盐透析体外组装核小体及检测方法

核小体是构成真核生物染色质的基本结构单位,体内研究核小体及染色质结构受到诸多因素限制,体外重构核小体结构是研究与核小体及染色质结构相关课题的一种重要的方法手段.实验将ES1,CS1以及601DNA序列克隆到载体中,通过PCR大量扩增回收得到目的DNA条带,表达纯化了4种组蛋白且装配成组蛋白八聚体,在盐透析的条件下组装形成核小体结构,利用EB染色以及Biotin标记的方法分析检测了形成核小体的效率.结果显示,在盐透析的条件下,可以有效的组装形成核小体结构,而且随着组蛋白八聚体与DNA的比例增加,核小体的形成效率显著提高.本实验为核小体定位、染色质重塑及组蛋白变体等表观遗传学以及结构生物学领域的研究奠定一定的基础.     

Crystal structure of the human centromeric nucleosome containing CENP-A

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg?80 and Gly?81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.     

Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA

The nucleosome subunits of chromatin are assembled from histones and DNA by an acidic protein which binds histones. The nucleosome assembly protein has been identified and purified from eggs of Xenopus laevis.     

Evidence for kinks in DNA folding in the nucleosome

The nucleosome subunit of chromatin consists of DNA folded around a histone core as a 1.8-turn left-handed solenoid. The crystal structure of the nucleosome core particle revealed that it has a dyad symmetry axis and that the minor helix groove faces outwards from the protein core. Richmond et al. noticed that the path traversed by the helix has severe bends at sites approximately one and four helix turns from the dyad axis. We have developed two photochemical methods to study the structure of DNA, and in particular that wrapped around the nucleosome core. One method depends on the sensitization of singlet oxygen production by an eosin analogue. We have monitored the rate at which excited state oxygen diffuses into contact with DNA base planes, and find that it attacks the nucleosome with high specificity. We have also mapped the DNA binding of the intercalating dye methylene blue, and conclude that it binds to the same sites accessible to oxygen by diffusion. On the basis of these results we suggest that the DNA in the nucleosome is bent or kinked at two sites, 1.5 helix turns from the dyad axis.     

Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. Eukaryotic cells repair DSBs by both non-homologous end joining and homologous recombination. How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important issues. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after DSB formation, spreads over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. In Saccharomyces cerevisiae, phosphorylation of the two principal H2A species is also signalled by DSB formation, which spreads approximately 40 kb in either direction from the DSB. Here we show that near a DSB phosphorylation of H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease; however, phosphorylation of H2A and nucleosome loss occur independently. The DNA damage sensor MRX is required for histone loss, which also depends on INO80, a nucleosome remodelling complex. The repair protein Rad51 (ref. 6) shows delayed recruitment to DSBs in the absence of histone loss, suggesting that MRX-dependent nucleosome remodelling regulates the accessibility of factors directly involved in DNA repair by homologous recombination. Thus, MRX may regulate two pathways of chromatin changes: nucleosome displacement for efficient recruitment of homologous recombination proteins; and phosphorylation of H2A, which modulates checkpoint responses to DNA damage.     

Determinants of nucleosome organization in primary human cells

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.     

Local protein-DNA interactions may determine nucleosome positions on yeast plasmids

The structure of the nucleosome core particle, the basic structural subunit of chromatin, is well known. Although nucleosomes often appear to be positioned randomly with respect to DNA sequences, in some cases they seem to occupy precisely defined positions on the DNA. The yeast plasmid TRP1ARS1 contains three precisely positioned, stable nucleosomes, I, II and III, which are flanked by nuclease-sensitive regions. Our aim in the present study was to determine whether the positions of these three nucleosomes relate to (1) protein-DNA interactions; (2) the limited space between nuclease-sensitive regions, which is just long enough to accommodate three yeast nucleosomes (that is, boundary conditions); or (3) proximity to the putative origin of replication in one of the nuclease-sensitive regions. We have tested these alternatives by analysing the positions of nucleosomes after insertion of various lengths of DNA into this region and assembly of chromatin in vivo. Our results suggest that specific protein-DNA interactions are the most likely determinants of these nucleosome positions.     

Structure and mechanism of the chromatin remodelling factor ISW1a

Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25?? and 3.60??, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.     

The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.     

A chromatin remodelling complex that loads cohesin onto human chromosomes

Nucleosomal DNA is arranged in a higher-order structure that presents a barrier to most cellular processes involving protein DNA interactions. The cellular machinery involved in sister chromatid cohesion, the cohesin complex, also requires access to the nucleosomal DNA to perform its function in chromosome segregation. The machineries that provide this accessibility are termed chromatin remodelling factors. Here, we report the isolation of a human ISWI (SNF2h)-containing chromatin remodelling complex that encompasses components of the cohesin and NuRD complexes. We show that the hRAD21 subunit of the cohesin complex directly interacts with the ATPase subunit SNF2h. Mapping of hRAD21, SNF2h and Mi2 binding sites by chromatin immunoprecipitation experiments reveals the specific association of these three proteins with human DNA elements containing Alu sequences. We find a correlation between modification of histone tails and association of the SNF2h/cohesin complex with chromatin. Moreover, we show that the association of the cohesin complex with chromatin can be regulated by the state of DNA methylation. Finally, we present evidence pointing to a role for the ATPase activity of SNF2h in the loading of hRAD21 on chromatin.     

Intrinsic coupling of lagging-strand synthesis to chromatin assembly

Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis--to our knowledge--of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.