BACKGROUND
drosophila x chromosomes show peculiar features in both gene expression  <cit>  and gene evolution  <cit> .
one of the most striking consequences of x chromosome hemizygosity in males, is dosage compensation, a process which brings x chromosome and autosome expression into balance  <cit> .
dosage compensation was probably acquired gradually in the course of sex chromosome evolution, as sex chromosomes are thought to arise by divergence of an ancestral autosome pair  <cit> .
gene loss from the y chromosome creates an increasingly aneuploid condition in males and is thought to be the driving force in the evolution of global x-chromosome dosage compensation.
in the absence of dosage compensation genomic imbalance results in male lethality.
it has long been known that selective pressures applied to just one of the sexes can effect change in the other  <cit> .
for example, the coloration of certain birds or the nipples of mammals are advantageous to one of the sexes and are likely to be present as an evolutionary side-effect in the other.
x chromosome dosage compensation might also show evidence of this type of sexual selection.
x chromosome dosage compensation requires both cis and trans components  <cit> .
cis changes resulting from selection of the compensation system in males will also be present in females, and might alter the character of the x chromosome in females as a secondary consequence  <cit> .
indeed, we have previously noted a slight over-expression of both male and female x chromosomes relative to autosomes  <cit> , which suggests that the x chromosome is inherently more active than autosomes.
we have therefore examined the structure of x chromatin in females in detail.
expression patterns and especially x chromosome dosage compensation are mediated by chromatin modification  <cit> .
histones are nucleosome subunits required for packing dna into the confines of the nucleus.
it has long been know that chromatin structure changes are associated with transcription  <cit> .
for example, when chromatin is physically sheared to small fragments by sonication or enzyme digestion, shearing-bias is associated with different chromatin structures across the genome  <cit> .
histones are also modified on n-terminal tail residues to generate an expanding repertoire of histone modifications that are important modulators of transcription  <cit> .
it has become increasingly clear that specific types of modification are associated with particular transcriptional outcomes.
for example, acetylation events are broadly associated with transcriptional activation, while methylation events can have either activating or repressing roles.
one of the best studied histone modifications is the acetylation of histone  <dig> on lysine  <dig> .
in organisms from yeast to humans, h4k16ac is broadly associated with active genes, and the histone acetyl transferase  that writes the modification is required for viability  <cit> .
in drosophila, h4k16ac is highly enriched on the x chromosomes of males  <cit> , and the responsible hat, males absent on first , is required for male viability  <cit> .
while mof is associated with some genes in both males and females  <cit> , mof is greatly enriched on the male x chromosome due to targeting by the male-specific-lethal  complex.
msl is composed of proteins  and two non-coding rnas encoded on the x   <cit> .
it is thought that the greatly increased h4k16ac levels act to increase x chromosome expression in males, although it is also possible that x chromosome enrichment depletes autosomes of h4k16ac  <cit> .
in either model, x chromosome and autosome expression are equilibrated to restore transcription balance.
another chromatin modifying enzyme, jil <dig>  is also enriched on the x chromosome of males  <cit> .
this kinase mediates phosphorylation of histone  <dig> at serine  <dig> .
jil <dig> is required for full dosage compensation and associates with the msl complex  <cit> .
h3s10ph is implicated in both chromosome condensation during mitosis and transcriptional activation during interphase, suggesting that jil <dig> has more general roles in addition to dosage compensation.
another mark associated with active transcription, dimethylation of histone h <dig> at lysine  <dig>   <cit>  is general, and thus likely to be msl complex independent.
we have performed chromatin-shearing experiments showing that x chromatin differs from autosomal chromatin in both males and females.
additionally, the histone marks associated with x chromosome dosage compensation in males are modestly enriched on female x chromosomes.
these data indicate that x chromatin is distinct even in the absence of dosage compensation.
we suggest that the pattern in females is a tolerated neutral side-effect of the evolution of x chromosome dosage compensation in males.
RESULTS
global analysis of chromatin structure in females and males
we took advantage of differential shearing to probe chromatin structure by deep dna sequencing   <cit> .
specifically, we sheared cross-linked chromatin, size selected for short  fragments, performed deep sequencing, and aligned the dna reads to the reference genome.
because these sequencing reactions generated reads from the ends of size-selected fragments, increased mapped read density occurs in regions of preferential shearing.
we constructed libraries of dna from sheared chromatin from female and male adult flies and obtained about  <dig> million uniquely mapping  <dig> bp reads for each sex.
we then parsed the mapped reads by chromosome arms.
the x chromosome and each arm of the 2nd and 3rd chromosomes bear about 20% of the genome, while the 4th chromosome bears <1%.
the average coverage of the different chromosomes  was similar  except the x chromosome in males  and the 4th chromosome in both males and females  .
the average coverage of the male x chromosome was 48% of the autosome coverage, which is consistent with the male karyotype.
permutation testing of chromosome arm sequence coverage relative to genome-wide coverage showed that the 4th chromosome had unusually low read density .
the under-representation of dna sequence coverage on the heterochromatin-rich, gene poor, the 4th chromosome  <cit>  suggests that this chromosome was resistant to shearing.
thus, the 4th chromosome data suggests that lower sequence coverage is associated with a more "closed" chromatin state in this largely heterochromatin chromosome.
we were interested in shearing at gene models, as chromatin alternations associated with transcription, and thus dosage compensation, should be evident at those sites.
to summarize the gene model read density patterns genome-wide, we calculated read coverage at multiple gene model features.
we clearly observed higher sequence coverage at exonic regions relative to intronic regions and elevated coverage flanking transcription start and termination sites on x chromosomes and autosomes .
we suggest that these signatures are due to association of different protein-complex types during transcription.
thus, like dnase hypersensitivity  <cit> , mechanically sheared chromatin breaks preferentially at some sites to provide information about chromatin structure genome-wide.
we then compared the coverage profiles of x chromosome and autosome gene model features inter se to determine if the structure of the x chromosome differed from the autosomes.
indeed, the average coverage for x chromosome genes was clearly higher than autosome arm genes at exons, transcription start sites, and transcription termination sites in both males and females .
the 4th chromosome always showed greatly reduced sequence coverage.
permutation testing clearly suggests that the x chromosome shearing at exons, transcription start sites, and termination sites is significantly greater than the pattern genome-wide in both males  and females .
these data indicate that x chromosome genes are susceptible to shearing, probably as result of a more open structure.
distribution of histone marks on female and male chromosomes
if cis features have evolved on the x chromosome to facilitate dosage compensation in conjunction with the trans-acting msl complex, then the x chromosome in females might show a "shadow" profile resembling the male x chromosome.
we therefore more specifically probed for the histone modifications that are associated with x chromosome dosage compensation.
to localize histone marks in the genome, we carried out chromatin immunoprecipitation coupled with microarray hybridization  to flygem arrays on sex-sorted adult flies.
the dna from chip and input chromatin were hybridized to the same slides, normalized, and averaged to generate a ratio for each gene .
in agreement with the dna-seq coverage data, the intensity of chip input x chromosome dna was significantly higher than the input for the autosomes in females and was significantly lower for the 4th chromosome .
thus, the input data also suggest that x chromatin is more open.
chip ratios  eliminate the effect of chromatin on the input channel when testing for histone modification enrichment.
as expected, we found a strong enrichment for h4k16ac on the male x chromosome relative to male autosomes .
the average h4k16ac chip ratio  on male x chromosome genes was  <dig>  fold higher than the value of all autosome gene.
these data show that we are able to easily score the high levels of h4k16ac present on the male x chromosomes in adult flies.
interestingly, we observed a modest  but significant enrichment for h4k16ac on female x chromosomes relative to all the autosomes .
in both males and females, the ratio of h4k16ac chip/input was similar for all major autosome arms  indicating that there are no differences in h4k16ac among the major autosome arms within either sex.
these data indicate that not only is x chromatin different from autosomal chromatin in females, but that the important dosage compensation mark h4k16ac is generally enriched on x chromosomes.
we observed a modest, but significant  <dig>  fold enrichment for the h3s10ph mark on the male x chromosome relative to the autosomes .
we also observed a  <dig>  fold enrichment of h3s10ph on the female x chromosome, but this difference was only modestly significant .
as observed for the other two histone marks, we found a significant enrichment for h3k4me <dig> on the x chromosome in males .
but again, we also found significant enrichment for h3k4me <dig> on the female x chromosome .
thus, the patterns of histone mark accumulation on the male x chromosome appear to be shadowed by similar distributions on the female x chromosome.
distributions of histone marks correlate with each other and active genes
if the female x chromosome shadows the pattern observed on the male x because of underlying changes in x chromosome sequence, then the histone marks should also correlate at the gene level.
to determine if the same genes show similar histone modifications on both the male and female x chromosome, we directly examined correlations by using the rank order of individual histone marks to normalize the results of the three distinct chip data sets and then subjected the rank order data to cluster analysis .
to highlight associations on the autosomes, we clustered the x chromosomes and autosomes separately.
we observed a strong positive correlation between h4k16ac and the other two histone marks on both x chromosomes and autosomes.
for example, spearman's rank correlation indicates that h4k16ac on male x chromosomes was positively correlated with h3k4me <dig>  and h3s10ph .
on the autosomes, h4k16ac enrichment was positively correlated with female h4k16ac  and with other two histone modifications .
more importantly, there was a good rank correlation between male and female x chromosomes for h4k16ac , h3s10ph , and h3k4me <dig> .
these data indicate that x chromosomes in the sexes differ by degree of modification, but the pattern of modifications along the male x chromosome is shadowed along the female x chromosome.
dosage compensation alters transcription relative to autosomes.
therefore, one might expect that genes showing particular modifications in males and females would be expressed.
to determine if the three histone marks were associated with transcribed genes, we examined gene expression data for male or female adult flies from a previous study  <cit>  for genes highly enriched or depleted for different histone marks .
the genes enriched for any of the three different histone marks all showed significantly higher expression levels compared to genes depleted for histone marks.
this observation held in both males and females tissues.
for example, the expression level of genes enriched for h4k16ac is  <dig> -fold higher in males and  <dig> -fold higher in females relative to genes depleted for h4k16ac  within each sex.
these data clearly indicate that the three different histone modifications are correlated inter se and with expression.
we suggest that all three marks contribute to the mild hypertranscription of female x chromosomes.
germline x chromosomes are dosage compensated by increased expression of the single x in males relative to autosomes  <cit> , but germline dosage compensation is not mediated by the msl complex  <cit> .
therefore the histone mark and expression correlations may not hold for the germline.
to determine if the shadow marks on the female x chromosome are restricted to genes expressed in the soma, we examined male-biased expression in adult flies, which is dominated by expression within the male germline  <cit> .
if h4k16ac is associated with active transcription only in somatic cells as predicted by our understanding of somatic msl function, then genes with male-biased expression in whole flies should show reduced h4k16ac levels.
indeed, this is what we observed .
x chromosome genes showing male-biased expression in whole flies show very low  h4k16ac levels in males, compared to x chromosome genes showing non-biased expression in males .
these data support the idea that dosage compensation marks occur at genes active in the male soma, but not the germline.
dosage compensation may be not being required for genes that should be differentially regulated in the female and male soma.
in support of this idea, we also observed lower h4k16ac among x chromosome genes showing male-biased expression in the soma .
these data are also consistent with the idea that genes with male-biased expression are poorly expressed on the x chromosome due to limited dosage compensation  <cit> .
interestingly, the same pattern is observed for genes with female-biased expression.
we observed lower h4k16ac among genes showing female-biased expression in females .
these data suggest that sex differentially regulated genes are not h4k16ac regulated.
discussion
our data indicate that the dosage compensation marks associated with x chromosomes in males are also enriched on x chromosomes more generally.
this is part of a growing body of data showing that the x chromosome is distinct from the autosomes.
for example, previous studies show that jil <dig> mutants cause accumulation of the h4k9me <dig> mark and the transcriptional negative regulator hp <dig> on the x chromosome of both males and females  <cit> .
additionally,  <dig> of  <dig> chromatin associated proteins surveyed in female drosophila kc <dig> cells are enriched on the x chromosome  <cit> .
finally, both mof and h4k16ac enrichment has been observed on the x chromosomes of female kc <dig> cells  <cit> , although female results served as controls in those manuscripts and the enrichment in females was not highlighted.
collectively, these data indicate that x chromatin differences from autosomes are not restricted to male-specific dosage compensation.
we have previously observed a modest elevation of gene expression from the female x chromosome relative to autosomes in d. melanogaster  <cit>  and this same modest  but highly significant elevation of female x chromosome expression is consistently observed in six other drosophila species  <cit> , suggesting that this modest elevation of expression on x chromosome is independent of microarray platform and species.
we suggest that specialized x chromatin contributes to elevated x chromosome gene expression in females in addition to males, albeit to a much more limited extent.
that there is enrichment for x chromosome marks associated with x chromosome dosage compensation in males is not de facto evidence of function within females.
indeed, the viability of female mutants for many of the msl components, indicates that the msl complex is not required.
it is possible that rather than being functional in females, the observed x chromosome enrichment is a consequence of the evolution of dosage compensation in males  <cit> .
in the sequence of sex chromosome evolution from an autosome pair, genes are gradually lost from the neo-y resulting in hemizygosity for those genes in males .
any sequence changes that would promote increased x chromosome expression in males, by for example opening chromatin structure, would be highly advantageous in males.
those sequence changes would also be present in females where they would be disadvantageous.
however, if the increase in male fitness does not result in a large negative affect on female fitness, then sequence changes supporting increased epigenetic enhancement of x chromosome expression would be favored.
as more genes were lost and the y chromosome was reduced to a gene desert, the need for balancing expression with the autosomes became critical, such that the general msl complex became specialized for the x. msl is highly deleterious for females, but msl formation is blocked by the action of sxl on the msl component msl <dig>  <cit> .
we suggest that sxl activity is sufficient to ensure female viability, but the cis changes on the x chromosome and any low level mof activity in the absence of msl result in a more open chromatin structure and slight over-expression from the female x chromosome.
while modest over expression of x chromosome genes in females may be of little evolutionary consequence, one can envision that more extensive over expression would be detrimental.
for example, large duplications are poorly tolerated in drosophila and  <cit>  trisomy is poorly tolerated in humans  <cit> .
if the x chromosome tends to be in a favourable state for transcription in both males and females, this could ultimately lead to counter measures in females to prevent functional tetrasomy.
this might be what has occurred in mammals, where one x chromosome is inactivated in females.
there is recent evidence that the active x chromosomes of mammals show increased expression  <cit>  raising the possibility that x inactivation is a consequence of x chromosome dosage compensation in males.
the main dosage compensation counter measure in drosophila females is inhibition of msl complex formation by sxl protein  <cit> .
however, if the observed over expression of x chromosomes in drosophila females  <cit>  was more extreme, then inactivating an x chromosome in females to counter x chromosome dosage compensation in males would be logical.
this may be how such a mechanism evolved in mammals.
CONCLUSIONS
in this study we examined the general chromatin structure and multiple histone marks in both drosophila males and females.
we found that x chromatin structure is different from autosome structure independent of sex.
histone marks associated with dosage compensation or active transcription that are highly enriched on the male x chromosome are also slightly enriched on the female x chromosome.
these findings suggest that the distinct female x chromatin structure is an evolutionary consequence of dosage compensation in males.

 drosophila x chromosomes show peculiar features in both gene expression and gene evolution. one of the most striking consequences of x chromosome hemizygosity in males, is dosage compensation, a process which brings x chromosome and autosome expression into balance. dosage compensation was probably acquired gradually in the course of sex chromosome evolution, as sex chromosomes are thought to arise by divergence of an ancestral autosome pair. gene loss from the y chromosome creates an increasingly aneuploid condition in males and is thought to be the driving force in the evolution of global x-chromosome dosage compensation. in the absence of dosage compensation genomic imbalance results in male lethality. it has long been known that selective pressures applied to just one of the sexes can effect change in the other. for example, the coloration of certain birds or the nipples of mammals are advantageous to one of the sexes and are likely to be present as an evolutionary side-effect in the other. x chromosome dosage compensation might also show evidence of this type of sexual selection. x chromosome dosage compensation requires both cis and trans components. cis changes resulting from selection of the compensation system in males will also be present in females, and might alter the character of the x chromosome in females as a secondary consequence. indeed, we have previously noted a slight over-expression of both male and female x chromosomes relative to autosomes, which suggests that the x chromosome is inherently more active than autosomes. we have therefore examined the structure of x chromatin in females in detail. expression patterns and especially x chromosome dosage compensation are mediated by chromatin modification. histones are nucleosome subunits required for packing dna into the confines of the nucleus. it has long been know that chromatin structure changes are associated with transcription. for example, when chromatin is physically sheared to small fragments by sonication or enzyme digestion, shearing-bias is associated with different chromatin structures across the genome. histones are also modified on n-terminal tail residues to generate an expanding repertoire of histone modifications that are important modulators of transcription. it has become increasingly clear that specific types of modification are associated with particular transcriptional outcomes. for example, acetylation events are broadly associated with transcriptional activation, while methylation events can have either activating or repressing roles. one of the best studied histone modifications is the acetylation of histone on lysine. in organisms from yeast to humans, h4k16ac is broadly associated with active genes, and the histone acetyl transferase that writes the modification is required for viability. in drosophila, h4k16ac is highly enriched on the x chromosomes of males, and the responsible hat, males absent on first, is required for male viability. while mof is associated with some genes in both males and females, mof is greatly enriched on the male x chromosome due to targeting by the male-specific-lethal complex. msl is composed of proteins and two non-coding rnas encoded on the x. it is thought that the greatly increased h4k16ac levels act to increase x chromosome expression in males, although it is also possible that x chromosome enrichment depletes autosomes of h4k16ac. in either model, x chromosome and autosome expression are equilibrated to restore transcription balance. another chromatin modifying enzyme, jil is also enriched on the x chromosome of males. this kinase mediates phosphorylation of histone at serine. jil is required for full dosage compensation and associates with the msl complex. h3s10ph is implicated in both chromosome condensation during mitosis and transcriptional activation during interphase, suggesting that jil has more general roles in addition to dosage compensation. another mark associated with active transcription, dimethylation of histone h at lysine is general, and thus likely to be msl complex independent. we have performed chromatin-shearing experiments showing that x chromatin differs from autosomal chromatin in both males and females. additionally, the histone marks associated with x chromosome dosage compensation in males are modestly enriched on female x chromosomes. these data indicate that x chromatin is distinct even in the absence of dosage compensation. we suggest that the pattern in females is a tolerated neutral side-effect of the evolution of x chromosome dosage compensation in males.  global analysis of chromatin structure in females and males we took advantage of differential shearing to probe chromatin structure by deep dna sequencing. specifically, we sheared cross-linked chromatin, size selected for short fragments, performed deep sequencing, and aligned the dna reads to the reference genome. because these sequencing reactions generated reads from the ends of size-selected fragments, increased mapped read density occurs in regions of preferential shearing. we constructed libraries of dna from sheared chromatin from female and male adult flies and obtained about million uniquely mapping bp reads for each sex. we then parsed the mapped reads by chromosome arms. the x chromosome and each arm of the 2nd and 3rd chromosomes bear about 20% of the genome, while the 4th chromosome bears <1%. the average coverage of the different chromosomes was similar except the x chromosome in males and the 4th chromosome in both males and females. the average coverage of the male x chromosome was 48% of the autosome coverage, which is consistent with the male karyotype. permutation testing of chromosome arm sequence coverage relative to genome-wide coverage showed that the 4th chromosome had unusually low read density. the under-representation of dna sequence coverage on the heterochromatin-rich, gene poor, the 4th chromosome suggests that this chromosome was resistant to shearing. thus, the 4th chromosome data suggests that lower sequence coverage is associated with a more "closed" chromatin state in this largely heterochromatin chromosome. we were interested in shearing at gene models, as chromatin alternations associated with transcription, and thus dosage compensation, should be evident at those sites. to summarize the gene model read density patterns genome-wide, we calculated read coverage at multiple gene model features. we clearly observed higher sequence coverage at exonic regions relative to intronic regions and elevated coverage flanking transcription start and termination sites on x chromosomes and autosomes. we suggest that these signatures are due to association of different protein-complex types during transcription. thus, like dnase hypersensitivity, mechanically sheared chromatin breaks preferentially at some sites to provide information about chromatin structure genome-wide. we then compared the coverage profiles of x chromosome and autosome gene model features inter se to determine if the structure of the x chromosome differed from the autosomes. indeed, the average coverage for x chromosome genes was clearly higher than autosome arm genes at exons, transcription start sites, and transcription termination sites in both males and females. the 4th chromosome always showed greatly reduced sequence coverage. permutation testing clearly suggests that the x chromosome shearing at exons, transcription start sites, and termination sites is significantly greater than the pattern genome-wide in both males and females. these data indicate that x chromosome genes are susceptible to shearing, probably as result of a more open structure. distribution of histone marks on female and male chromosomes if cis features have evolved on the x chromosome to facilitate dosage compensation in conjunction with the trans-acting msl complex, then the x chromosome in females might show a "shadow" profile resembling the male x chromosome. we therefore more specifically probed for the histone modifications that are associated with x chromosome dosage compensation. to localize histone marks in the genome, we carried out chromatin immunoprecipitation coupled with microarray hybridization to flygem arrays on sex-sorted adult flies. the dna from chip and input chromatin were hybridized to the same slides, normalized, and averaged to generate a ratio for each gene. in agreement with the dna-seq coverage data, the intensity of chip input x chromosome dna was significantly higher than the input for the autosomes in females and was significantly lower for the 4th chromosome. thus, the input data also suggest that x chromatin is more open. chip ratios eliminate the effect of chromatin on the input channel when testing for histone modification enrichment. as expected, we found a strong enrichment for h4k16ac on the male x chromosome relative to male autosomes. the average h4k16ac chip ratio on male x chromosome genes was fold higher than the value of all autosome gene. these data show that we are able to easily score the high levels of h4k16ac present on the male x chromosomes in adult flies. interestingly, we observed a modest but significant enrichment for h4k16ac on female x chromosomes relative to all the autosomes. in both males and females, the ratio of h4k16ac chip/input was similar for all major autosome arms indicating that there are no differences in h4k16ac among the major autosome arms within either sex. these data indicate that not only is x chromatin different from autosomal chromatin in females, but that the important dosage compensation mark h4k16ac is generally enriched on x chromosomes. we observed a modest, but significant fold enrichment for the h3s10ph mark on the male x chromosome relative to the autosomes. we also observed a fold enrichment of h3s10ph on the female x chromosome, but this difference was only modestly significant. as observed for the other two histone marks, we found a significant enrichment for h3k4me on the x chromosome in males. but again, we also found significant enrichment for h3k4me on the female x chromosome. thus, the patterns of histone mark accumulation on the male x chromosome appear to be shadowed by similar distributions on the female x chromosome. distributions of histone marks correlate with each other and active genes if the female x chromosome shadows the pattern observed on the male x because of underlying changes in x chromosome sequence, then the histone marks should also correlate at the gene level. to determine if the same genes show similar histone modifications on both the male and female x chromosome, we directly examined correlations by using the rank order of individual histone marks to normalize the results of the three distinct chip data sets and then subjected the rank order data to cluster analysis. to highlight associations on the autosomes, we clustered the x chromosomes and autosomes separately. we observed a strong positive correlation between h4k16ac and the other two histone marks on both x chromosomes and autosomes. for example, spearman's rank correlation indicates that h4k16ac on male x chromosomes was positively correlated with h3k4me and h3s10ph. on the autosomes, h4k16ac enrichment was positively correlated with female h4k16ac and with other two histone modifications. more importantly, there was a good rank correlation between male and female x chromosomes for h4k16ac, h3s10ph, and h3k4me. these data indicate that x chromosomes in the sexes differ by degree of modification, but the pattern of modifications along the male x chromosome is shadowed along the female x chromosome. dosage compensation alters transcription relative to autosomes. therefore, one might expect that genes showing particular modifications in males and females would be expressed. to determine if the three histone marks were associated with transcribed genes, we examined gene expression data for male or female adult flies from a previous study for genes highly enriched or depleted for different histone marks. the genes enriched for any of the three different histone marks all showed significantly higher expression levels compared to genes depleted for histone marks. this observation held in both males and females tissues. for example, the expression level of genes enriched for h4k16ac is -fold higher in males and -fold higher in females relative to genes depleted for h4k16ac within each sex. these data clearly indicate that the three different histone modifications are correlated inter se and with expression. we suggest that all three marks contribute to the mild hypertranscription of female x chromosomes. germline x chromosomes are dosage compensated by increased expression of the single x in males relative to autosomes, but germline dosage compensation is not mediated by the msl complex. therefore the histone mark and expression correlations may not hold for the germline. to determine if the shadow marks on the female x chromosome are restricted to genes expressed in the soma, we examined male-biased expression in adult flies, which is dominated by expression within the male germline. if h4k16ac is associated with active transcription only in somatic cells as predicted by our understanding of somatic msl function, then genes with male-biased expression in whole flies should show reduced h4k16ac levels. indeed, this is what we observed. x chromosome genes showing male-biased expression in whole flies show very low h4k16ac levels in males, compared to x chromosome genes showing non-biased expression in males. these data support the idea that dosage compensation marks occur at genes active in the male soma, but not the germline. dosage compensation may be not being required for genes that should be differentially regulated in the female and male soma. in support of this idea, we also observed lower h4k16ac among x chromosome genes showing male-biased expression in the soma. these data are also consistent with the idea that genes with male-biased expression are poorly expressed on the x chromosome due to limited dosage compensation. interestingly, the same pattern is observed for genes with female-biased expression. we observed lower h4k16ac among genes showing female-biased expression in females. these data suggest that sex differentially regulated genes are not h4k16ac regulated. discussion our data indicate that the dosage compensation marks associated with x chromosomes in males are also enriched on x chromosomes more generally. this is part of a growing body of data showing that the x chromosome is distinct from the autosomes. for example, previous studies show that jil mutants cause accumulation of the h4k9me mark and the transcriptional negative regulator hp on the x chromosome of both males and females. additionally, of chromatin associated proteins surveyed in female drosophila kc cells are enriched on the x chromosome. finally, both mof and h4k16ac enrichment has been observed on the x chromosomes of female kc cells, although female results served as controls in those manuscripts and the enrichment in females was not highlighted. collectively, these data indicate that x chromatin differences from autosomes are not restricted to male-specific dosage compensation. we have previously observed a modest elevation of gene expression from the female x chromosome relative to autosomes in d. melanogaster and this same modest but highly significant elevation of female x chromosome expression is consistently observed in six other drosophila species, suggesting that this modest elevation of expression on x chromosome is independent of microarray platform and species. we suggest that specialized x chromatin contributes to elevated x chromosome gene expression in females in addition to males, albeit to a much more limited extent. that there is enrichment for x chromosome marks associated with x chromosome dosage compensation in males is not de facto evidence of function within females. indeed, the viability of female mutants for many of the msl components, indicates that the msl complex is not required. it is possible that rather than being functional in females, the observed x chromosome enrichment is a consequence of the evolution of dosage compensation in males. in the sequence of sex chromosome evolution from an autosome pair, genes are gradually lost from the neo-y resulting in hemizygosity for those genes in males. any sequence changes that would promote increased x chromosome expression in males, by for example opening chromatin structure, would be highly advantageous in males. those sequence changes would also be present in females where they would be disadvantageous. however, if the increase in male fitness does not result in a large negative affect on female fitness, then sequence changes supporting increased epigenetic enhancement of x chromosome expression would be favored. as more genes were lost and the y chromosome was reduced to a gene desert, the need for balancing expression with the autosomes became critical, such that the general msl complex became specialized for the x. msl is highly deleterious for females, but msl formation is blocked by the action of sxl on the msl component msl. we suggest that sxl activity is sufficient to ensure female viability, but the cis changes on the x chromosome and any low level mof activity in the absence of msl result in a more open chromatin structure and slight over-expression from the female x chromosome. while modest over expression of x chromosome genes in females may be of little evolutionary consequence, one can envision that more extensive over expression would be detrimental. for example, large duplications are poorly tolerated in drosophila and trisomy is poorly tolerated in humans. if the x chromosome tends to be in a favourable state for transcription in both males and females, this could ultimately lead to counter measures in females to prevent functional tetrasomy. this might be what has occurred in mammals, where one x chromosome is inactivated in females. there is recent evidence that the active x chromosomes of mammals show increased expression raising the possibility that x inactivation is a consequence of x chromosome dosage compensation in males. the main dosage compensation counter measure in drosophila females is inhibition of msl complex formation by sxl protein. however, if the observed over expression of x chromosomes in drosophila females was more extreme, then inactivating an x chromosome in females to counter x chromosome dosage compensation in males would be logical. this may be how such a mechanism evolved in mammals.  in this study we examined the general chromatin structure and multiple histone marks in both drosophila males and females. we found that x chromatin structure is different from autosome structure independent of sex. histone marks associated with dosage compensation or active transcription that are highly enriched on the male x chromosome are also slightly enriched on the female x chromosome. these findings suggest that the distinct female x chromatin structure is an evolutionary consequence of dosage compensation in males. 
x chromosomes are subject to dosage compensation in drosophila males. our data indicate that x chromatin structure is fundamentally different from autosome structure in both sexes. we find that the general chromatin structure of female x chromosomes is distinct from autosomes. additionally, specific histone marks associated with dosage compensation and active chromatin marks on the male x chromosome are also enriched on the x chromosomes of females, albeit to a lesser degree. we suggest that the differences between the x chromosomes and autosomes in females are a consequence of mechanisms that have evolved to ensure sufficient x chromosome expression in the soma of males. dosage compensation requires cis sequence features of the x chromosome that are present in both sexes by definition and trans acting factors that target chromatin modifying machinery to the x specifically in males.
 x chromosomes are subject to dosage compensation in drosophila males. dosage compensation requires cis sequence features of the x chromosome that are present in both sexes by definition and trans acting factors that target chromatin modifying machinery to the x specifically in males. the evolution of this system could result in neutral x chromatin changes that will be apparent in females.  we find that the general chromatin structure of female x chromosomes is distinct from autosomes. additionally, specific histone marks associated with dosage compensation and active chromatin marks on the male x chromosome are also enriched on the x chromosomes of females, albeit to a lesser degree.  our data indicate that x chromatin structure is fundamentally different from autosome structure in both sexes. we suggest that the differences between the x chromosomes and autosomes in females are a consequence of mechanisms that have evolved to ensure sufficient x chromosome expression in the soma of males. 