ssrs present among the mars are represented as percent of total genomic occurrence and fold enrichment. sequence motifs found to be enriched in mars based on mumod search. transposable elements that associate with numat * *based on blast sequence comparison of mar sequence hit region with euchromatic genome sequence carried out on flybase release annotation including partial tes. mars as chromatin domain boundaries as mars are proposed to be responsible for compartmentalization of genome into functional domains, we looked if these sequences overlap with chromatin domain boundaries. based on modencode chip data, two types of boundaries - class i and class ii were identified earlier. of the mars, only overlap with class i and class ii boundaries. about 90% of the experimentally determined sites bound with beaf, cp ctcf or su do not map to the mars identified in this study. along with this, we also looked into a list of predicted boundaries obtained by an in silico prediction tool cdbest, that uses clusters of binding sites of boundary interacting proteins for prediction. of the cdbest predicted boundaries, overlap with mars. we, therefore, conclude that only a small subset of mars function as chromatin domain boundaries. for a closer look, we analysed the drosophila bithorax complex for overlap of boundaries and mars. the region has four identified and predicted boundaries. out of these boundaries only one coincides with one of the thirteen mars present in this region. discussion sequencing of the genome of higher eukaryotes promises insight into the complexity of how genetic information is contained and regulated. to achieve that, however, annotation of various functional elements of the genome needs to be accomplished. other than protein coding genes, some rna coding genes and few regulatory elements, much of the genomic content remains to be explored. in the present study, we sequenced mars from drosophila embryo and mapped to annotate ~ % of the euchromatic genome that opens up ways to understand structural basis of genomic packaging and its functional implications. sequence analysis of the mars from our study shows abundance of several known mar motifs including at-rich stretches and ori site. in addition to the known mar features, our analysis shows enrichment of certain ssrs. ability of several repeats to influence nucleosome positioning and chromatin level regulatory functions gives structural context of numat to these functional repeats. we also found retrotransposon sequences associated with mars. transposons are implicated in shaping up of genome architecture, occupy ~5% of euchromatic genome and many occurrences are restraint by transcriptional inactivation. while functional link between mar and retrotransposons remains be investigated, our findings and earlier report that gypsy retrotransposon, which functions as chromatin insulator element, is also associated with nuclear matrix, provide possible structural basis to it. mars link the genomic loci to the nuclear architecture and by that are proposed to define topologically independent domains. numat proteins that bind to dna may have a role in the process. for example, lamin b, an abundant structural protein in the numat has been shown to bind to s/mars. we earlier identified numat proteins from drosophila embryo, where ~13% were related to dna binding or chromatin remodelling and, possibly, bridge numat to chromatin in vivo. we find an average inter-mar distance and, thereby, chromatin loop size to be 16 kb, which supports the earlier reports showing the loops in active chromatin regions to range from to 13 kb. this also raises the possibility of a cohabitation of chromatin domain boundary and mar in agreement with the mechanism where boundaries interact with nuclear lamina, nucleolus, and numat as well. however, we found only ~5% overlap between boundaries and mars. this shows that most of the boundaries do not associate with numat and insulator bodies may be a numat independent feature. it remains, however, to be formally ruled out if only a subset of boundaries are functional in a given cell type and partial overlap with mar reflects that. we find several mars in coding regions that are actively transcribed or are poised for transcription. it has been shown that association of chromosomes with numat determines its transcriptional activity. we expect transcription related mars to be dynamic and dependent on the transcription profile of the cell in question, although it remains to be firmly established. interestingly, we find that x chromosome, that is known to be hyper-activated in drosophila males for dosage compensation, has double the mar density compared to the rest of the genome. a direct link between mar and dosage compensation remains to be explored. another striking feature, in the context of transcription and mar emerged as the association of stalled pol ii promoters with mar. this indicates that transcriptionally engaged pol ii accumulates just downstream of the promoters and structural basis for this state is provided by the association with numat. in the actively transcribed genes, we also noticed that only exons were associated with numat. it has become increasingly clear that transcription and splicing are coupled events as both the processes are executed in numat in a co-ordinated manner. it has also been shown that pol ii accumulates mostly at exons during transcription and only exons are tethered to pol ii transcription machinery excluding introns by looping out. such an arrangement helps in accurate splice site selection especially across large introns that may have many alternative splice sites. as pol ii transcription machinery is closely associated with numat, enrichment of exonic sequences in mars provides the structural link between the two. we also noticed that the average mar size measures to 2– nucleosomal space. this incidentally is the number of nucleosomes that get displaced during many nuclear processes like transcription, and double stranded break repair. as these processes are executed in close proximity of numat, mars of this size may be the reflection of functional dna in action at a given instance. mars in coding regions were, however, relatively longer than those in non-coding regions. while the relevance of this remains to be investigated, this observation is in agreement with earlier report of a similar difference in size of mars from coding and non-coding regions that was observed in hela cell. we observed very little overlap between the output of mar prediction tools as well as the mars identified in this study. one possibility is that various ‘rules’ may not have been given appropriate weightage or these rules apply less to drosophila genome. while further studies will be required to settle this issue, mar prediction tools will need to incorporate functional/epigenetic state of the region and its dynamic nature among the parameters. several observations in our study indicate functional links of mar, viz., association with exons of highly expressed genes, longer mars from exons and association with sites of paused rna pol ii which adds conditional mar situation in addition to generally considered facultative mars.  in conclusion, mapping of nuclear matrix attachment regions across 120 mb of euchromatic drosophila genome reveals strong link between mar and transcriptional status of the locus. we also find mars cohabiting with regulatory functions, viz., ori sequences, paused pol ii sites, domain boundaries, etc. these observations reflect the structural perspective of nuclear architecture in context of functioning of these elements. it is also possible that there are two kinds of mars, one that function as structural elements – attach genome to the nuclear matrix, for example, to define chromatin loops and the other that associate with components of nuclear architecture as a consequence being part of a regulatory mechanism, for example, pausing of transcription. while the first kind is expected to be constant or static mar, the second one is likely to be transient or dynamic which will depend on cell type or functional state of the associated loci. our study leads to a better understanding of the elements that define the chromatin landscape and co-ordinate packaging and regulation of genome in nuclear space. mapping of mars at genome scale offers a structural look at the high throughput epigenetic modifications now available in public domain and an understanding of the structural basis of epigenetic regulation of gene expression. further studies will be needed to examine such regulatory elements in the context of specific cell types. 
we find significant association of mars with the origins of replication, transcription start sites, paused rna polymerase ii sites and exons, but not introns, of highly expressed genes. we also identified sequence motifs and repeats that constitute mars. this process is facilitated by the structural framework of nuclear matrix, a nucleo-proteinaceous meshwork. nuclear matrixmatrix attachment regionsgenome packagingissue-copyright-statement© the author 2014 our data reveal the contact points of genome to the nuclear architecture and provide a link between nuclear functions and genomic packaging.
 eukaryotic genome acquires functionality upon proper packaging within the nucleus. this process is facilitated by the structural framework of nuclear matrix, a nucleo-proteinaceous meshwork. matrix attachment regions in the genome serve as anchoring sites to this framework.  here we report direct sequencing of the mar preparation from drosophila melanogaster embryos and identify > mars. this amounts to ~ % of the fly genome and often coincide with at rich non-coding regions. we find significant association of mars with the origins of replication, transcription start sites, paused rna polymerase ii sites and exons, but not introns, of highly expressed genes. we also identified sequence motifs and repeats that constitute mars.  our data reveal the contact points of genome to the nuclear architecture and provide a link between nuclear functions and genomic packaging. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nuclear matrixmatrix attachment regionsgenome packagingissue-copyright-statement© the author 2014 