泛素化核小体复合物的结构机制研究进展

核小体组蛋白(H2A、H2B、H3、H4)的泛素化修饰介导了一系列效应蛋白与核小体发生特异性的相互作用,并调控细胞的表观遗传.近年来得益于一些新的化学方法与生物方法的发展,如蛋白质化学修饰等样品获取技术以及冷冻电子显微镜等结构观测技术,一些泛素化核小体与效应蛋白的复合物结构得以被解析,揭示了泛素调控染色质表观遗传过程的分子机制.本文对近期报道的六种泛素化核小体与效应蛋白的复合物进行了归纳,对比分析了泛素化核小体对效应蛋白的特异性识别与活化机制,并总结了组蛋白上的关键相互作用位点.     

组蛋白去乙酰化酶抑制剂的分子对接与构效关系研究

肿瘤是仅次于心血管疾病的第二大"杀手",肿瘤的发生与核心组蛋白的乙酰化及去乙酰化的失衡有密切的关系.这两种酶是一对功能相互拮抗的蛋白酶,分别对组蛋白氮端氨基酸残基进行乙酰化和去乙酰化调节染色质的结构,进而调控基因转录.组蛋白去乙酰酶抑制剂在体外和体内实验中均能引起乙酰化核小体组蛋白的堆积,提高p21基因的表达水平,抑制肿瘤细胞的增殖,诱导细胞分化或凋亡.研究组蛋白去乙酰化酶抑制剂和酶的分子对接可以阐明抑制剂与蛋白酶的作用模式,揭示抗癌机理.同时利用CoMFA方法研究了抑制剂结构和活性之间的关系,得到了预测能力较好的定量模型.     

鲱鱼精DNA的电化学氧化及与组蛋白的相互作用

应用循环伏安法、微分脉冲伏安法和荧光光谱法研究了鲱鱼精DNA的电化学氧化及其与组蛋白的相互作用.结果发现,在0.20~1.25 V电位区间内,DNA在酸性溶液中呈现一个明显的不可逆氧化峰,在中性及碱性溶液中呈现两个不可逆氧化峰.氧化峰电位随溶液pH值增大而负移,变化幅度为-57 mV.pH-1.氧化峰电流与DNA浓度(0.45~8.20 mmol.L-1)成线性关系.DNA能与组蛋白结合,导致氧化电位正移,氧化电流减小,并减弱钌配合物指示剂和DNA相互作用的荧光强度以及减少DNA的氧化损伤.     

非细胞体系重建核染色质组装、结构和DNA复制

用lambda DNA和蛙卵提取物进行非细胞体系核重建实验,结果显示,lambda DNA参与构成了重建核的染色质;染色质组装是逐步进行的;组装的染色质含有典型的核小体串珠状一级结构、300的螺线管和染色质的更高级结构;重建核能够复制DNA。     

未受精鸡蛋表层卵黄颗粒的染色质和DNA

本文通过电子显微镜的研究,证明未受精鸡蛋表层卵黄颗粒的染色质同受精未孵育鸡蛋的胚下表层卵黄颗粒的染色质一样,都与核染色质的基本结构相类似;其DNA的分子形态,也同受精未孵育鸡蛋的胚下表层卵黄颗粒一样,都与核的DNA相类似,同为线型DNA。根据这些观察,说明染色质和DNA在一定部位和发育时相的卵黄颗粒中,是广泛存在的。实验结果还进一步指出,卵黄颗粒具有就地合成DNA的能力,而且卵黄颗粒中的DNA可能与组蛋白结合,通过白组织构成染色质。     

利用实时荧光PCR鉴定小体鲟物种的快速方法

从小体鲟线粒体基因筛选出位于COⅠ(细胞色素氧化酶)基因中的一段保守序列,针对其设计引物,优化SYBR Green实时荧光PCR反应体系,建立了一种鉴定小体鲟的实时荧光PCR定性检测方法.该方法检测小体鲟DNA灵敏度为0.04mg/L.通过对采集和市售小体鲟鱼样品检测,该方法可检测出样品中的小体鲟成分.实验证明该方法可对血液样品和组织样品中的小体鲟进行种源鉴别.     

使用原子力显微镜观察DNA与PEI的层层自组装过程

选择了DNA与聚乙烯亚胺(PEI)进行相互作用,使用AFM直观地观察DNA与PEI层层自组装时其分子形貌的变化过程,总结了自组装过程中薄膜表面粗糙度的变化规律.同时详细地分析了离子浓度、DNA浓度、基底和固定方法对层层自组装膜的影响,以此来探讨DNA与PEI之间的相互作用机理.研究结果表明上述因素都会对膜的形貌产生影响,其中以云母作为基底,PEI处理基底表面后,再进行交替组装时,膜的表面粗糙度变化呈现出锯齿状的增长趋势,而用其他方法会影响膜的形貌以及粗糙度的变化规律.     

科学家研制出速测组蛋白甲基化试纸条

<正>中科院广州生物医药与健康研究院曾令文研究组,研制出一种快速灵敏检测组蛋白甲基化的试纸条。相关成果发表在《分析化学》。据介绍,组蛋白甲基化是一种重要的表观遗传学修饰,通常发生在氨基末端的赖氨酸或者精氨酸上,同其他调节蛋白和DNA相互作用,参与基因的调节和染色质高级结构的形成。细胞染色质中不同类型的组蛋白甲基化具有不同的意义,这往往取决于组蛋白被甲基化的位置和甲基化的程度。常用的     

组蛋白乙酰化的耦合增强拉曼散射生物传感方法

提出了一种组蛋白乙酰化修饰检测的耦合增强拉曼散射生物传感新方法. 该方法以金纳米粒子为表面增强拉曼散射(SERS)基底, 表面修饰乙酰化组蛋白H3多肽为识别探针, 对甲氧基苯硫酚(4-MTP)为拉曼标记物, 制备了组蛋白乙酰化修饰检测的SERS纳米探针. 通过紫外可见吸收光谱与动态光散射分析, 证实了组蛋白乙酰化抗体可介导SERS纳米粒子发生可控组装与聚集, 使SERS纳米探针间发生局域电场共振耦合, 产生显著增强的SERS信号. 基于此, 通过待测抗原与SERS纳米探针对抗体的竞争性相互作用, 我们设计了组蛋白乙酰化修饰检测的竞争免疫SERS生物传感方法. 该法操作简便、快速、重现性好, 且裸眼即能进行可视化鉴定. 通过设计不同染料标记的SERS纳米探针, 该法有望实现多种组蛋白修饰的复合检测.     

秋水仙碱与DNA相互作用的电化学研究

秋水仙碱(COLC)临床上用于乳腺癌、皮肤癌、宫颈癌、白血病、霍奇金病及AIDS病的治疗,并能控制和预防急性痛风症~([1])的发作.DNA贮存着生物体生命活动的所有信息,指导着细胞一系列的活动,也是体内抗癌药物主要的作用靶.而电化学研究可以得到DNA与识别分子相互作用的宏观信息,有助于进一步阐明它们之间的作用机理.本实验利用电化学方法研究了两者相互作用的电化学行为,获得了它们相互作用的基本信息,为其与DNA相互作用的机理研究提供了依据.     

Examining histone modification crosstalk using immobilized libraries established from ligation-ready nucleosomes

Chromatin signaling relies on a plethora of posttranslational modifications (PTM) of the histone proteins which package the long DNA molecules of our cells in reoccurring units of nucleosomes. Determining the biological function and molecular working mechanisms of different patterns of histone PTMs requires access to various chromatin substrates of defined modification status. Traditionally, these are achieved by individual reconstitution of single nucleosomes or arrays of nucleosomes in conjunction with modified histones produced by means of chemical biology. Here, we report an alternative strategy for establishing a library of differentially modified nucleosomes that bypasses the need for many individual syntheses, purification and assembly reactions by installing modified histone tails on ligation-ready, immobilized nucleosomes reconstituted in a single batch. Using the ligation-ready nucleosome strategy with sortase-mediated ligation for histone H3 and intein splicing for histone H2A, we generated libraries of up to 280 individually modified nucleosomes in 96-well plate format. Screening these libraries for the effects of patterns of PTMs onto the recruitment of a well-known chromatin factor, HP1 revealed a previously unknown long-range cross-talk between two modifications. H3S28 phosphorylation enhances recruitment of the HP1 protein to the H3K9 methylated H3-tail only in nucleosomal context. Detailed structural analysis by NMR measurements implies negative charges at position 28 to increase nucleosomal H3-tail dynamics and flexibility. Our work shows that ligation-ready nucleosomes enable unprecedented access to the ample space and complexity of histone modification patterns for the discovery and dissection of chromatin regulatory principles.

280 different patterns of histone modifications were installed in preassembled nucleosomes using PTS and SML enabling screening of readout crosstalk.     

Histone-catalyzed cleavage of nucleosomal DNA containing 2-deoxyribonolactone

Oxidized abasic sites such as 2-deoxyribonolactone (L) are produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor natural products. 2-Deoxyribonolactone is labile under alkaline conditions, but its half-life in free DNA at pH 7.5 is approximately 1 week. Independent generation of L at defined positions within nucleosomes reveals that the histone proteins catalyze strand scission and increase the rate between 11- and ～43-fold. Mechanistic studies indicate that DNA-protein cross-links are not intermediates en route to strand scission and that C2 deprotonation is the rate-determining step. The use of mutant histone H4 proteins demonstrates that the lysine-rich tail that is often post-translationally modified in cells contributes to the cleavage of L but is not the sole source of the enhanced cleavage rates. Consideration of DNA repair in cells suggests that L formation in nucleosomal DNA as part of bistranded lesions by antitumor antibiotics results in de facto double strand breaks, the most deleterious form of DNA damage.     

THE EFFECTS OF ULTRAVIOLET C ON in vitro NUCLEOSOME ASSEMBLY AND STABILITY

Abstract The effects of ultraviolet C (UVC) irradiation on nucleosome assembly and its stability were investigated quantitatively using an in vitro nucleosome assembly system comprising a plasmid DNA of pBR322 and core histones isolated from rat ascites hepatoma cells. Nucleosomal formation was estimated by analyzing the resulting DNA supercoils. When UVC-irradiated (3000 J/m²) DNA was used as a substrate for the nucleosome assembly system, the nucleosomal formation efficiency was reduced by half compared with nonirradiated DNA. On the other hand, when the reconstituted nucleosomes (minichromosomes) on the nonirradiated DNA were irradiated with UVC (3000 J/m²), about half each were disrupted and retained. These results indicate that it is difficult for UV-damaged DNA regions to supercoil around the histone octamers to form nucleosomes and that the histone octamers in the UV-damaged nucleosomes tend to be dissociated from DNA.     

Differentially Isotope‐Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time‐Resolved NMR Spectroscopy

Post‐translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core histones, the establishment of different PTMs on individual “sister” histones in the same nucleosomal context, that is, asymmetric histone PTMs, are difficult to analyze. Here, we generated differentially isotope‐labeled nucleosomes to study asymmetric histone modification crosstalk by time‐resolved NMR spectroscopy. Specifically, we present mechanistic insights into nucleosomal histone H3 modification reactions in cis and in trans, that is, within individual H3 copies or between them. We validated our approach by using the H3S10phK14ac crosstalk mechanism, which is mediated by the Gcn5 acetyltransferase. Moreover, phosphorylation assays on methylated substrates showed that, under certain conditions, Haspin kinase is able to produce nucleosomes decorated asymmetrically with two distinct types of PTMs.     

Cisplatin damage overrides the predefined rotational setting of positioned nucleosomes

Cisplatin and carboplatin are used successfully to treat various types of cancer. The drugs target the nucleosomes of cancer cells and form intrastrand DNA cross-links that are located in the major groove. We constructed two site-specifically modified nucleosomes containing defined intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links. Histones from HeLa-S3 cancer cells were transferred onto synthetic DNA duplexes having nucleosome positioning sequences. The structures of these complexes were investigated by hydroxyl radical footprinting. Employing nucleosome positioning sequences allowed us to quantify the structural deviation induced by the cisplatin adduct. Our experiments demonstrate that a platinum cross-link locally overrides the rotational setting predefined in the nucleosome positioning sequence such that the lesion faces toward the histone core. Identical results were obtained for two DNA duplexes in which the sites of platination differed by approximately half a helical turn. Additionally, we determined that cisplatin unwinds nucleosomal DNA globally by approximately 24 degree. The intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links are located in an area of the nucleosome that contains locally overwound DNA in undamaged reference nucleosomes. Because most nucleosome positions in vivo are defined by the intrinsic DNA sequence, the ability of cisplatin to influence the structure of these positioned nucleosomes may be of physiological relevance.     

Structural variability of nucleosomes detected by single-pair Förster resonance energy transfer: histone acetylation, sequence variation, and salt effects

Nucleosomes were reconstituted from 170 bp long fragments of 5S rDNA and an optimal positioning sequence, the Selex 601, with recombinant histones. In free-solution single pair F?rster resonance energy transfer (spFRET) measurements of the distance between fluorescently labeled bases in the nucleosomal DNA, the samples exhibited structural diversity. The structural heterogeneity correlated with the stability of the complexes and depended on the DNA sequence and histone acetylation. The stability of the nucleosomes was assessed via dilution-driven disruption: histone acetylation decreased nucleosome stability. The spFRET experiments used a new approach for data acquisition and analysis that we term "deliberately detuned detection" (D3). This permits the separation of subpopulations in the samples even for the low-FRET regime characteristic for the linker-DNA labeled nucleosomes. Thus, it became possible to study in more detail histone acetylation- and salt-dependent structural variations using either end- or internally labeled DNAs on the nucleosome. We found that the distance distribution of the fluorophore pairs on the linker DNA ends was much more sensitive to histone acetylation or sequence variation than that of labels on the internal part of the DNA, which was more tightly associated with the histone core. spFRET on freely diffusing nucleosomes allows us therefore to localize the influence of histone modifications and DNA sequence variations on the nucleosome structure and dynamics.     

Rigid assembly and Monte Carlo models of stable and unstable chromatin structures: the effect of nucleosomal spacing

Coarse-grained models are used to assess the packing of the 30-nm chromatin fiber. First, rigid assembly models for nucleosomal repeats from 155 to 211 bp are built using the crystal structure of the mononucleosome and attached straight stretches of B-DNA. The resulting fiber conformations are analyzed for static clashes and classified into stable and unstable structures. The effect of flexibility and thermal fluctuations is then taken into account by conducting Monte Carlo simulations of chromatin fiber models. Here the DNA is approximated by a flexible polymer chain with Debye–Hückel electrostatics, the geometry of the linker DNA connecting the nucleosomes is based on a two-angle zigzag model, and nucleosomes are represented by flat ellipsoids interacting via an attractive Gay–Berne potential. Unstable fibers occur at a particular repeat length period of 10 bp. Also, the regions of densely compacted fibers repeat at intervals of 10 bp. Besides one- and two-start helical zigzag structures, we show evidence for possible three-start structures, which have not been reported in experiments yet. Finally, we show that a local opening of the linker DNA at the nucleosome core—as probably occurs upon histone acetylation—leads to more open and flexible structures.     

A 1,2-d(GpG) cisplatin intrastrand cross-link influences the rotational and translational setting of DNA in nucleosomes

The mechanism of action of platinum-based anticancer drugs such as cis-diamminedichloroplatinum(II), or cisplatin, involves three early steps: cell entry, drug activation, and target binding. A major target in the cell, responsible for the anticancer activity, is nuclear DNA, which is packaged in nucleosomes that comprise chromatin. It is important to understand the nature of platinum-DNA interactions at the level of the nucleosome. The cis-{Pt(NH3)2}2+ 1,2-d(GpG) intrastrand cross-link is the DNA lesion most commonly encountered following cisplatin treatment. We therefore assembled two site-specifically platinated nucleosomes using synthetic DNA containing defined cis-{Pt(NH3)2}2+ 1,2-d(GpG) cross-links and core histones from HeLa-S3 cancer cells. The structures of these complexes were investigated by hydroxyl radical footprinting and exonuclease III mapping. Our experiments demonstrate that the 1,2-d(GpG) cross-link alters the rotational setting of the DNA on the histone octamer core such that the lesion faces inward, with disposition angles of the major groove relative to the core of xi approximately -20 degrees and xi approximately 40 degrees . We observe increased solvent accessibility of the platinated DNA strand, which may be caused by a structural perturbation in proximity of the 1,2-d(GpG) cisplatin lesion. The effect of the 1,2-d(GpG) cisplatin adduct on the translational setting of the nucleosomal DNA depends strongly on the position of the adduct within the sequence. If the cross-link is located at a site that is in phase with the preferred rotational setting of the unplatinated nucleosomal DNA, the effect on the translational position is negligible. Minor exonuclease III digestion products in this substrate indicate that the cisplatin adduct permits only those translational settings that differ from one another by integral numbers of DNA helical turns. If the lesion is located out of phase with the preferred rotational setting, the translational position of the main conformation was shifted by 5 bp. Additionally, a fraction of platinated nucleosomes with widely distributed translational positions was observed, suggesting increased nucleosome sliding relative to platinated nucleosomes containing the 1,3-intrastrand d(GpTpG) cross-link investigated previously (Ober, M.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 6278-6286).