Single-epitope recognition imaging of native chromatin.
Single-epitope recognition imaging of native chromatin.
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DOI：
10.1186/1756-8935-1-10
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发表时间：
2008-12-17
影响因子：
3.9
通讯作者：
Lindsay S
中科院分区：
文献类型：
--
作者：
Wang H;Dalal Y;Henikoff S;Lindsay S
染色质的直接可视化有可能为表观遗传过程提供重要的见解。特别是，原子力显微镜（AFM）可以在生理离子条件下可视化单个核小体。然而，AFM 主要应用于体外重构的染色质，其作为解剖天然核小体工具的潜力尚未得到探索。最近，我们将 AFM 应用于含有着丝粒特异性组蛋白 3 (CenH3) 的天然果蝇染色质，表明它在较小的颗粒中大量富集。结合 CenH3 核小体的生化分析，我们提出着丝粒核小体是半体，一圈 DNA 包裹在一个颗粒上，该颗粒由着丝粒特异性 CenH3、H4、H2A 和 H2B 各一个分子组成。在这里，我们应用 AFM 成像的识别模式来直接识别从天然着丝粒染色质释放的组蛋白核心颗粒内的 CenH3。发现这些颗粒中超过 90% 的高度为四聚体。通过用 CenH3 肽封闭来确认识别的特异性，并通过力测量来量化相互作用的强度。这些结果表明 AFM 成像的颗粒确实是成熟的含有 CenH3 的半体。对从天然着丝粒染色质分离的组蛋白核心颗粒中的 CenH3 进行高效且高度特异性的识别表明，四聚体是成熟四聚体中着丝粒核小体的主要形式。我们的研究结果提供了原理证明，即这种方法可以通过直接快速检测生理盐浓度中的天然核小体来深入了解染色质生物学。
Direct visualization of chromatin has the potential to provide important insights into epigenetic processes. In particular, atomic force microscopy (AFM) can visualize single nucleosomes under physiological ionic conditions. However, AFM has mostly been applied to chromatin that has been reconstituted in vitro, and its potential as a tool for the dissection of native nucleosomes has not been explored. Recently we applied AFM to native Drosophila chromatin containing the centromere-specific histone 3 (CenH3), showing that it is greatly enriched in smaller particles. Taken together with biochemical analyses of CenH3 nucleosomes, we propose that centromeric nucleosomes are hemisomes, with one turn of DNA wrapped around a particle consisting of one molecule each of centromere-specific CenH3, H4, H2A and H2B. Here we apply a recognition mode of AFM imaging to directly identify CenH3 within histone core particles released from native centromeric chromatin. More than 90% of these particles were found to be tetrameric in height. The specificity of recognition was confirmed by blocking with a CenH3 peptide, and the strength of the interaction was quantified by force measurements. These results imply that the particles imaged by AFM are indeed mature CenH3-containing hemisomes. Efficient and highly specific recognition of CenH3 in histone core particles isolated from native centromeric chromatin demonstrates that tetramers are the predominant form of centromeric nucleosomes in mature tetramers. Our findings provide proof of principle that this approach can yield insights into chromatin biology using direct and rapid detection of native nucleosomes in physiological salt concentrations.
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影响因子：
9.8
作者：
Dalal Y;Wang H;Lindsay S;Henikoff S
通讯作者：
Henikoff S
影响因子：
3.4
作者：
Lin, Liyun;Wang, Hongda;Lindsay, Stuart
通讯作者：
Lindsay, Stuart
DOI：
10.1073/pnas.0701459104
发表时间：
2007-08-21
影响因子：
11.1
作者：
Ranjith, Padinhateeri;Yan, Jie;Marko, John F.
通讯作者：
Marko, John F.
影响因子：
5.7
作者：
Tomschik, M;Karymov, MA;Leuba, SH
通讯作者：
Leuba, SH
影响因子：
7.5
作者：
Black, Ben E.;Bassett, Emily A.
通讯作者：
Bassett, Emily A.