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Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

Packaging of DNA into chromatin regulates DNA accessibility and, consequently, all DNA-dependent processes, such as transcription, recombination, repair, and replication. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This defined organization of chromatin has been recently questioned using microscopy techniques, proposing a rather irregular structure. To gain more insight into the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. Thus, we assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions by utilizing statistical data analyses and computer modelling. We found no difference in the degree of compaction between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially compacted domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

Eukaryotic genomes are packaged into nucleosomes that occlude DNA from interacting with most DNA-binding proteins. Nucleosome positioning and chromatin organization is critical for gene regulation. We have investigated the mechanism by which nucleosomes are positioned at the promoters of active and silent rRNA genes (rDNA). The reconstitution of nucleosomes on rDNA results in sequence-dependent nucleosome positioning at the rDNA promoter that mimics the chromatin structure of silent rRNA genes in vivo, suggesting that active mechanisms are required to reorganize chromatin structure upon gene activation. Nucleosomes are excluded from positions observed at active rRNA genes, resulting in transcriptional repression on chromatin. We suggest that the repressed state is the default chromatin organization of the rDNA and gene activation requires ATP-dependent chromatin remodelling activities that move the promoter-bound nucleosome about 22-bp upstream. We suggest that nucleosome remodelling precedes promoter-dependent transcriptional activation as specific inhibition of ATP-dependent chromatin remodelling suppresses the initiation of RNA Polymerase I transcription in vitro. Once initiated, RNA Polymerase I is capable of elongating through reconstituted chromatin without apparent displacement of the nucleosomes. The results reveal the functional cooperation of DNA sequence and chromatin remodelling complexes in nucleosome positioning and in establishing the epigenetic active or silent state of rRNA genes.

DNA in eukaryotes is highly compacted. However, it is essential that proteins can have controlled access to the DNA during a number of fundamental cellular processes such as replication, transcription or repair. A large family of protein complexes commonly referred to as Swi2/Snf2 complexes allows for the transient remodelling of the essential compaction unit, the nucleosome and thus assures the access to, or repression of certain segments of DNA. In spite of a large number of research efforts using bio-chemical, structural and theoretical approaches the molecular mechanism of the ATP dependent nucleosome remodelling is currently not well understood. We therefore performed single molecule FRET experiments aimed to unravel details of the remodelling kinetics and pathway.We present remodelling data obtained by the remodelling complex ACF from drosophila on mono-nucleosomal constructs that use the 601- localisation sequence in combination with a biochemically well characterised linker DNA. The remodelling data was obtained, both, on nucleosomes immobilised onto surfaces of micro-fluidic chambers as well as from solution measurements using pulsed interleaved excitation and multi-parameter fluorescence detection.

Nucleosomes, the primary repeating unit of chromatin, package DNA by wrapping 147 base pairs tightly around an octamer of histone proteins in approximately 1.7 helical turns. In order to allow central nuclear processes to happen, such as DNA replication, recombination, repair and transcription, regulated exposure of the template DNA is required. Therefore, the nucleosome has to undergo certain conformational changes. One possible strategy for such a regulated exposure is the translocation or ‘sliding’ of nucleosomes along the DNA by the class of ATP-hydrolyzing enzymatic machines called chromatin remodellers.We apply single pair-FRET for direct observation of intrinsic nucleosome dynamics as well as conformational changes of mononucleosomes induced by chromatin-remodeling complexes. The movement of DNA around the nucleosome surface is tracked in the presence and absence of remodeller by using mononucleosomes, which are reconstituted with a 200 bp long DNA containing the nucleosome positioning sequence 601 and a donor- and acceptor-dye pair at well-defined positions. Mononucleosomes are immobilized on DOPC-bilayer functionalized quartz slides and visualized by Total Internal Reflection Microscopy.