Advanced Biomolecular Research Cluster, University of the Free State, Bloemfontein, South Africa.
BMC Biochem. 2011 Jul 4;12:34. doi: 10.1186/1471-2091-12-34.
The involvement of histone acetylation in facilitating gene expression is well-established, particularly in the case of histones H3 and H4. It was previously shown in Saccharomyces cerevisiae that gene expression was significantly down-regulated and chromatin more condensed in stationary phase compared to exponential phase. We were therefore interested in establishing the acetylation state of histone H3 and H4 in stationary and in exponential phase, since the regulation of this modification could contribute to transcriptional shut-down and chromatin compaction during semi-quiescence.
We made use of nano-spray tandem mass spectrometry to perform a precursor ion scan to detect an m/z 126 immonium ion, diagnostic of an Nε-acetylated lysine residue that allowed unambiguous identification of acetylated as opposed to tri-methylated lysine. The fragmentation spectra of peptides thus identified were searched with Mascot against the Swiss-Prot database, and the y-ion and b-ion fragmentation series subsequently analyzed for mass shifts compatible with acetylated lysine residues. We found that K9, K14 and K36 of histone H3 and K12 and K16 of histone H4 were acetylated in exponential phase (bulk histones), but could not detect these modifications in histones isolated from stationary phase cells at the sensitivity level of the mass spectrometer. The corresponding un-acetylated peptides were, however, observed. A significantly higher level of acetylation of these residues in exponential phase was confirmed by immuno-blotting.
H4K16 acetylation was previously shown to disrupt formation of condensed chromatin in vitro. We propose that de-acetylation of H4K16 allowed formation of condensed chromatin in stationary phase, and that acetylation of H3K9, H3K14, H3K36, and H4K12 reflected the active transcriptional state of the yeast genome in exponential phase.
组蛋白乙酰化在促进基因表达方面的作用已得到充分证实，尤其是组蛋白 H3 和 H4。先前在酿酒酵母中已表明，与指数生长期相比，静止期的基因表达显著下调，染色质更加浓缩。因此，我们对静止期和指数生长期组蛋白 H3 和 H4 的乙酰化状态很感兴趣，因为这种修饰的调节可能有助于转录关闭和半静止期染色质紧缩。
我们利用纳喷雾串联质谱法进行前体离子扫描，以检测到 m/z 126 的亚稳离子，这是一个 Nε-乙酰化赖氨酸残基的特征，可明确鉴定出乙酰化的赖氨酸与三甲基化的赖氨酸。因此鉴定的肽的碎裂谱与 Mascot 一起在 Swiss-Prot 数据库中进行搜索，然后分析 y-离子和 b-离子碎裂系列，以寻找与乙酰化赖氨酸残基相容的质量位移。我们发现，组蛋白 H3 的 K9、K14 和 K36 以及组蛋白 H4 的 K12 和 K16 在指数生长期（大量组蛋白）中被乙酰化，但在质谱仪的灵敏度水平下，无法检测到从静止期细胞中分离的组蛋白中的这些修饰。然而，观察到了相应的未乙酰化肽。通过免疫印迹进一步证实了这些残基在指数生长期的乙酰化水平显著提高。
先前已证明 H4K16 乙酰化会破坏体外形成的浓缩染色质。我们提出，H4K16 的去乙酰化允许在静止期形成浓缩染色质，而 H3K9、H3K14、H3K36 和 H4K12 的乙酰化反映了酵母基因组在指数生长期的活跃转录状态。
