Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy.
Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy.
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DOI：
10.1083/jcb.131.6.1365
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发表时间：
1995-12
期刊：
影响因子：
--
通讯作者：
Woodcock CL
中科院分区：
文献类型：
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作者：
Bednar J;Horowitz RA;Dubochet J;Woodcock CL
用低温电子显微镜观察了鸡红细胞核在不同离子强度溶液中玻璃化后的三维构象。从倾斜的显微照片对，可以获得核小体圆盘的三维位置和取向，以及暴露的连接子片段的路径。在“低盐”条件下(5 mM氯化钠，1 mM EDTA，pH 7.5)，平均三核小体呈等边三角形，顶端有核小体，连续核小体之间暴露的接头DNA长度约相当于46bp。两个连接子DNA片段在中央核小体汇合。去除组蛋白H1和H5会导致更不同的三核小体形态，并且这两个连接DNA片段通常在不同的位置连接中央核小体。三核小体在20 mM氯化钠、1 mM EDTA(产生最大沉降增加的盐浓度)中玻璃化，表明压实是通过减少中央核小体上连接DNA片段的夹角而发生的，而不涉及连续核小体之间距离的减少。通常，连接子进出部位的形态也会发生变化。在40 mM的氯化钠中，三核小体的形态没有进一步的变化，但多核小体明显更致密。然而，在低盐下观察到的多核小体中的三维之字形构象在40 mM的氯化钠中保持不变，单个核小体的圆盘仍然彼此分离。没有证据表明多核小体内形成螺线管状排列。对单个寡核小体的溶液构象与大量染色质样品的物理测量数据的比较表明，后者应该被重新解释。新的数据支持在一定离子强度范围内溶液中染色质的不规则之字形构象的概念，这与其他原位研究人员(McDowall，A.W.，J.M.Smith和J.Dubochet)的观点一致。1986年，EMBO(欧元摩尔。比奥尔。器官。)J.5：1395-1402；Horowitz，R.A.，D.A.Agard，J.W.Sedat和C.L.Woodcock，1994。J.细胞生物学。125：1-10)，以及体外结论(van Holde，K.和J.Zlatanova)。1995年。J.Biol.化学。270：8373-8376)。低温电子显微镜还提供了一种确定自然产生的染色质的三维构象的方法，其中精确的核小体定位在转录调控中发挥着作用。
Cryoelectron microscopy has been used to examine the three-dimensional (3-D) conformation of small oligonucleosomes from chicken erythrocyte nuclei after vitrification in solutions of differing ionic strength. From tilt pairs of micrographs, the 3-D location and orientation of the nucleosomal disks, and the paths of segments of exposed linker can be obtained. In "low-salt" conditions (5 mM NaCl, 1 mM EDTA, pH 7.5), the average trinucleosome assumes the shape of an equilateral triangle, with nucleosomes at the vertices, and a length of exposed linker DNA between consecutive nucleosomes equivalent to approximately 46 bp. The two linker DNA segments converge at the central nucleosome. Removal of histones H1 and H5 results in a much more variable trinucleosome morphology, and the two linker DNA segments usually join the central nucleosome at different locations. Trinucleosomes vitrified in 20 mM NaCl, 1 mM EDTA, (the salt concentration producing the maximal increase in sedimentation), reveal that compaction occurs by a reduction in the included angle made by the linker DNA segments at the central nucleosome, and does not involve a reduction in the distance between consecutive nucleosomes. Frequently, there is also a change in morphology at the linker entry-exit site. At 40 mM NaCl, there is no further change in trinucleosome morphology, but polynucleosomes are appreciably more compact. Nevertheless, the 3-D zig-zag conformation observed in polynucleosomes at low salt is retained at 40 mM NaCl, and individual nucleosome disks remain separated from each other. There is no evidence for the formation of solenoidal arrangements within polynucleosomes. Comparison of the solution conformation of individual oligonucleosomes with data from physical measurements on bulk chromatin samples suggests that the latter should be reinterpreted. The new data support the concept of an irregular zig-zag chromatin conformation in solution over a range of ionic strengths, in agreement with other in situ (McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395-1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1-10), and in vitro conclusions (van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373-8376). Cryoelectron microscopy also provides a way to determine the 3-D conformation of naturally occurring chromatins in which precise nucleosome positioning plays a role in transcriptional regulation.