Histone octamer instability under single molecule experiment conditions

We have studied the sample concentration-dependent and external stress-dependent stability of native and reconstituted nucleosomal arrays. Whereas upon stretching a single chromatin fiber in a solution of very low chromatin concentration the statistical distribution of DNA length released upon nucleosome unfolding shows only one population centered around approximately 25 nm, in nucleosome stabilizing conditions a second population with average length of approximately 50 nm was observed. Using radioactively labeled histone H3 and H2B, we demonstrate that upon lowering the chromatin concentration to very low values, first the linker histones are released, followed by the H2A-H2B dimer, whereas the H3-H4 tetramer remains stably attached to DNA even at the lowest concentration studied. The nucleosomal arrays reconstituted on a 5 S rDNA tandem repeat exhibited similar behavior. This suggests that the 25-nm disruption length is a consequence of the histone H2A-H2B dimer dissociation from the histone octamer. In nucleosome stabilizing conditions, a full approximately 145 bp is constrained in the nucleosome. Our data demonstrate that the nucleosome stability and histone octamer integrity can be severely degraded in experiments where the sample concentration is low.     

Histone Octamer Helical Tubes Suggest that an Internucleosomal Four-Helix Bundle Stabilizes the Chromatin Fiber

A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-α3 and H2B-αC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed.     

体外装配核小体过程组蛋白浓度依赖的动力学模型

为探索组蛋白浓度对核小体体外装配的影响,本研究表达纯化了4种组蛋白,通过控制实验反应体系中组蛋白的浓度,利用盐透析法在体外装配了核小体,检测分析了组蛋白浓度与核小体组装效率的关系。以此实验数据为基础,提出了核小体组装过程组蛋白浓度依赖性的动力学模型。实验结果发现,反应体系中组蛋白浓度与核小体生成量呈典型的线性关系。依据动力学理论模型,进行线性回归拟合,回归系数达到0.963;经计算601 DNA序列组装核小体的反应速率常数k为1.49×10^-5mL·h·μg^-1。CS1序列验证动力学模型的线性回归相关系数为0.989,反应速率常数为1.52×10^-5mL·h·μg^-1。该实验方法及动力学模型中反应速率常数k可用于评价相同长度的DNA序列组装核小体的能力、组蛋白与其突变体以及组蛋白变体之间形成核小体结构能力的差异。该动力学模型的建立为理解核小体装配、核小体定位、染色质结构等相关问题提供了理论指导。     

Localization of linker histone in chromatosomes by cryo-atomic force microscopy

Linker histones play a fundamental role in determining higher order chromatin structure as a consequence of their association with nucelosomal DNA. Yet the locations and structural consequences of linker histone binding are still enigmatic. Here, using cryo-atomic force microscopy, we show that the linker histone H5 in native chromatin and in chromatosomes reconstituted on the 5S rDNA template is located at the dyad of the nucleosome core particle, within the "stem" structure. Direct measurement also indicates that the length of free linker DNA between chromatosomes in native chromatin is approximately 30 bp, slightly shorter than that estimated from nuclease digestion assays.     

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.