BACKGROUND
eukaryotic dna is packaged into chromatin, a highly compacted structure made up of repeating nucleosome units.
nucleosomes consist of 147 bp of dna wrapped around an octamer of core histones  and are connected to each other by short stretches of linker dna  <cit> .
in most organisms, this short extra-nucleosomal linker region is bound by an additional histone, known as h <dig>  h <dig> belongs to a class of chromatin architectural proteins  that are responsible for maintaining, modulating and stabilizing chromatin architecture.
h <dig> binds to the dna as it enters/exits the nucleosome and may compact chromatin into a higher-order fiber structure  <cit> .
this is supported by in vivo studies that have shown that reducing the total amount of cellular h <dig> results in a less compact chromatin structure  <cit> .
the ability of h <dig> to compact chromatin may be antagonized by other caps, such as the highly abundant high mobility group proteins .
hmgs decompact higher-order chromatin structures to promote the binding of nuclear regulatory factors to their binding sites  <cit> .
hmgs have similar dna and chromatin binding properties to h <dig>  <cit> , bind to sites at the entry/exit dyad of the nucleosome and linker dna  <cit> , and may out-compete h <dig> in order to activate specific transcriptional programs  <cit> .
furthermore, changes in the concentration of h <dig> and hmg proteins alter transcriptional programs important for normal cell development and viability  <cit> .
thus, h <dig> and hmg proteins are both chromatin architectural proteins that may serve as active regulators of transcription.
similar chromatin binding of h <dig> and hmgs suggests that they may be functionally linked and act in opposition with respect to the stability of chromatin structure  <cit> .
potentially, access to the genome by transcription regulatory machinery could be mediated by competition between h <dig> and hmg.
support for this hypothesis comes from early embryogenesis, where initially hmgd <dig>  or hmg-b  is highly abundant and h <dig> is barely detectable.
as development progresses, h <dig> is expressed and replaces hmgd1/hmgb <dig> at some regions of the genome  <cit> .
this replacement is thought to silence specific genes and thus contribute to programmed development.
despite suggestive evidence of a relationship between hmgs and h <dig>  their genome-wide distributions are not known and a clear understanding of how they are related to chromatin structure and gene expression is lacking.
here we apply genome-wide profiling and gene specific approaches to study these proteins in d. melanogaster s <dig> cells and to better understand their roles in gene regulation and chromatin structural changes.
as drosophila only encodes one isoform each of histone h <dig> and hmgd <dig>  and there exists a wealth of other genomics data for this cell line, s <dig> cells are an excellent model system for studying this problem.
here, we report detailed experiments and analyses that show that h <dig> and hmgd <dig> are associated with specific genomic regions with specific transcriptional activity.
we show that these proteins bind reciprocally with each other and affect gene regulation and local chromatin structures.
the data we have generated serves as a useful resource for understanding the interplay between histone modifications, chromatin architectural proteins, chromatin structure and gene expression.
RESULTS
h <dig> and hmgd are enriched in different chromatin regions
to test whether hmgd <dig> and h <dig> are associated with distinct chromatin states, we first determined the relative abundance of these proteins in different chromatin fractions.
we isolated nuclei from d. melanogaster s <dig> cells and digested their chromatin with micrococcal nuclease .
we subjected the solubilized chromatin to salt fractionation analyses  and analyzed the resulting euchromatic  and heterochromatic  fractions by agarose gel  and western blot .
the majority of hmgd <dig> protein is associated with the active euchromatic fraction as typified by the presence of h4k16ac , while most of h <dig> is present in the heterochromatic fraction .
we confirmed these results using a slightly modified version of the sucrose gradient fractionation method  <cit>  .
here too, h <dig> sedimented with the highly dense, longer and inactive chromatin and hmgd <dig> associated with the lighter, shorter and active chromatin fractions.
while both results show that hmgd <dig> and h <dig> associate preferentially with euchromatin and heterochromatin respectively, a non-negligible proportion of each protein is present in both fractions.
this could be due to post translational modifications of these proteins, which may change their targets and cellular localization.
to gain further insight into the functional consequences of hmgd <dig> and histone h <dig> binding to chromatin, we used nucleosome-chip-seq to obtain genome-wide maps of hmgd1- and h <dig> -bound nucleosomes in d. melanogaster s <dig> cells.
we first digested chromatin with mnase to yield ~450 bp fragments, which are long enough to include linker dna bound by either h <dig> or hmgd <dig>  we then immunoprecipitated chromatin fragments using chip-grade antibodies against histone h <dig>  hmgd <dig>  and igg  and adapted the immunoprecipitated mononucleosomal fragments for solid sequencing.
after sequencing the reads, we aligned them to the reference genome and discarded those that did not map uniquely.
in total, we mapped 12– <dig> million unique reads, equivalent to ~18-22× fold coverage per nucleosome .
total nucleosomal input dna from s <dig> cells was also sequenced and used to correct for background nucleosome occupancy.
we examined the broad distribution of h1– and hmgd1-bound nucleosomes across chromosomes.
after normalizing the total number of mapped reads in the h <dig> and hmgd <dig> datasets, we found that hmgd1-bound nucleosomes are consistently depleted on the heterochromatic chromosome arms compared to both h <dig> and total nucleosomes .
interestingly, hmgd <dig> is highly abundant on the x chromosome compared to total nucleosomes.
potentially this could be related to dosage compensation in male flies , which results in a doubling of gene expression on the x-chromosome .
these results are consistent with those from the chromatin fractionation experiments and indicate that hmgd <dig> is enriched in the euchromatic fraction of the genome.
hmgd <dig> and h <dig> presence at transcription start sites correlates with gene expression
the above results prompted us to ask whether the occupancy of hmgd <dig> and h <dig> vary across different chromosomal regions and features that are associated with transcription.
we used the flybase gene annotations to define genomic regions as intergenic, intronic, or exonic, and we labeled regions within 1000 bp of annotated transcription start sites  as promoters.
to estimate the relative abundance of hmgd <dig> and h <dig>  we counted the number of chip-seq nucleosome centers within each region, and divided by the total number of mapped reads from each experiment.
compared to total nucleosomes, hmgd <dig> is depleted in intergenic regions and is highly enriched at promoters.
conversely, h <dig> is depleted in promoter regions .
to further understand the hmgd <dig> promoter enrichment, we split promoters into regions that are downstream and upstream of the tss.
hmgd <dig> is enriched within both upstream and downstream regions of tsss, which suggests that it may be associated with both transcriptional initiation  and elongation.
we next asked whether differences in hmgd <dig> and h <dig> binding correlate with transcriptional activity.
we utilized rna-seq data from s <dig> cells  <cit>  and grouped genes by their expression levels.
we then aggregated data across genes in each expression group and compared the density of hmgd <dig> and histone h <dig> to the density of total nucleosomes.
total nucleosome reads from mnase-seq were used to subtract out effects that are solely attributable to nucleosome positioning .
we find that hmgd <dig> is highly enriched around the promoters of genes with high expression , while in contrast h <dig> is preferentially enriched in the promoters of genes that are silenced or have low expression .
we further validated the presence of hmgd <dig> and h <dig> at a subset of promoters with high and low expression genes using chip followed by rt-qpcr .
to quantify the relationship between gene expression, hmgd <dig>  and h <dig>  we calculated the pearson correlation between gene expression and chip-seq read depth across promoter regions .
hmgd <dig> showed a strong positive correlation  and h <dig> had a negative correlation  with gene expression .
we next divided promoters into several non-overlapping regions corresponding to the approximate locations of well-positioned nucleosomes and the nucleosome-depleted region  .
for each region we then calculated the correlation across all promoters.
hmgd <dig> has a moderately strong positive correlation with gene expression both upstream and downstream of the promoter, with a maximum correlation at the + <dig> nucleosome  .
h <dig> has a negative correlation with gene expression, which is strongest at the nucleosome-depleted region immediately upstream of the tss  .
these data suggest that while chromatin binding of hmgd <dig> is associated with transcription activation, h <dig> may be involved in gene silencing or repression.
if hmgd <dig> and h <dig> bind mutually exclusively and reciprocally to the same sequences, then the ratio of hmgd <dig> to h <dig> should be more strongly associated with gene expression than either measure alone.
to assess this we computed the correlation between gene expression and the ratio of hmgd <dig> to h <dig> density.
the correlations obtained with this ratio are substantially and significantly stronger than those with h <dig> or hmgd <dig> , and reach a maximum of r =  <dig>  at the + <dig> nucleosome.
this argues that the relative levels of these proteins are more important than their individual absolute levels .
hmgd <dig> is associated with dnase i hypersensitive sites
hmgn <dig>  the human homolog of hmgd <dig>  is known to co-localize with dnasei hypersensitive sites  <cit> , which are sensitive indicators of open chromatin  <cit> .
we wondered whether hmgd <dig> would be generally associated with open chromatin regions, including those outside of promoter regions.
to address this question, we obtained a set of dhss for drosophila s <dig> cells  <cit> .
we then divided the drosophila genome into non-overlapping 1 kb regions and computed the distance of each region to the nearest dhs.
the density of hmgd <dig> nucleosome midpoints is much higher near dhss, and there is a negative correlation between dhs distance and hmgd <dig> density  .
h <dig> density has only a very weak positive correlation with dhs distance  .
collectively our data suggest a high degree of correspondence between hmgd <dig> and regions of open chromatin in s <dig> cells.
in contrast, chromatin regions containing h <dig> are less accessible and perhaps refractory to dnase i. previous studies have argued that hmgd <dig> and h <dig> compete for binding to the same linker dna regions  <cit> ; thus the accessibility of chromatin to regulatory factors could be mediated by competition between these proteins.
if chromatin accessibility is driven by competition between these proteins, then their relative abundance should be more predictive than the level of either protein on its own.
we tested this prediction by computing the ratio of hmgd <dig> to h <dig> with respect to the distance to dhss .
the correlation with the ratio is significantly stronger than obtained by either protein on its own , even after excluding all regions that are within 2 kb of a known tss, the hmgd1/h <dig> ratio is highest near to dhss .
our data suggest that the relative levels of hmgd <dig> to h <dig> binding may be a primary determinant of chromatin openness and are consistent with previous experimental results that suggest these proteins bind competitively in vivo.
hmgd <dig> and h <dig> genome-wide binding regions align with transcriptionally active and inactive epigenetic marks respectively
histone posttranslational modifications  direct many important processes such as gene activation and repression  <cit> .
to determine whether h <dig> and hmgd <dig> are associated with specific core histone ptms, we downloaded a large set of s <dig> cell line histone modification data from the modencode project .
using 2 kb windows surrounding each annotated tsss, we computed the mean signal for each ptm and estimated the density of hmgd1- and h1–bound nucleosomes.
we then calculated the pairwise correlations across tss regions for all possible combinations of ptms, hmgd <dig> and h <dig> and performed hierarchical clustering to visualize the results .
we found that hmgd <dig> clusters with a large number of activating ptms including h3k36me <dig>  h3k79me1/ <dig>  h3k27ac, h3k18ac, h3k9acs10p, h3k9ac, h3k4me2/ <dig>  h4k16ac, and h4k8ac and with elongating marks h3k36me <dig>  h2bubi  <cit> .
in contrast, h <dig> clusters with several repressive marks, namely h3k27me2/ <dig>  h3k9me <dig>  and h4k20me <dig>  which are known to be associated with gene silencing and heterochromatin.
hmgd <dig> and h <dig> are associated with local nucleosome spacing
the average distance between two nucleosomes, the nucleosome repeat length , is an important parameter that describes primary chromatin organization.
since h1’s expression level is a key determinant of the genome-wide nrl  <cit> , we sought to determine whether the relative protein levels of hmgd <dig> and h <dig> are associated with local differences in nucleosome spacing.
we divided the genome into non-overlapping 2 kb regions and computed the ratio of hmgd <dig> to h <dig> in each region.
we then calculated the density of nucleosome midpoints as a function of distance from an “anchor” nucleosome .
the resulting plots show well-defined peaks in nucleosome occupancy particularly for regions with the highest hmgd1/h <dig> ratio .
these peaks were then used to estimate the nrl, which we found to be better correlated with the ratio of hmgd <dig> to h <dig>  than either h <dig> or hmgd <dig> alone, .
the nrl varies from 174 bp in regions with a high hmgd1/h <dig> ratio to 187 bp in regions with a low hmgd1/h <dig> ratio.
these results suggest that the stoichiometry of h <dig> is important, not only for the average nrl across the genome, but also for defining the local spacing of nucleosomes.
potentially, the spacing between nucleosomes may be specified by which cap is bound to the linker region .
under this hypothesis the average nrl across cells would reflect the relative concentrations of these proteins.
h <dig> and hmgd <dig> bind reciprocally and affect gene expression and nucleosome repeat length
to directly test the hypothesis that reciprocal binding of hmgd <dig> and h <dig> has functional consequences for gene regulation and nucleosome spacing, we performed sirna knockdown of both hmgd <dig> and h <dig>   <cit> .
following both knockdowns, we assayed the occupancy of h <dig> and hmgd <dig> in representative promoters using chip-qpcr .
for the hmgd <dig> knockdown, the subset of promoters with high initial levels of hmgd <dig> showed a remarkable two- to three-fold increase in h <dig> binding and a significant reduction in gene expression .
conversely, h <dig> knockdown resulted in only modest hmgd <dig> promoter occupancy increases at  <dig> of the  <dig> target h <dig> promoters examined and no change was observed at one promoter, hsp <dig>  likewise, observed changes in gene expression were varied at these promoters with only two genes showing significant increases in expression .
this suggests that h <dig> depletion alone may not be sufficient to initiate gene expression in all heterochromatic regions.
on the other hand, the reduction in gene expression following hmgd <dig> knockdown suggests that hmgd <dig> facilitates transcriptional initiation or elongation.
interestingly, knockdown of h <dig> decreased the average nrl in bulk chromatin by ~24 bp, whereas hmgd <dig> knockdown increased the average nrl by ~7 bp .
h <dig> knockdown may have a greater effect on the repeat length of bulk chromatin because it is present in most regions of the genome, while hmgd <dig> is most abundant at highly transcribed regions that comprise a smaller fraction of the genome.
these results suggest that nucleosome spacing may be dictated by the binding of these  caps.
discussion
the role of the core histones and their modifications in gene regulation has been widely studied, however, the function of chromatin architectural proteins such as histone h <dig> and the high mobility group proteins  are less well understood.
in this study we used genomic, gene specific and biochemical approaches to characterize two highly abundant chromatin architectural proteins: h <dig> and hmgd <dig>  additionally, by using a homogenous cell population and high-throughput sequencing, we were able to directly correlate h <dig> and hmgd <dig> protein binding with gene activity and chromatin structure on a genome-wide scale.
we found that hmgd <dig> is associated with active transcription and euchromatin, while h <dig> is associated with repressed genes and heterochromatin.
more specifically, hmgd <dig> occupancy is highest in regions with active transcription, activating histone ptms, and dhss, while h <dig> is enriched in inaccessible regions with low gene expression, repressive histone ptms and low dnase i sensitivity.
our results for hmgd <dig> are consistent with those from recent studies of hmgn <dig> , which found that this protein is enriched around dnasei hypersensitive sites at the promoters of actively transcribed genes  <cit> .
this suggests that the hmgd <dig> distribution at promoter regions is likely a broadly conserved feature of hmg proteins.
recently, genome-wide distributions of the histone h <dig> variants h1d and h1c have been obtained from human embryonic stem cells  <cit> .
as with histone h <dig> in drosophila s <dig> cells, these histone variants are depleted from regions of active chromatin and are enriched in heterochromatic regions  <cit> .
our results suggest that h <dig> and hmgd <dig> are part of a mechanism that establishes or maintains repressed and active chromatin states.
how hmgd <dig> or h <dig> are recruited to distinct chromatin regions remains unknown, but our data argue that reciprocal binding of these architectural proteins to the same chromatin region is important  <cit> .
the high on/off rates of these proteins’ chromatin association may provide a window of opportunity where one cap can replace the other to cause a change in chromatin state.
for instance, specific hmgs may be able to displace h <dig> and locally destabilize chromatin so that other proteins can be recruited to initiate transcriptional activation  <cit> .
consistent with this notion, we found that depletion of hmgd <dig> results in h <dig> binding to previously hmgd1-bound promoters , with a subsequent decrease in gene expression.
on the other hand, depletion of h <dig> only slightly increases in hmgd <dig> occupancy at a subset of previously bound h <dig> promoters, and had varied effects on gene expression, with  <dig> of  <dig> genes showing a significant increase in expression .
the expression level of h <dig> was previously known to affect the genome-wide average nrl  <cit> .
we found additionally that depletion of hmgd <dig> affects the genome-wide nrl.
we hypothesize that hmgs may be responsible for the reduction in nucleosome repeat lengths in active chromatin regions  <cit> .
changes in nrl are likely very important in gene regulation, because even a single base pair  shift in linker length changes the dna helical twist, resulting in a 36° torsional angle change in the neighboring nucleosome position  <cit> .
it is therefore possible that changes in nucleosome spacing introduced by caps such as hmgd <dig> modulate the accessibility of regulatory factor binding sites and have downstream consequences on transcription factor binding and gene expression.
CONCLUSIONS
in summary, we have shown that hmgd <dig> is localized to genomic loci with transcriptionally active histone modifications and dnase i hypersensitive sites, whereas h <dig> is primarily associated with heterochromatic regions.
both proteins contribute to the spacing of nucleosomes and it is likely that the depletion of h <dig> in transcriptionally active regions reflects the competitive and mutually exclusive binding of hmgs to linker sequences.
while we have studied the genomic localization of h <dig> and hmgd <dig>  other caps are also likely to play important roles in gene regulation, nucleosome spacing and chromatin compaction.
for example, competition between h <dig> and poly-adp-ribose polymerase can elicit specific transcriptional outcomes  <cit> .
an important future direction will be to study how caps compete with each other and whether their different isoforms and post-translational modifications affect their function.
already some data suggest that h <dig> and hmgd <dig> act differently depending on their post-translational modifications or binding partners  <cit> .
finally with this data, we provide a platform to determine in fine detail, the interplay between chromatin architectural proteins, epigenetic factors  and gene expression.

 eukaryotic dna is packaged into chromatin, a highly compacted structure made up of repeating nucleosome units. nucleosomes consist of 147 bp of dna wrapped around an octamer of core histones and are connected to each other by short stretches of linker dna. in most organisms, this short extra-nucleosomal linker region is bound by an additional histone, known as h h belongs to a class of chromatin architectural proteins that are responsible for maintaining, modulating and stabilizing chromatin architecture. h binds to the dna as it enters/exits the nucleosome and may compact chromatin into a higher-order fiber structure. this is supported by in vivo studies that have shown that reducing the total amount of cellular h results in a less compact chromatin structure. the ability of h to compact chromatin may be antagonized by other caps, such as the highly abundant high mobility group proteins. hmgs decompact higher-order chromatin structures to promote the binding of nuclear regulatory factors to their binding sites. hmgs have similar dna and chromatin binding properties to h, bind to sites at the entry/exit dyad of the nucleosome and linker dna, and may out-compete h in order to activate specific transcriptional programs. furthermore, changes in the concentration of h and hmg proteins alter transcriptional programs important for normal cell development and viability. thus, h and hmg proteins are both chromatin architectural proteins that may serve as active regulators of transcription. similar chromatin binding of h and hmgs suggests that they may be functionally linked and act in opposition with respect to the stability of chromatin structure. potentially, access to the genome by transcription regulatory machinery could be mediated by competition between h and hmg. support for this hypothesis comes from early embryogenesis, where initially hmgd or hmg-b is highly abundant and h is barely detectable. as development progresses, h is expressed and replaces hmgd1/hmgb at some regions of the genome. this replacement is thought to silence specific genes and thus contribute to programmed development. despite suggestive evidence of a relationship between hmgs and h their genome-wide distributions are not known and a clear understanding of how they are related to chromatin structure and gene expression is lacking. here we apply genome-wide profiling and gene specific approaches to study these proteins in d. melanogaster s cells and to better understand their roles in gene regulation and chromatin structural changes. as drosophila only encodes one isoform each of histone h and hmgd and there exists a wealth of other genomics data for this cell line, s cells are an excellent model system for studying this problem. here, we report detailed experiments and analyses that show that h and hmgd are associated with specific genomic regions with specific transcriptional activity. we show that these proteins bind reciprocally with each other and affect gene regulation and local chromatin structures. the data we have generated serves as a useful resource for understanding the interplay between histone modifications, chromatin architectural proteins, chromatin structure and gene expression.  h and hmgd are enriched in different chromatin regions to test whether hmgd and h are associated with distinct chromatin states, we first determined the relative abundance of these proteins in different chromatin fractions. we isolated nuclei from d. melanogaster s cells and digested their chromatin with micrococcal nuclease. we subjected the solubilized chromatin to salt fractionation analyses and analyzed the resulting euchromatic and heterochromatic fractions by agarose gel and western blot. the majority of hmgd protein is associated with the active euchromatic fraction as typified by the presence of h4k16ac, while most of h is present in the heterochromatic fraction. we confirmed these results using a slightly modified version of the sucrose gradient fractionation method. here too, h sedimented with the highly dense, longer and inactive chromatin and hmgd associated with the lighter, shorter and active chromatin fractions. while both results show that hmgd and h associate preferentially with euchromatin and heterochromatin respectively, a non-negligible proportion of each protein is present in both fractions. this could be due to post translational modifications of these proteins, which may change their targets and cellular localization. to gain further insight into the functional consequences of hmgd and histone h binding to chromatin, we used nucleosome-chip-seq to obtain genome-wide maps of hmgd1- and h -bound nucleosomes in d. melanogaster s cells. we first digested chromatin with mnase to yield ~450 bp fragments, which are long enough to include linker dna bound by either h or hmgd we then immunoprecipitated chromatin fragments using chip-grade antibodies against histone h hmgd and igg and adapted the immunoprecipitated mononucleosomal fragments for solid sequencing. after sequencing the reads, we aligned them to the reference genome and discarded those that did not map uniquely. in total, we mapped 12– million unique reads, equivalent to ~18-22× fold coverage per nucleosome. total nucleosomal input dna from s cells was also sequenced and used to correct for background nucleosome occupancy. we examined the broad distribution of h1– and hmgd1-bound nucleosomes across chromosomes. after normalizing the total number of mapped reads in the h and hmgd datasets, we found that hmgd1-bound nucleosomes are consistently depleted on the heterochromatic chromosome arms compared to both h and total nucleosomes. interestingly, hmgd is highly abundant on the x chromosome compared to total nucleosomes. potentially this could be related to dosage compensation in male flies, which results in a doubling of gene expression on the x-chromosome. these results are consistent with those from the chromatin fractionation experiments and indicate that hmgd is enriched in the euchromatic fraction of the genome. hmgd and h presence at transcription start sites correlates with gene expression the above results prompted us to ask whether the occupancy of hmgd and h vary across different chromosomal regions and features that are associated with transcription. we used the flybase gene annotations to define genomic regions as intergenic, intronic, or exonic, and we labeled regions within 1000 bp of annotated transcription start sites as promoters. to estimate the relative abundance of hmgd and h we counted the number of chip-seq nucleosome centers within each region, and divided by the total number of mapped reads from each experiment. compared to total nucleosomes, hmgd is depleted in intergenic regions and is highly enriched at promoters. conversely, h is depleted in promoter regions. to further understand the hmgd promoter enrichment, we split promoters into regions that are downstream and upstream of the tss. hmgd is enriched within both upstream and downstream regions of tsss, which suggests that it may be associated with both transcriptional initiation and elongation. we next asked whether differences in hmgd and h binding correlate with transcriptional activity. we utilized rna-seq data from s cells and grouped genes by their expression levels. we then aggregated data across genes in each expression group and compared the density of hmgd and histone h to the density of total nucleosomes. total nucleosome reads from mnase-seq were used to subtract out effects that are solely attributable to nucleosome positioning. we find that hmgd is highly enriched around the promoters of genes with high expression, while in contrast h is preferentially enriched in the promoters of genes that are silenced or have low expression. we further validated the presence of hmgd and h at a subset of promoters with high and low expression genes using chip followed by rt-qpcr. to quantify the relationship between gene expression, hmgd and h we calculated the pearson correlation between gene expression and chip-seq read depth across promoter regions. hmgd showed a strong positive correlation and h had a negative correlation with gene expression. we next divided promoters into several non-overlapping regions corresponding to the approximate locations of well-positioned nucleosomes and the nucleosome-depleted region. for each region we then calculated the correlation across all promoters. hmgd has a moderately strong positive correlation with gene expression both upstream and downstream of the promoter, with a maximum correlation at the + nucleosome. h has a negative correlation with gene expression, which is strongest at the nucleosome-depleted region immediately upstream of the tss. these data suggest that while chromatin binding of hmgd is associated with transcription activation, h may be involved in gene silencing or repression. if hmgd and h bind mutually exclusively and reciprocally to the same sequences, then the ratio of hmgd to h should be more strongly associated with gene expression than either measure alone. to assess this we computed the correlation between gene expression and the ratio of hmgd to h density. the correlations obtained with this ratio are substantially and significantly stronger than those with h or hmgd, and reach a maximum of r =  at the + nucleosome. this argues that the relative levels of these proteins are more important than their individual absolute levels. hmgd is associated with dnase i hypersensitive sites hmgn the human homolog of hmgd is known to co-localize with dnasei hypersensitive sites, which are sensitive indicators of open chromatin. we wondered whether hmgd would be generally associated with open chromatin regions, including those outside of promoter regions. to address this question, we obtained a set of dhss for drosophila s cells. we then divided the drosophila genome into non-overlapping 1 kb regions and computed the distance of each region to the nearest dhs. the density of hmgd nucleosome midpoints is much higher near dhss, and there is a negative correlation between dhs distance and hmgd density. h density has only a very weak positive correlation with dhs distance. collectively our data suggest a high degree of correspondence between hmgd and regions of open chromatin in s cells. in contrast, chromatin regions containing h are less accessible and perhaps refractory to dnase i. previous studies have argued that hmgd and h compete for binding to the same linker dna regions; thus the accessibility of chromatin to regulatory factors could be mediated by competition between these proteins. if chromatin accessibility is driven by competition between these proteins, then their relative abundance should be more predictive than the level of either protein on its own. we tested this prediction by computing the ratio of hmgd to h with respect to the distance to dhss. the correlation with the ratio is significantly stronger than obtained by either protein on its own, even after excluding all regions that are within 2 kb of a known tss, the hmgd1/h ratio is highest near to dhss. our data suggest that the relative levels of hmgd to h binding may be a primary determinant of chromatin openness and are consistent with previous experimental results that suggest these proteins bind competitively in vivo. hmgd and h genome-wide binding regions align with transcriptionally active and inactive epigenetic marks respectively histone posttranslational modifications direct many important processes such as gene activation and repression. to determine whether h and hmgd are associated with specific core histone ptms, we downloaded a large set of s cell line histone modification data from the modencode project. using 2 kb windows surrounding each annotated tsss, we computed the mean signal for each ptm and estimated the density of hmgd1- and h1–bound nucleosomes. we then calculated the pairwise correlations across tss regions for all possible combinations of ptms, hmgd and h and performed hierarchical clustering to visualize the results. we found that hmgd clusters with a large number of activating ptms including h3k36me h3k79me1/ h3k27ac, h3k18ac, h3k9acs10p, h3k9ac, h3k4me2/ h4k16ac, and h4k8ac and with elongating marks h3k36me h2bubi. in contrast, h clusters with several repressive marks, namely h3k27me2/ h3k9me and h4k20me which are known to be associated with gene silencing and heterochromatin. hmgd and h are associated with local nucleosome spacing the average distance between two nucleosomes, the nucleosome repeat length, is an important parameter that describes primary chromatin organization. since h1’s expression level is a key determinant of the genome-wide nrl, we sought to determine whether the relative protein levels of hmgd and h are associated with local differences in nucleosome spacing. we divided the genome into non-overlapping 2 kb regions and computed the ratio of hmgd to h in each region. we then calculated the density of nucleosome midpoints as a function of distance from an “anchor” nucleosome. the resulting plots show well-defined peaks in nucleosome occupancy particularly for regions with the highest hmgd1/h ratio. these peaks were then used to estimate the nrl, which we found to be better correlated with the ratio of hmgd to h than either h or hmgd alone,. the nrl varies from 174 bp in regions with a high hmgd1/h ratio to 187 bp in regions with a low hmgd1/h ratio. these results suggest that the stoichiometry of h is important, not only for the average nrl across the genome, but also for defining the local spacing of nucleosomes. potentially, the spacing between nucleosomes may be specified by which cap is bound to the linker region. under this hypothesis the average nrl across cells would reflect the relative concentrations of these proteins. h and hmgd bind reciprocally and affect gene expression and nucleosome repeat length to directly test the hypothesis that reciprocal binding of hmgd and h has functional consequences for gene regulation and nucleosome spacing, we performed sirna knockdown of both hmgd and h. following both knockdowns, we assayed the occupancy of h and hmgd in representative promoters using chip-qpcr. for the hmgd knockdown, the subset of promoters with high initial levels of hmgd showed a remarkable two- to three-fold increase in h binding and a significant reduction in gene expression. conversely, h knockdown resulted in only modest hmgd promoter occupancy increases at of the target h promoters examined and no change was observed at one promoter, hsp likewise, observed changes in gene expression were varied at these promoters with only two genes showing significant increases in expression. this suggests that h depletion alone may not be sufficient to initiate gene expression in all heterochromatic regions. on the other hand, the reduction in gene expression following hmgd knockdown suggests that hmgd facilitates transcriptional initiation or elongation. interestingly, knockdown of h decreased the average nrl in bulk chromatin by ~24 bp, whereas hmgd knockdown increased the average nrl by ~7 bp. h knockdown may have a greater effect on the repeat length of bulk chromatin because it is present in most regions of the genome, while hmgd is most abundant at highly transcribed regions that comprise a smaller fraction of the genome. these results suggest that nucleosome spacing may be dictated by the binding of these caps. discussion the role of the core histones and their modifications in gene regulation has been widely studied, however, the function of chromatin architectural proteins such as histone h and the high mobility group proteins are less well understood. in this study we used genomic, gene specific and biochemical approaches to characterize two highly abundant chromatin architectural proteins: h and hmgd additionally, by using a homogenous cell population and high-throughput sequencing, we were able to directly correlate h and hmgd protein binding with gene activity and chromatin structure on a genome-wide scale. we found that hmgd is associated with active transcription and euchromatin, while h is associated with repressed genes and heterochromatin. more specifically, hmgd occupancy is highest in regions with active transcription, activating histone ptms, and dhss, while h is enriched in inaccessible regions with low gene expression, repressive histone ptms and low dnase i sensitivity. our results for hmgd are consistent with those from recent studies of hmgn, which found that this protein is enriched around dnasei hypersensitive sites at the promoters of actively transcribed genes. this suggests that the hmgd distribution at promoter regions is likely a broadly conserved feature of hmg proteins. recently, genome-wide distributions of the histone h variants h1d and h1c have been obtained from human embryonic stem cells. as with histone h in drosophila s cells, these histone variants are depleted from regions of active chromatin and are enriched in heterochromatic regions. our results suggest that h and hmgd are part of a mechanism that establishes or maintains repressed and active chromatin states. how hmgd or h are recruited to distinct chromatin regions remains unknown, but our data argue that reciprocal binding of these architectural proteins to the same chromatin region is important. the high on/off rates of these proteins’ chromatin association may provide a window of opportunity where one cap can replace the other to cause a change in chromatin state. for instance, specific hmgs may be able to displace h and locally destabilize chromatin so that other proteins can be recruited to initiate transcriptional activation. consistent with this notion, we found that depletion of hmgd results in h binding to previously hmgd1-bound promoters, with a subsequent decrease in gene expression. on the other hand, depletion of h only slightly increases in hmgd occupancy at a subset of previously bound h promoters, and had varied effects on gene expression, with of genes showing a significant increase in expression. the expression level of h was previously known to affect the genome-wide average nrl. we found additionally that depletion of hmgd affects the genome-wide nrl. we hypothesize that hmgs may be responsible for the reduction in nucleosome repeat lengths in active chromatin regions. changes in nrl are likely very important in gene regulation, because even a single base pair shift in linker length changes the dna helical twist, resulting in a 36° torsional angle change in the neighboring nucleosome position. it is therefore possible that changes in nucleosome spacing introduced by caps such as hmgd modulate the accessibility of regulatory factor binding sites and have downstream consequences on transcription factor binding and gene expression.  in summary, we have shown that hmgd is localized to genomic loci with transcriptionally active histone modifications and dnase i hypersensitive sites, whereas h is primarily associated with heterochromatic regions. both proteins contribute to the spacing of nucleosomes and it is likely that the depletion of h in transcriptionally active regions reflects the competitive and mutually exclusive binding of hmgs to linker sequences. while we have studied the genomic localization of h and hmgd other caps are also likely to play important roles in gene regulation, nucleosome spacing and chromatin compaction. for example, competition between h and poly-adp-ribose polymerase can elicit specific transcriptional outcomes. an important future direction will be to study how caps compete with each other and whether their different isoforms and post-translational modifications affect their function. already some data suggest that h and hmgd act differently depending on their post-translational modifications or binding partners. finally with this data, we provide a platform to determine in fine detail, the interplay between chromatin architectural proteins, epigenetic factors and gene expression. 
chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone h and the high mobility group protein hmgd in drosophila s cells. this study provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation. in contrast, h is primarily associated with heterochromatic regions marked with repressive histone marks. collectively, our data suggest that dynamic and mutually exclusive binding of h and hmgd to nucleosomes and their linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. we find that the ratio of hmgd to h binding is a better predictor of gene activity than either protein by itself, which suggests that reciprocal binding between these proteins is important for gene regulation. using chip-seq, biochemical and gene specific approaches, we find that hmgd binds to highly accessible regulatory chromatin and active promoters. using knockdown experiments, we show that hmgd and h affect the occupancy of the other protein, change nucleosome repeat length and modulate gene expression. chromatin structuretranscriptional regulationhistone h1high mobility group proteinnucleosome repeat length while these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins.
 chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. while these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins.  here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone h and the high mobility group protein hmgd in drosophila s cells. using chip-seq, biochemical and gene specific approaches, we find that hmgd binds to highly accessible regulatory chromatin and active promoters. in contrast, h is primarily associated with heterochromatic regions marked with repressive histone marks. we find that the ratio of hmgd to h binding is a better predictor of gene activity than either protein by itself, which suggests that reciprocal binding between these proteins is important for gene regulation. using knockdown experiments, we show that hmgd and h affect the occupancy of the other protein, change nucleosome repeat length and modulate gene expression.  collectively, our data suggest that dynamic and mutually exclusive binding of h and hmgd to nucleosomes and their linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. this study provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation. chromatin structuretranscriptional regulationhistone h1high mobility group proteinnucleosome repeat length 