Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA

SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.     

A dominant role for DNA secondary structure in forming hypersensitive structures in chromatin

Previous work has suggested that potential information in DNA secondary structure might be used by cells to define DNAase I- and S1-sensitive chromatin structures associated with promoter and terminator regions. To test this hypothesis, supercoiled pBR322 was cotransfected into L cells. For the majority of transfected clones supercoil-induced S1-sensitive sites in pure pBR322 DNA are also S1-sensitive in L-cell nuclei. These results suggest that the potential of certain DNA sequences to form specific secondary structures in chromatin can be a dominant characteristic. A recombinant chicken β^A-lobin supercoiled plasmid was reconstituted in vitro with histones. The reconstituted chromatin also retained the ability to form S1-sensitive sites. Evidence suggests that DNA sequences capable of forming S1-sensitive sites in supercoiled plasmids may bind nucleosomes poorly after reconstitution with histones.     

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants

Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA-histone interactions in vivo. The procedure includes DNA-histone cross-linking in chromatin, shearing DNA into smaller fragments, immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which provides a good yield of high-quality DNA resulting in at least 15 mug of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g of starting tissue material) sufficient to test > or =25 genes of interest. This simpler and cost-efficient protocol has been applied for histone-modification studies of various Arabidopsis thaliana tissues and is easy to adapt for other systems as well.     

Correlation between molecular clock ticking, codon usage fidelity of DNA repair, chromosome banding and chromatin compactness in germline cells

The vertebrate genome is built of long DNA regions, relatively homogeneous in GC content, which likely correspond to bands on stained chromosomes. Large differences in composition have been found among DNA regions belonging to the same genome. They are paralleled by differences in codon usage in genes differently localized. The hypothesis presented here asserts that these differences in composition are caused by different mutational bias of alpha and beta DNA polymerases, these polymerases being involved to different extents in the repair of DNA lesions in compact and relaxed chromatin, respectively, in germline cells.     

Benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide modification of DNA: Relation to chromatin structure and reconstitution

Purified duck reticulocyte DNA was incubated in vitro with a 7,8-dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease S₁ than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by Staphylococcal nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested that the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the ‘open’ regions of chromatin, and 35% to ‘closed’ regions.These results indicate that although covalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does not appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones.     

A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis

Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function.     

Condensin binding at distinct and specific chromosomal sites in the Saccharomyces cerevisiae genome

Mitotic chromosome condensation is chiefly driven by the condensin complex. The specific recognition (targeting) of chromosomal sites by condensin is an important component of its in vivo activity. We previously identified the rRNA gene cluster in Saccharomyces cerevisiae as an important condensin-binding site, but both genetic and cell biology data suggested that condensin also acts elsewhere. In order to characterize the genomic distribution of condensin-binding sites and to assess the specificity of condensin targeting, we analyzed condensin-bound sites using chromatin immunoprecipitation and hybridization to whole-genome microarrays. The genomic condensin-binding map shows preferential binding sites over the length of every chromosome. This analysis and quantitative PCR validation confirmed condensin-occupied sites across the genome and in the specialized chromatin regions: near centromeres and telomeres and in heterochromatic regions. Condensin sites were also enriched in the zones of converging DNA replication. Comparison of condensin binding in cells arrested in G(1) and mitosis revealed a cell cycle dependence of condensin binding at some sites. In mitotic cells, condensin was depleted at some sites while enriched at rRNA gene cluster, subtelomeric, and pericentromeric regions.     

Intergenic Locations of Rice Centromeric Chromatin

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions.     

Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome

The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.     

Transition-transversion bias is not universal: a counter example from grasshopper pseudogenes

Comparisons of the DNA sequences of metazoa show an excess of transitional over transversional substitutions. Part of this bias is due to the relatively high rate of mutation of methylated cytosines to thymine. Postmutation processes also introduce a bias, particularly selection for codon-usage bias in coding regions. It is generally assumed, however, that there is a universal bias in favour of transitions over transversions, possibly as a result of the underlying chemistry of mutation. Surprisingly, this underlying trend has been evaluated only in two types of metazoan, namely Drosophila and the Mammalia. Here, we investigate a third group, and find no such bias. We characterize the point substitution spectrum in Podisma pedestris, a grasshopper species with a very large genome. The accumulation of mutations was surveyed in two pseudogene families, nuclear mitochondrial and ribosomal DNA sequences. The cytosine-guanine (CpG) dinucleotides exhibit the high transition frequencies expected of methylated sites. The transition rate at other cytosine residues is significantly lower. After accounting for this methylation effect, there is no significant difference between transition and transversion rates. These results contrast with reports from other taxa and lead us to reject the hypothesis of a universal transition/transversion bias. Instead we suggest fundamental interspecific differences in point substitution processes.