Characteristic low density and shear sensitivity of cross-linked chromatin containing polycomb complexes
Characteristic low density and shear sensitivity of cross-linked chromatin containing polycomb complexes
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DOI：
10.1128/mcb.25.1.432-439.2005
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发表时间：
2005-01-01
影响因子：
5.3
通讯作者：
Pirrotta, V
中科院分区：
文献类型：
--
作者：
Schwartz, YB;Kahn, TG;Pirrotta, V
染色质交联被广泛用于通过免疫沉淀来绘制染色体蛋白质的分布，但是我们对染色质复合物的物理性质的了解仍然是基本的。密度梯度长期以来一直用于将交联染色质片段与其结合的蛋白质从游离蛋白质或游离DNA中分离。我们发现，非常高分子量的蛋白质复合物的DNA片段的协会转移其浮力密度值低得多，然后散装染色质。我们发现，在氯化铯梯度，Polycomb响应元件，启动子的活性基因，绝缘体或边界元素被发现在浮力密度类似于那些游离蛋白质和耗尽的散装染色质组分。在这些区域中，低密度与大蛋白复合物的存在和对超声处理的高灵敏度相关。我们的研究结果表明，不同的染色质区域的分离，根据其浮力密度可能会偏向染色质免疫沉淀的结果。交联染色质的密度离心可以提供一种简单的方法来研究体内大染色质复合物的性质。
Chromatin cross-linking is widely used for mapping the distribution of chromosomal proteins by immunoprecipitation, but our knowledge of the physical properties of chromatin complexes remains rudimentary. Density gradients have been long used to separate fragments of cross-linked chromatin with their bound proteins from free protein or free DNA. We find that the association of DNA fragments with very-high-molecular-weight protein complexes shifts their buoyant density to values much lower then that of bulk chromatin. We show that in a CsCl gradient, Polycomb response elements, promoters of active genes, and insulator or boundary elements are found at buoyant densities similar to those of free protein and are depleted from the bulk chromatin fractions. In these regions, the low density is associated with the presence of large protein complexes and with high sensitivity to sonication. Our results suggest that separation of different chromatin regions according to their buoyant density may bias chromatin immunoprecipitation results. Density centrifugation of cross-linked chromatin may provide a simple approach to investigate the properties of large chromatin complexes in vivo.