Department of Biology, University of Rochester, Rochester, New York 14627, USA.
Genetics. 2011 Jun;188(2):291-308. doi: 10.1534/genetics.111.127886. Epub 2011 Mar 24.
Transcriptional silencing in Saccharomyces cerevisiae is mediated by heterochromatin. There is a plethora of information regarding the roles of histone residues in transcriptional silencing, but exactly how histone residues contribute to heterochromatin structure is not resolved. We address this question by testing the effects of a series of histone H3 and H4 mutations involving residues in their aminoterminal tails, on the solvent-accessible and lateral surfaces of the nucleosome, and at the interface of the H3/H4 tetramer and H2A/H2B dimer on heterochromatin structure and transcriptional silencing. The general state, stability, and conformational heterogeneity of chromatin are examined with a DNA topology-based assay, and the primary chromatin structure is probed by micrococcal nuclease. We demonstrate that the histone mutations differentially affect heterochromatin. Mutations of lysine 16 of histone H4 (H4-K16) and residues in the LRS (loss of rDNA silencing) domain of nucleosome surface markedly alter heterochromatin structure, supporting the notion that H4-K16 and LRS play key roles in heterochromatin formation. Deletion of the aminoterminal tail of H3 moderately alters heterochromatin structure. Interestingly, a group of mutations in the globular domains of H3 and H4 that abrogate or greatly reduce transcriptional silencing increase the conformational heterogeneity and/or reduce the stability of heterochromatin without affecting its overall structure. Surprisingly, yet another series of mutations abolish or reduce silencing without significantly affecting the structure, stability, or conformational heterogeneity of heterochromatin. Therefore, histone residues may contribute to the structure, stability, conformational heterogeneity, or other yet-to-be-characterized features of heterochromatin important for transcriptional silencing.
酿酒酵母中的转录沉默是由异染色质介导的。关于组蛋白残基在转录沉默中的作用有很多信息，但组蛋白残基如何有助于异染色质结构尚未解决。我们通过测试一系列涉及组蛋白 H3 和 H4 氨基末端尾部残基、核小体的溶剂可及和侧表面以及 H3/H4 四聚体和 H2A/H2B 二聚体界面的组蛋白 H3 和 H4 突变对异染色质结构和转录沉默的影响来解决这个问题。通过基于 DNA 拓扑的测定来检查染色质的一般状态、稳定性和构象异质性，并通过微球菌核酸酶探测主要染色质结构。我们证明组蛋白突变会以不同的方式影响异染色质。组蛋白 H4 的赖氨酸 16（H4-K16）和核小体表面 LRS（丧失 rDNA 沉默）结构域中的残基突变显著改变异染色质结构，支持 H4-K16 和 LRS 在异染色质形成中起关键作用的观点。H3 的氨基末端尾部缺失会适度改变异染色质结构。有趣的是，一组在 H3 和 H4 的球状结构域中突变的组蛋白突变会破坏或大大降低转录沉默，增加构象异质性和/或降低异染色质的稳定性，而不影响其整体结构。令人惊讶的是，另一组突变会破坏或降低沉默，而不会显著影响异染色质的结构、稳定性或构象异质性。因此，组蛋白残基可能有助于异染色质的结构、稳定性、构象异质性或其他尚未确定的对转录沉默重要的特征。
