Istomina Natalia E, Shushanov Sain S, Springhetti Evelyn M, Karpov Vadim L, Krasheninnikov Igor A, Stevens Kimberly, Zaret Kenneth S, Singh Prim B, Grigoryev Sergei A
Department of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Mol Cell Biol. 2003 Sep;23(18):6455-68. doi: 10.1128/MCB.23.18.6455-6468.2003.
Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.
在终末分化细胞中，活跃基因与发育调控的染色质浓缩相互隔离。我们绘制了终末阶段特异性染色质浓缩蛋白MENT在活跃的鸡β-珠蛋白结构域上的分布图谱。我们观察到MENT浓度的两个急剧转变，这与β-珠蛋白边界元件相吻合。MENT的分布模式与乙酰化核心组蛋白的分布模式相反，但与赖氨酸9二甲基化的组蛋白H3（H3me2K9）的分布模式相关。在NIH 3T3细胞中异位表达MENT会导致以H3me2K9为特征的大规模特异性染色质重塑。MENT在鸡红细胞和NIH 3T3细胞中均与H3me2K9共定位。MENT的突变分析以及与去乙酰化酶抑制剂的实验揭示了反应中心环结构域的重要作用以及组蛋白高乙酰化对体内MENT诱导的染色质重塑的抑制作用。在体外，用胰蛋白酶去除含有赖氨酸9的组蛋白H3 N端肽可阻断MENT介导的染色质自缔合，而用组蛋白H3的二甲基化而非乙酰化N端结构域进行重组可特异性恢复MENT介导的染色质自缔合。我们认为，赖氨酸9处的组蛋白H3修饰通过将MENT招募到染色质上，以一种在空间上受基因边界元件和组蛋白高乙酰化限制而远离活跃基因的方式，直接调节染色质浓缩。
