Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus(MIC) and macr...Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus(MIC) and macronucleus(MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease(MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of^200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.展开更多
基金supported by the Natural Science Foundation of China(31522051,31470064)the funding awarded to Weibo Song(15-12-1-1-jch)+3 种基金the Qingdao National Laboratory for Marine Science and Technology,ChinaYifan Liu was supported by National Sanitation Foundation(MCB 1411565)National Institute of Health(R01 GM087343)the Department of Pathology at the University of Michigan
文摘Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus(MIC) and macronucleus(MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease(MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of^200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.
