Robinson Karen M, Schultz Michael C
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Biochemistry. 2005 Mar 22;44(11):4551-61. doi: 10.1021/bi047523u.
Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays. Biochemical analysis reveals that the activity tightly associates with chromatin and reorganizes nucleosome arrays by a mechanism which is insensitive to ATP depletion after nucleosome assembly. It generates a chromatin organization in which a nucleosome is stably positioned immediately adjacent to Gal4p binding sites in the template DNA. Individual deletion of genes previously implicated in chromatin assembly and remodeling, namely, the histone chaperones NAP1, ASF1, and CAC1 and the SNF2-like DEAD/H ATPases SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20, does not significantly perturb reorganization. Therefore, Gal4-VP16-directed chromatin reorganization in yeast can occur by an ATP-independent mechanism that does not require SAGA, SWI/SNF, Isw1, or Isw2 chromatin remodeling complexes.
对染色质组织调控的重大见解源于使用Gal4-VP16的生化研究，Gal4-VP16是一种嵌合转录激活因子，其中Gal4p的DNA结合结构域与病毒蛋白VP16的激活结构域融合。出乎意料的是，尽管之前为了解Gal4-VP16在染色质环境中的功能付出了巨大努力，但我们发现了一种依赖于Gal4-VP16的染色质重组新模式。这种重组由来自芽殖酵母的粗DEAE（CD）组分中的一种活性完成，该组分也支持生理间隔核小体阵列的ATP依赖性组装。生化分析表明，该活性与染色质紧密结合，并通过一种对核小体组装后ATP消耗不敏感的机制重组核小体阵列。它产生一种染色质组织，其中核小体稳定地定位在模板DNA中Gal4p结合位点的紧邻位置。先前涉及染色质组装和重塑的基因，即组蛋白伴侣NAP1、ASF1和CAC1以及SNF2样DEAD/H ATP酶SNF2、ISW1、ISW2、CHD1、SWR1、YFR038w和SPT20的单个缺失，不会显著干扰重组。因此，酵母中Gal4-VP16指导的染色质重组可以通过一种不依赖ATP的机制发生，该机制不需要SAGA、SWI/SNF、Iswl或Iswz染色质重塑复合物。
