ical line extends from a promoter region to note a corresponding position in the respective lower charts. Red circles and blue rectangles denote the Up/Lo values of active and repressed MRT-67307 site reference loci, respectively, which are abbreviated as TU, AC, GA, OR, MY, or IL for the TUBB, ACTB, GAPDH, OR1A1, MYT1, or IL2RA loci, respectively. To compare assessed regions of the CYP19 locus to these references, red and blue belts are placed on the charts to indicate the ranges of the Up/Lo values of the references. The positions referred to in the text are labeled as “enrichment in upper fractions of the SEVENS assay ” in the charts. doi:10.1371/journal.pone.0128282.g003 9 / 20 Chromatin Structures for Activity of the CYP19 Promoters Fig 4. The enrichment of the 
reference loci and the EUS regions of the CYP19 locus in individual SEVENS fractions. The fractional distribution of the active reference loci, the repressed reference loci, and the EUS regions in HepG2, KGN, or HeLa cells was represented as the fold enrichment in each fraction relative to an average calculated from total fractions by using a log2 scale. doi:10.1371/journal.pone.0128282.g004 every 5 kb in the CYP19 locus were used to further characterize the landscape of chromatin structure at the locus. We first assessed the CYP19 locus in HepG2 cells by using the SEVENS assay. The distribution of Up/Lo ratios throughout the CYP19 locus mostly fell on the upper edge of the blue belt representing the range of the ratios of the repressed reference loci. This finding indicated that a major portion of the locus in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698015 HepG2 cells was moderately occupied by closed chromatin like the repressed reference loci. The assay also identified five regions relatively enriched in the upper fractions, which were designated as EUS . The EUS peaks in the figure did not reach the red belt defined by the ratios of the active reference loci, but were above the blue belt. To further elucidate chromatin structure at these EUS regions, the distribution of the regions PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19699128 in individual fractions was analyzed. Hg-EUS-1, which was shown as the highest peak among the five in Fig 3A, was enriched in the two upper fractions, and excluded from the four lower fractions. However, the magnitude of the biased distribution of Hg-EUS-1 was 10 / 20 Chromatin Structures for Activity of the CYP19 Promoters less than that of the active reference loci. This appeared to be the reason why the peak of HgEUS-1 did not reach the red belt in Fig 3A. Likewise, the upper enrichment of Hg-EUS-2 to -5 was reduced compared to the active reference loci, but still higher than that of the repressed reference loci. Such fractional distributions indicate an intermediate proportion of open chromatin between the active and the repressed reference loci. Importantly, Hg-EUS-1 and -4 coincided with the position of the Ib and Ic promoters, respectively. Since the proportion of open chromatin at Hg-EUS-1 was more than at Hg-EUS-4, this difference was likely to be reflected in the higher activity of the Ib promoter than the Ic promoter. In addition, the inactive Ia promoter was plotted within the blue belt, suggesting a large proportion of closed chromatin and a small proportion of open chromatin, like the repressed reference loci. Therefore, the strength of the CYP19 promoters in HepG2 cells was well reflected in the chromatin proportion as determined via the SEVENS assay. When chromatin at the CYP19 locus in KGN cells was examined in the SEVENS assay, regions