ABSTRACT The chromatin observed by conventional electron microscopy under the nuclear envelope constitutes a single layer of dense 30–35 nm granules, while ∼30 nm fibrils laterally attached to them, form large patches of lamin-associated domains (LADs). This particular surface “epichromatin” can be discerned by specific (H2A+H2B+DNA) conformational antibody at the inner nuclear envelope and around mitotic chromosomes. In order to differentiate the DNA conformation of the peripheral chromatin we applied an Acridine orange (AO) DNA structural test involving RNAse treatment and the addition of AO after acid pre-treatment. MCF-7 cells treated in this way revealed yellow/red patches of LADs attached to a thin green nuclear rim and with mitotic chromosomes outlined in green, topologically corresponding to epichromatin epitope staining by immunofluorescence. Differentially from LADs, the epichromatin was unable to provide metachromatic staining by AO, unless thermally denatured at 94oC. DNA enrichment in GC stretches has been recently reported for immunoprecipitated ∼ 1Kb epichromatin domains. Together these data suggest that certain epichromatin segments assume the relatively hydrophobic DNA A-conformation at the nuclear envelope and surface of mitotic chromosomes, preventing AO side dimerisation. We hypothesize that epichromatin domains form nucleosome superbeads. Hydrophobic interactions stack these superbeads and align them at the nuclear envelope, while repulsing the hydrophilic LADs. The hydrophobicity of epichromatin explains its location at the surface of mitotic chromosomes and its function in mediating chromosome attachment to the restituting nuclear envelope during telophase.

中文翻译：

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用吖啶橙对外周核染色质进行差异染色意味着 DNA 的 A 型表染色质构象

摘要通过常规电子显微镜观察到的核包膜下的染色质构成单层致密的 30-35 nm 颗粒，而 30 nm 的纤维横向附着在它们上面，形成大片的核纤层蛋白相关域 (LAD)。这种特殊的表面“表染色质”可以通过内部核膜和有丝分裂染色体周围的特定 (H2A+H2B+DNA) 构象抗体来辨别。为了区分外周染色质的 DNA 构象，我们应用了吖啶橙 (AO) DNA 结构测试，包括 RNA 酶处理和酸预处理后添加 AO。以这种方式处理的 MCF-7 细胞显示出黄色/红色的 LAD 斑块附着在薄的绿色核边缘上，有丝分裂的染色体以绿色勾勒出来，拓扑对应于免疫荧光染色的表染色质表位。与 LAD 不同的是，表染色质无法通过 AO 提供异染染色，除非在 94oC 下热变性。最近报道了免疫沉淀的 ∼ 1Kb 表染色质结构域在 GC 序列中的 DNA 富集。这些数据一起表明，某些表染色质片段在核膜和有丝分裂染色体表面呈现相对疏水的 DNA A 构象，防止 AO 侧二聚化。我们假设表染色质域形成核小体超级珠。疏水相互作用将这些超级珠子堆叠起来，并将它们排列在核膜上，同时排斥亲水性 LAD。