Wunsch Ann, Jackson Vaughn
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Biochemistry. 2005 Dec 13;44(49):16351-64. doi: 10.1021/bi050876o.
A high level of the post-translational modification, acetylation, is found on the N-terminal regions of the core histones H2A, H2B, H3, and H4 and is primarily located in the nucleosomes of active genes. An in vitro transcription system was applied, which utilizes T7 RNA polymerase and template DNAs that are either moderately or highly positively coiled, to determine whether acetylation alters the dynamics of histone displacement from these templates during transcription. To measure displacement, an excess of a competitor (negatively coiled DNA reconstituted with unlabeled H3-H4) was included during the transcription process. Acetylated but not unacetylated (3)H-labeled H3-H4 was found to displace with high frequency from the moderately positively coiled template. This displacement of acetylated H3-H4 was not observed when the template was highly positively coiled. Acetylated (3)H-labeled H2A-H2B also preferentially displaced to the competitor, but in this instance, transcription-induced stress on the highly positively coiled template was required. The histone chaperone, NAP1, was found to facilitate the displacement of both H3-H4 and H2A-H2B. Surprisingly, when acetylated H2A-H2B and acetylated H3-H4 were reconstituted together in the same nucleosomes, the displacement of acetylated H2A-H2B was much reduced during transcription. We conclude that acetylation alters nucleosome stability by enhancing displacement of H3-H4, while decreasing the displacement of H2A-H2B. These results are discussed with regard to potential in vivo conditions in which these observations may be relevant.
在核心组蛋白H2A、H2B、H3和H4的N端区域发现了高水平的翻译后修饰——乙酰化，且主要位于活性基因的核小体中。应用了一种体外转录系统，该系统利用T7 RNA聚合酶和中度或高度正超螺旋的模板DNA，以确定乙酰化是否会改变转录过程中组蛋白从这些模板上的置换动力学。为了测量置换情况，在转录过程中加入了过量的竞争者（用未标记的H3-H4重构的负超螺旋DNA）。发现乙酰化而非未乙酰化的（3）H标记的H3-H4能高频地从中度正超螺旋模板上置换下来。当模板为高度正超螺旋时，未观察到乙酰化H3-H4的这种置换。乙酰化的（3）H标记的H2A-H2B也优先置换到竞争者上，但在这种情况下，需要高度正超螺旋模板上的转录诱导应激。发现组蛋白伴侣NAP1促进了H3-H4和H2A-H2B的置换。令人惊讶的是，当乙酰化的H2A-H2B和乙酰化的H3-H4在同一核小体中一起重构时，转录过程中乙酰化H2A-H2B的置换大大减少。我们得出结论，乙酰化通过增强H3-H4的置换同时减少H2A-H2B的置换来改变核小体稳定性。讨论了这些结果与这些观察结果可能相关的潜在体内条件。
