actual values of Up/Lo in HepG2 and HeLa cells were larger than those in KGN cells; for example, the Up/Lo of the TUBB gene in HepG2 and HeLa cells was around +1.6, while KGN cells showed +0.5. To determine the reason for the difference, we also analyzed the enrichment of each reference locus in AZ-3146 individual fractions. The active reference loci 7 / 20 Chromatin Structures for Activity of the CYP19 Promoters Fig 2. The principle of the SEVENS assay. Neighboring nucleosomes are crosslinked to each other by formaldehyde. Because this crosslinking reagent has a short arm of a single carbon, it preferentially reacts to condensed nucleosomes in closed chromatin. Thus, following solubilization with a detergent and mild sonication, condensed nucleosomes that are sufficiently crosslinked form a large particle, while nucleosomes in open chromatin escape from the crosslinking reaction to be a small particle. When such a preparation is subjected to sedimentation velocity centrifugation with a sucrose density gradient, chromatin is fractionated with regard to the size of sheared particles, namely to the crowdedness of nucleosomes. Chromatin structure comprises nucleosomes in equilibrium between a condensed and a dispersed state. Because a preparation for the assay contains multiple fragments of crosslinked chromatin obtained from more than a million cells, data analyzed for a locus-of-interest are represented as the proportion of chromatin graded between open and closed chromatin. Thus, the fractional distribution of a locus in closed chromatin gradually increases toward PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698015 lower fractions. Conversely, a locus in open chromatin increases toward upper fractions. doi:10.1371/journal.pone.0128282.g002 in HepG2 cells were greatly enriched in the two upper fractions and excluded from the three lower fractions; although the repressed reference loci were excluded from the uppermost fraction, the concentration in the other fractions was close to 
the average. This tendency was also seen in HeLa cells. KGN cells, however, showed a distinct pattern; the repressed reference loci were apparently excluded from the two upper fractions and enriched in the four lower fractions, but the enrichment of the active reference loci in the upper fractions was not clear, although a decrease in the lower fractions was observed. These observations suggest that the efficiency of chromatin crosslinking differed among the cell lines even when the same conditions were used; chromatin in HepG2 and HeLa cells was unlikely to be crosslinked enough to sediment the repressed reference loci to lower fractions; in contrast, the poor upper enrichment of the active reference loci in KGN cells could result from undesirable excess crosslinking. However, because the chromatin structure of the active and the repressed reference loci within each cell line was distinguished in the SEVENS assay, these reference loci were useful as internal controls to assess chromatin structure in the CYP19 locus. PCR performed with primer PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697345 sets that anneal 8 / 20 Chromatin Structures for Activity of the CYP19 Promoters Fig 3. Chromatin structure observed in the SEVENS assay. The SEVENS assays revealed distinct chromatin structures in the CYP19 locus of HepG2, KGN, and HeLa cells. The proportion of open and closed chromatin is represented as an enrichment ratio of a given sequence in upper fractions to that in lower fractions using a log2 scale. The gene structure of the CYP19 locus is drawn above each chart. Each vert
