Byrd Keith, Corces Victor G
Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
J Cell Biol. 2003 Aug 18;162(4):565-74. doi: 10.1083/jcb.200305013.
Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.
绝缘子可能通过建立和维持细胞核内染色质纤维的组织来调节基因表达。对裂解细胞进行生化分级分离和原位高盐提取表明，吉普赛绝缘子的两种已知蛋白质成分存在于核基质中。使用位于两个内源性Su(Hw)结合位点之间的DNA探针进行荧光原位杂交，我们发现中间的DNA呈环状排列，两个绝缘子位于环的基部。绝缘子蛋白的突变、使细胞经受短暂热休克或破坏核基质会导致环的破坏。在环的中心插入额外的吉普赛绝缘子会通过插入序列与核基质的附着导致成对环的形成。这些结果表明，吉普赛绝缘子可能通过将DNA拴系到核基质并形成染色质环来建立染色质结构的高阶结构域并调节核组织。
