Kerrigan L A, Kadonaga J T
Department of Biology, University of California, San Diego, La Jolla 92093.
Nucleic Acids Res. 1992 Dec 25;20(24):6673-80. doi: 10.1093/nar/20.24.6673.
By using a DNase I footprinting assay, we have purified a factor by DNA affinity chromatography that binds to the minimal enhancer region of the Drosophila knirps gene and subsequently identified the protein as the core histone H2B. This inadvertent purification of a core histone as a putative sequence-specific DNA binding protein was due to a previously unknown property of H2B to interact with DNA in a periodic manner. Moreover, we found that each of the individual core histones, but not histone H1 or high mobility group protein 1, bound to the knirps enhancer to give a repetitive DNase I footprint pattern with a periodicity of about 10 base pairs, which is approximately one turn of the DNA helix. In addition, preparations containing the core histones H2A-H2B or H3-H4 yielded identical periodic DNase I footprint patterns on several different promoter and enhancer regions. These findings suggest that there are periodic, homotypic interactions between DNA-bound core histones that result from an alteration of the overall DNA structure such as the curvature rather than a specific sequence. We have also shown that histones H2A-H2B can repress initiation of transcription by RNA polymerase II. The phenomena described here may reflect histone-DNA interactions in non-nucleosomal stretches of chromatin and could be involved in some aspects of either rotational or translational positioning of nucleosomes. Furthermore, these findings indicate that a repeated 10 bp DNase I ladder, which has previously been considered to be a property of an intact nucleosome, can also be generated with subnucleosomal components. It will thus be necessary to reevaluate the criteria applied to the analysis of nucleosomes both in vivo and in vitro.
通过使用DNA酶I足迹分析，我们通过DNA亲和色谱法纯化了一种与果蝇克尼普斯基因的最小增强子区域结合的因子，随后鉴定该蛋白质为核心组蛋白H2B。将核心组蛋白意外纯化为假定的序列特异性DNA结合蛋白，是由于H2B以前未知的以周期性方式与DNA相互作用的特性。此外，我们发现每个单独的核心组蛋白，而不是组蛋白H1或高迁移率族蛋白1，都与克尼普斯增强子结合，产生具有约10个碱基对周期性的重复DNA酶I足迹模式，这大约是DNA螺旋的一圈。此外，含有核心组蛋白H2A - H2B或H3 - H4的制剂在几个不同的启动子和增强子区域产生相同的周期性DNA酶I足迹模式。这些发现表明，DNA结合的核心组蛋白之间存在周期性的同型相互作用，这是由整体DNA结构的改变（如曲率）而非特定序列引起的。我们还表明，组蛋白H2A - H2B可以抑制RNA聚合酶II的转录起始。这里描述的现象可能反映了染色质非核小体区域中的组蛋白 - DNA相互作用，并且可能参与核小体的旋转或平移定位的某些方面。此外，这些发现表明，先前被认为是完整核小体特性的重复10 bp DNA酶I梯带，也可以由亚核小体成分产生。因此，有必要重新评估体内和体外分析核小体所应用的标准。
