This conclusion is consistent with previous studies using the Caco-2 cell culture model

with a relatively high Up/Lo ratio were evident at Kg-EUS-1 to -7. Because the Up/Lo ratio at Kg-EUS-7 reaches the red belt in the figure, chromatin structure at this region appeared to be mostly open like the active reference loci. In fact, the panel of Kg-EUS-7 in Fig 4B shows a similar fractional distribution to that of the active reference loci, in which Kg-EUS-7 was excluded from the two lower fractions although the upper enrichment was not clear. The Up/Lo ratios at the other EUS peaks fell between the blue and the red belts, suggesting that these regions had an intermediate proportion of open and closed chromatin. Interestingly, one peak, Kg-EUS-6, coincided with the position of the Ic promoter, which is the only active promoter in KGN cells. This intermediate proportion seemed to be required for the active state of the Ic promoter even though its activity was quite weak. Notably, the regions of the Ia and the Ib promoters fell into the blue belt in Fig 3B, indicating that they were both mostly occupied by closed chromatin like the repressed reference loci. Thus, the absence of open chromatin could be responsible for the repressed state of these promoters. Finally, chromatin structure in HeLa cells was also analyzed in the SEVENS assay. A single region with a high Up/Lo ratio was observed and labeled as HlEUS-1. When the fractional distribution of this region was analyzed, a AMI-1 site moderate enrichment in upper fractions accompanied by a moderate exclusion from lower fractions was observed. This observation indicates that the proportion of open chromatin at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698015 Hl-EUS-1 was relatively large, but less than that of the active reference loci. Importantly, the Up/Lo ratio in the upstream three-quarters of the CYP19 locus in HeLa cells fell almost entirely in the blue belt. A high proportion of closed chromatin, which was also interpreted as a low proportion of open chromatin, appeared to contribute to the repression of the CYP19 promoters in HeLa cells. Relationship between nucleosome occupancy and chromatin structure observed in the SEVENS assays Nucleosomes are omitted from the promoter region of a highly expressed gene; such promoter regions are known as nucleosome-depleted regions . To assess whether the EUS regions indicated by the SEVENS assay were correlated to a low occupancy of nucleosomes, we performed ChIP assays with a pan-antibody that recognizes modified and unmodified histone H3. We first examined nucleosomes at the CYP19 locus in HepG2 cells. Three regions that corresponded to Hg-EUS-1, -3, and -4 were observed with relatively lower nucleosome occupancy. Because the occupancy at these regions was still above the background level seen at the NDR in the TUBB promoter, in a subpopulation of the cells, nucleosomes 11 / 20 Chromatin Structures for Activity of the CYP19 Promoters Fig 5. The nucleosome occupancy in the CYP19 locus. ChIP assays with anti-pan-histone H3 revealed the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698988 amount of nucleosomes in the CYP19 locus in HepG2, KGN, or HeLa cells. The nucleosome level is expressed as a percentage of total input chromatin. The solid line in the respective charts represents the average value of IP/Input. The level at the OR1A1 or the TUBB locus as a reference is also plotted at the right of each chart. To compare a given region in the CYP19 locus to the references, broken lines are drawn from the reference marks. The gene structure of CYP19 and the EUS regions are also represented on each chart. Since Hg-EUS-1 and -4 coincided with the