Chan S, Attisano L, Lewis P N
Department of Biochemistry, University of Toronto, Canada.
J Biol Chem. 1988 Oct 25;263(30):15643-51.
Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-[3H]lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size. The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting. The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury. This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction. In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled. Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively. Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion. High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled. Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet. Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I. For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes. The enzyme may be localized in the globin domain in erythrocytes and maintains that region in a hyperacetylated state which results in an altered linker histone binding reflected in a change in the reactivity of the usually inaccessible H3 cysteine 110.
先前分别与两种新型汞化合物（N - 氯汞苯甲酰基） - 生物素和双（对 - （氯汞苯甲酰基）） - [³H]赖氨酸二酰胺一起孵育的鸡红细胞核，用微球菌核酸酶消化，消化产物根据其在0.15 M NaCl中的溶解度和分子大小进行分级分离。通过蛋白质印迹法、放射自显影和闪烁计数来确定染色质组分和含有共价结合汞的蛋白质的身份和定量。在0.15 M NaCl可溶的多核小体组分中，组蛋白H3的乙酰化程度最高的物种也含有最高比例的结合汞。该组分含有高度乙酰化的核心组蛋白，缺乏连接组蛋白，并且富含非组蛋白。在0.15 M NaCl可溶的单核小体中，组蛋白H3未乙酰化且缺乏连接组蛋白，其标记程度比0.15 M NaCl可溶的多核小体组分中的组蛋白H3低45%。在0.15 M NaCl不溶的多核小体中，含有未乙酰化的组蛋白和摩尔比例的连接组蛋白，组蛋白H3的标记程度低63%。考虑到这些亚组分在细胞核中的丰度差异，对于0.15 M NaCl可溶的多核小体、单核小体和0.15 M NaCl不溶的多核小体，相对H3反应性分别为50、7和1。因此，存在与超乙酰化增加和连接组蛋白缺失相关的反应性梯度。在0.15 M NaCl可溶组分的亚核小体颗粒中发现的高迁移率族蛋白1和2被大量汞标记。盐溶性和大小分辨的微球菌核酸酶产生的组分之间，组蛋白乙酰转移酶活性在0.15 M NaCl可溶的上清液中的分布几乎比0.15 M NaCl不溶的沉淀中的高5倍。此外，紧密结合于未消化染色质的乙酰转移酶活性可被微球菌核酸酶和DNase I迅速释放。对于短消化时间，该酶与盐溶性多核小体相关，但在较长消化时间时，该酶似乎与完整的核小体分离。该酶可能定位于红细胞中的珠蛋白结构域，并使该区域保持在超乙酰化状态，这导致连接组蛋白结合改变，反映在通常难以接近的H3半胱氨酸110的反应性变化上。
