Parlow M H, Haas A L, Lough J
Department of Anatomy, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1990 May 5;265(13):7507-12.
We recently demonstrated that ubiquitinated histone H2A (uH2A) declines 2.5-fold during terminal skeletal muscle differentiation, coincident with reductions in transcriptional activity (Wunsch, A. M., Haas, A. L., and Lough, J. (1987) Dev. Biol. 119, 85-93). To assess whether this indicates an association of uH2A with transcriptionally active genes, we have used micrococcal nuclease digestion and salt extraction to fractionate myotube nuclei. An oligonucleosomal fraction obtained by micrococcal nuclease digestion and extraction in low salt (100 mM NaCl) comprising only 25% of the nuclear DNA contained 90% of the total uH2A, as revealed by Western blotting. Further fractionation of this 100 mM salt extract by sucrose gradient centrifugation revealed that virtually all of the uH2A was localized in monomer to heptamer-sized oligonucleosomes. A second ubiquitinated species of 57-kDa (u57) was also localized in the 100 mM salt extract. In contrast, an 18-kD band (u18) was associated with fractions that were resistant to micrococcal nuclease digestion and salt extraction. Although micrococcal nuclease recognized a unique structural feature of active myotube chromatin, as evidenced by the appearance of hybridized skeletal alpha-actin sequences as a smear rather than the nucleosomal ladder exhibited by inactive and bulk sequences, neither the skeletal alpha-actin gene nor the inactive alpha D-globin gene was exclusively localized in the 100 mM salt fraction. Moreover, further fractionation of the 100 mM salt extract on a sucrose gradient failed to separate active from inactive genes. These findings suggest that uH2A is localized in a fraction of myotube chromatin which, although nuclease-sensitive and relatively soluble, is not enriched in active or inactive genes.
我们最近证明，在终末骨骼肌分化过程中，泛素化组蛋白H2A（uH2A）下降了2.5倍，同时转录活性也降低（Wunsch, A. M., Haas, A. L., and Lough, J. (1987) Dev. Biol. 119, 85 - 93）。为了评估这是否表明uH2A与转录活性基因相关联，我们使用微球菌核酸酶消化和盐提取法对肌管细胞核进行分级分离。通过微球菌核酸酶消化并在低盐（100 mM NaCl）条件下提取得到的寡核小体组分，仅占核DNA的25%，但Western印迹显示其包含了90%的总uH2A。对该100 mM盐提取物进行蔗糖梯度离心进一步分级分离，结果表明几乎所有的uH2A都定位于单体至七聚体大小的寡核小体中。另一种57 kDa的泛素化蛋白（u57）也定位于100 mM盐提取物中。相比之下，一条18-kD条带（u18）与对微球菌核酸酶消化和盐提取具有抗性的组分相关联。尽管微球菌核酸酶识别出活跃肌管染色质的独特结构特征，这表现为杂交的骨骼肌α-肌动蛋白序列呈现为涂抹状而非非活跃和大量序列所呈现的核小体阶梯状，但骨骼肌α-肌动蛋白基因和非活跃的α D-珠蛋白基因都没有专门定位于100 mM盐组分中。此外，对100 mM盐提取物在蔗糖梯度上进行进一步分级分离，未能将活跃基因与非活跃基因分开。这些发现表明，uH2A定位于肌管染色质的一部分，该部分虽然对核酸酶敏感且相对可溶，但在活跃或非活跃基因中并未富集。
