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.
Togashi
Tadayuki
T
Department of Molecular Genetics, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Niigata 951-8510, Japan.
Obata
Miki
M
Aoyagi
Yutaka
Y
Kominami
Ryo
R
Mishima
Yukio
Y
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division; D045744:Cell Line, Tumor; D002498:Centrifugation / Q000379:methods; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D006657:Histones / Q000737:chemistry*; D008099:Liver / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D051379:Mice; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D013950:Thymus Gland / Q000378:metabolism; D014018:Tissue Distribution; D018631:ras Proteins / Q000235:genetics*
D000906:Antibodies; D002843:Chromatin; D006657:Histones; D004247:DNA; C472865:Rit1 protein, mouse; D018631:ras Proteins; D008239:Lysine