Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene.

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.

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