the data on the expression levels of human genes were taken from the gene expression atlas ( 35 ) .
the uniform platform for all tissues was affymetrix u95a . only probes that presented the characterized genes ( i.e. those with links to the refseq database ) were used , with signals from probes on the chip corresponding to the same gene being averaged ( with total of 7708 genes ) .
only data for normal tissues were used , samples and replicates representing the same tissue were averaged ( with total of 32 tissues ) . as was recommended ,
a gene was regarded as expressed if its signal level exceeded a conservative threshold of 200 arbitrary units ( 35 ) .
it should also be noted that there is a strong correlation ( spearman r = 0.89 ,
p < 10 ) between the expression breadth ( number of tissues where a given gene is expressed ) and the expression level averaged over all tissues studied ( 34 ) .
the intronic sequences were found for 6874 genes , and the intergenic sequences ( both upstream and downstream ones ) , for 5104 genes . in the case of alternative splicing variants , the longest coding sequence was taken .
the average length and gc content of upstream and downstream intergenic sequences were taken as the corresponding values of the intergenic spacer for a given gene .
the nucleosome formation potential of nucleotide sequences was determined using the method by levitsky and co - workers ( 3739 ) , constructed and verified on the basis of large experimental datasets of nucleosome positioning sequences ( 4042 ) .
the nucleosome formation potential was averaged over each sequence length ; for introns , exons and intergenic spacers it was also weight - averaged for each gene .
the nucleosome formation potential shows a negative correlation with gc content ( figure 1 ) , which is in accordance with the expectations from the physical properties of dna helix associated with gc content ( 16 ) .
it was previously reported that gc content of both intronic and intergenic sequences should be included as covariate into multifactor statistical analyses for the correlation between the  gene nest proportion and expression breadth to disappear completely ( 34 ) .
this fact was interpreted in the sense that either both intronic and intergenic gc content taken together better reflects the isochore affiliation of a given gene ( i.e. the regional effect ) or that the local variation of gc content among intronic and intergenic sequences is also an important property of the  gene nest. the negative correlation between nucleosome formation potential and gc content ( figure 1 ) suggests that the combined impact of both intergenic and intronic gc content on the expression breadth is a local effect related to nucleosome formation .
introns of broadly expressed genes show a lower nucleosome formation potential compared with introns of tissue - specific genes ( figure 2 ) .
intergenic spacers show a similar picture ( not shown in figure 2 because they mostly overlap with introns ) .
exons show a similar trend with introns ' ( and intergenic spacers ' ) reduction of nucleosome formation potential with increasing gene expression breadth
. however , their mean potential is lower than potential of introns and intergenic spacers for all gene expression groups ( figure 2 ) . in the intergenic spacers , the nucleosome formation potential drops similarly to introns with the increase of gc content , whereas in exons it drops significantly faster ( confer slopes in figure 1 ) .
it is noteworthy that in the gc - poor sequences , exons have a similar or even a higher nucleosome formation potential compared with noncoding dna , but the picture is reversing with the elevation of gc content ( figure 1 ) . in genes located in the heavy isochores [ i.e. with gc content above 50% ( 1 ) ] , exons have a higher gc content than introns by 5% ( absolute percentage ) ( 16 ) .
therefore , judging by the data shown in figure 1 , introns seem to be the main source of nucleosome formation potential in the genes of heavy isochores . a greater scatter of exon points compared with noncoding sequences ( figure 1 ) can be explained by the shorter length of exons ( and thus , higher statistical noise ) and/or by exons ' informational load that may interfere with requirements of nucleosome formation .
the nucleosome formation potential correlates positively with intronic and intergenic sequence lengths ( for log - transformed lengths : for introns , r = 0.39 , p < 10 ; for intergenic spacers , r = 0.43 , p < 10 ) .
( for exons , the partial correlation between nucleosome formation potential and sequence length independently of gc content was not significant . ) in turn , the negative correlation between nucleosome formation potential and gc content is partially independent of sequence length ( figure 3b ) .
these facts indicate that both the gc content and the length of introns and intergenic spacers are related to chromatin condensation in the human genome .
it was previously reported that nucleosome formation potential is higher in introns compared with exons ; however , no statistical analysis was made , and the authors ' histograms of nucleosome formation potential for introns and exons mostly overlapped [ figure 1a in ( 38 ) ] .
this overlap is probably due to a large number of gc - poor introns and exons in the authors ' dataset .
it was also found ( on a limited dataset of 200 genes ) that nucleosome formation potential is higher in the promoter regions of tissue - specific genes compared with the promoters of housekeeping genes ( 37 ) .
the data presented here show that the same is true not only for the promoters but also for the bulk of noncoding dna , which suggests its involvement in chromatin - mediated suppression of tissue - specific genes .
it is now well recognized that epigenetic mechanisms , operating on the large domains rather than on individual promoters , are used in maintaining and stably transmitting chromatin states through the cell cycle ( 43,44 ) .
a high local concentration of nucleosomes is necessary for the higher - order chromatin condensation , which is a distinct level of transcriptional regulation ( 44,45 ) .
the present data also help explain why gc content rises near gene transcription start site [ figure 1a in ( 46 ) ] .
the elevation of gc content is associated with the reduction of nucleosome formation potential that should facilitate the binding of transcription factors ( after the higher levels of chromatin condensation are unfolded ) .
it was long argued that noncoding dna might be necessary for correct chromatin structure because exons are under strong selection pressure for informational content ( 38,4749 ) .
it was even shown in several cases that after experimentally removing the introns , genes lose the ability to form nucleosomes ( 50,51 ) .
the present data suggest that both the length and the gc content of noncoding dna ( and thus the isochoric structure ) in the human genome can be relevant to chromatin - mediated suppression of tissue - specific genes .
it should be noted that the existence of the isochores in the genomes of warm - blooded vertebrates , which was initially discovered using the cscl density gradient ultracentrifugation , was questioned after the completion of the human genome ( 52 ) .
however , the statistical test for isochoric structure used in ( 52 ) was criticized , and the later works confirmed the existence of isochores ( 5355 ) . while too complicated tests may be prone to unexpected errors and interpretation problems , even a simple analysis of intron  exon contrasts demonstrated that notwithstanding a high global compositional heterogeneity , genomes of warm - blooded vertebrates show an unusual local homogeneity : the mean absolute intron  exon difference in gc content is more than 2-fold lower in them than in the genomes of other organisms [ figure 1a in ( 56 ) ] .
the greater length of mammal introns compared with introns of other studied organisms makes this small intron - exon contrast even more unusual .
buffering role , damping the effect of solvent fluctuations on the nuclear machinery ( 57 ) .
recently , it was shown in comparison of two closely related amphibian species differing in genome size that chromatin condensation was steadier and its reaction to changes in solvent composition ( caused by elevated extracellular salinity ) was more inertial in the species with the larger genome , which is in agreement with the 
the ability of dna to act as a  buffer to control the concentration ( more exactly , the activity ) of dna - binding proteins was even used for the development of experimental methods for the investigation of histone  dna interactions [ reviewed in ( 59 ) ] .
it is also possible that there can be local buffering in regard to a given locus .
a large amount of noncoding dna within and around a tissue - specific gene may furnish high local histone concentration and thus secure suppression of transcriptional noise .
[ transcriptional background noise is believed to be a great threat to cellular function as the number of genes increases during evolution ( 60,61 ) . ]
the role of dna in providing high local concentration of sequence - specific dna - tropic proteins is discussed in ( 62 ) .
( a ) exons ( intercept = 4.6  0.1 , slope = 0.08  0.00 , r = 0.75 , p < 10 ) .
( b ) introns ( intercept = 2.3  0.0 , slope = 0.02  0.00 , r = 0.78 , p < 10 ) .
( c ) intergenic spacers ( intercept = 2.4  0.0 , slope = 0.03  0.00 , r = 0.73 , p < 10 ) .
dashed lines , confidence limits ; dotted lines , prediction limits , both for p = 0.95 ; and a.u .
the nucleosome formation potential in human exons ( squares ) and introns ( circles ) of genes expressed in a different number of tissues ( anova and kruskal  wallis , in both cases p < 10 ) .
the data for intergenic spacers are not shown because they mostly overlap with introns ; a.u .
( a ) regression on ( log - transformed ) sequence length at fixed gc - content ( partial r = 0.23 , p < 10 ; for introns , the picture was similar , partial r = 0.14 , p < 10 ) .
( b ) regression on gc - content at fixed sequence length ( r = 0.68 , p < 10 ; for introns , partial r = 0.74 , p < 10 ) . dashed lines , confidence limits ; dotted lines , prediction limits , both for p = 0.95 ; and nfp , nucleosome formation potential .
 the nucleosome formation potential of introns , intergenic spacers and exons of human genes is shown here to negatively correlate with among - tissues breadth of gene expression . 
 the nucleosome formation potential is also found to negatively correlate with the gc content of genomic sequences ; the slope of regression line is steeper in exons compared with noncoding dna ( introns and intergenic spacers ) . 
 the correlation with gc content is independent of sequence length ; in turn , the nucleosome formation potential of introns and intergenic spacers positively ( albeit weakly ) correlates with sequence length independently of gc content . 
 these findings help explain the functional significance of the isochores ( regions differing in gc content ) in the human genome as a result of optimization of genomic structure for epigenetic complexity and support the notion that noncoding dna is important for orderly chromatin condensation and chromatin - mediated suppression of tissue - specific genes . 
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Figures and Tables