The vast majority of methods available to characterize genome-wide chromatin structure exploit differences in DNA accessibility to nucleases or chemical crosslinking. We developed a novel method to gauge genome-wide accessibility of histone protein surfaces within nucleosomes by assessing reactivity of engineered cysteine residues with a thiol-specific reagent, biotin-maleimide (BM). Yeast nuclei were obtained from cells expressing the histone mutant H2B S116C, in which a cysteine resides near the center of the external flat protein surface of the nucleosome. BM modification revealed that nucleosomes are generally equivalently accessible throughout the S. cerevisiae genome, including heterochromatic regions, suggesting limited, higher-order chromatin structures in which this surface is obstructed by tight nucleosome packing. However, we find that nucleosomes within 500 bp of transcription start sites exhibit the greatest range of accessibility, which correlates with the density of chromatin remodelers. Interestingly, accessibility is not well correlated with RNA polymerase density and thus the level of gene expression. We also investigated the accessibility of cysteine mutations designed to detect exposure of histone surfaces internal to the nucleosome thought to be accessible in actively transcribed genes: H3 102, is at the H2A–H2B dimer/H3–H4 tetramer interface, and H3 A110C, resides at the H3–H3 interface. However, in contrast to the external surface site, we find that neither of these internal sites were found to be appreciably exposed. Overall, our finding that nucleosomes surfaces within S. cerevisiae chromatin are equivalently accessible genome-wide is consistent with a globally uncompacted chromatin structure lacking substantial higher-order organization. However, we find modest differences in accessibility that correlate with chromatin remodelers but not transcription, suggesting chromatin poised for transcription is more accessible than actively transcribed or intergenic regions. In contrast, we find that two internal sites remain inaccessible, suggesting that such non-canonical nucleosome species generated during transcription are rapidly and efficiently converted to canonical nucleosome structure and thus not widely present in native chromatin.

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酵母中的全局组蛋白表面可及性表明具有典型核小体的均匀松散包装的基因组

绝大多数可用于表征全基因组染色质结构的方法都利用了 DNA 对核酸酶或化学交联的可及性的差异。我们开发了一种新方法，通过评估工程半胱氨酸残基与硫醇特异性试剂生物素-马来酰亚胺 (BM) 的反应性，来衡量核小体内组蛋白表面的全基因组可及性。酵母细胞核是从表达组蛋白突变体 H2B S116C 的细胞中获得的，其中半胱氨酸位于核小体外部扁平蛋白表面的中心附近。BM 修饰表明，核小体通常在整个酿酒酵母基因组中均等价可及，包括异染色质区域，这表明染色质结构受限、高阶，其中该表面被紧密的核小体堆积所阻碍。然而，我们发现转录起始位点 500 bp 以内的核小体表现出最大的可及性范围，这与染色质重塑因子的密度相关。有趣的是，可访问性与 RNA 聚合酶密度以及基因表达水平没有很好的相关性。我们还研究了半胱氨酸突变的可及性，旨在检测核小体内部组蛋白表面的暴露，这些核小体被认为在活跃转录的基因中是可接近的：H3 102，位于 H2A-H2B 二聚体/H3-H4 四聚体界面，H3 A110C，位于在 H3-H3 接口处。然而，与外部表面站点相比，我们发现这些内部站点都没有明显暴露。总的来说，我们发现核小体在 S. 酿酒酵母染色质在全基因组范围内是等效的，这与缺乏实质性高阶组织的全球未压缩染色质结构一致。然而，我们发现可及性的适度差异与染色质重塑相关，但与转录无关，这表明准备转录的染色质比主动转录或基因间区域更容易接近。相比之下，我们发现两个内部位点仍然无法访问，这表明在转录过程中产生的此类非规范核小体种类会快速有效地转化为规范核小体结构，因此不会广泛存在于天然染色质中。我们发现可及性的适度差异与染色质重塑相关，但与转录无关，这表明准备转录的染色质比主动转录或基因间区域更容易接近。相比之下，我们发现两个内部位点仍然无法访问，这表明在转录过程中产生的此类非规范核小体种类会快速有效地转化为规范核小体结构，因此不会广泛存在于天然染色质中。我们发现可及性的适度差异与染色质重塑相关，但与转录无关，这表明准备转录的染色质比主动转录或基因间区域更容易接近。相比之下，我们发现两个内部位点仍然无法访问，这表明在转录过程中产生的此类非规范核小体种类会快速有效地转化为规范核小体结构，因此不会广泛存在于天然染色质中。