Nick Kepper

Folding and organization of a contiguous chromosome region according to the gene distribution pattern in primary genomic sequence

Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene “deserts.” In nuclei, this region forms multiple, nonrandom “higher order” structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization.

Nucleosome Geometry and Internucleosomal Interactions Control the Chromatin Fiber Conformation

Based on model structures with atomic resolution, a coarse-grained model for the nucleosome geometry was implemented. The dependence of the chromatin fiber conformation on the spatial orientation of nucleosomes and the path and length of the linker DNA was systematically explored by Monte Carlo simulations. Two fiber types were analyzed in detail that represent nucleosome chains without and with linker histones, respectively: two-start helices with crossed-linker DNA (CL conformation) and interdigitated one-start helices (ID conformation) with different nucleosome tilt angles. The CL conformation was derived from a tetranucleosome crystal structure that was extended into a fiber. At thermal equilibrium, the fiber shape persisted but relaxed into a structure with a somewhat lower linear mass density of 3.1 +/- 0.1 nucleosomes/11 nm fiber. Stable ID fibers required local nucleosome tilt angles between 40 degrees and 60 degrees. For these configurations, much higher mass densities of up to 7.9 +/- 0.2 nucleosomes/11 nm fiber were obtained. A model is proposed, in which the transition between a CL and ID fiber is mediated by relatively small changes of the local nucleosome geometry. These were found to be in very good agreement with changes induced by linker histone H1 binding as predicted from the high resolution model structures.

The Effect of Internucleosomal Interaction on Folding of the Chromatin Fiber

The folding of the nucleosome chain into a chromatin fiber modulates DNA accessibility and is therefore an important factor for the control of gene expression. The fiber conformation depends crucially on the interaction between individual nucleosomes. However, this parameter has not been accurately determined experimentally, and it is affected by posttranslational histone modifications and binding of chromosomal proteins. Here, the effect of different internucleosomal interaction strengths on the fiber conformation was investigated by Monte Carlo computer simulations. The fiber geometry was modeled to fit that of chicken erythrocyte chromatin, which has been examined in numerous experimental studies. In the Monte Carlo simulation, the nucleosome shape was described as an oblate spherocylinder, and a replica exchange protocol was developed to reach thermal equilibrium for a broad range of internucleosomal interaction energies. The simulations revealed the large impact of the nucleosome geometry and the nucleosome repeat length on the compaction of the chromatin fiber. At high internucleosomal interaction energies, a lateral self-association of distant fiber parts and an interdigitation of nucleosomes were apparent. These results identify key factors for the control of the compaction and higher order folding of the chromatin fiber.

Targeted Chromatin Capture (T2C): a novel high resolution high throughput method to detect genomic interactions and regulatory elements

BackgroundSignificant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort. Techniques like Hi-C provide a genome-wide analysis. However, it requires massive sequencing effort with considerable costs. Here we describe a new technique termed Targeted Chromatin Capture (T2C), to interrogate large selected regions of the genome. T2C provides an unbiased view of the spatial organization of selected loci at superior resolution (single restriction fragment resolution, from 2 to 6 kbp) at much lower costs than Hi-C due to the lower sequencing effort.ResultsWe applied T2C on well-known model regions, the mouse β-globin locus and the human H19/IGF2 locus. In both cases we identified all known chromatin interactions. Furthermore, we compared the human H19/IGF2 locus data obtained from different chromatin conformation capturing methods with T2C data. We observed the same compartmentalization of the locus, but at a much higher resolution (single restriction fragments vs. the common 40 kbp bins) and higher coverage. Moreover, we compared the β-globin locus in two different biological samples (mouse primary erythroid cells and mouse fetal brain), where it is either actively transcribed or not, to identify possible transcriptional dependent interactions. We identified the known interactions in the β-globin locus and the same topological domains in both mouse primary erythroid cells and in mouse fetal brain with the latter having fewer interactions probably due to the inactivity of the locus. Furthermore, we show that interactions due to the important chromatin proteins, Ldb1 and Ctcf, in both tissues can be analyzed easily to reveal their role on transcriptional interactions and genome folding.ConclusionsT2C is an efficient, easy, and affordable with high (restriction fragment) resolution tool to address both genome compartmentalization and chromatin-interaction networks for specific genomic regions at high resolution for both clinical and non-clinical research.

Dissecting DNA-Histone Interactions in the Nucleosome by Molecular Dynamics Simulations of DNA Unwrapping

The nucleosome complex of DNA wrapped around a histone protein octamer organizes the genome of eukaryotes and regulates the access of protein factors to the DNA. We performed molecular dynamics simulations of the nucleosome in explicit water to study the dynamics of its histone-DNA interactions. A high-resolution histone-DNA interaction map was derived that revealed a five-nucleotide periodicity, in which the two DNA strands of the double helix made alternating contacts. On the 100-ns timescale, the histone tails mostly maintained their initial positions relative to the DNA, and the spontaneous unwrapping of DNA was limited to 1-2 basepairs. In steered molecular dynamics simulations, external forces were applied to the linker DNA to investigate the unwrapping pathway of the nucleosomal DNA. In comparison with a nucleosome without the unstructured N-terminal histone tails, the following findings were obtained: 1), Two main barriers during unwrapping were identified at DNA position ±70 and ±45 basepairs relative to the central DNA basepair at the dyad axis. 2), DNA interactions of the histone H3 N-terminus and the histone H2A C-terminus opposed the initiation of unwrapping. 3), The N-terminal tails of H2A, H2B, and H4 counteracted the unwrapping process at later stages and were essential determinants of nucleosome dynamics. Our detailed analysis of DNA-histone interactions revealed molecular mechanisms for modulating access to nucleosomal DNA via conformational rearrangements of its structure.

Force spectroscopy of chromatin fibers: Extracting energetics and structural information from Monte Carlo simulations

The folding of the nucleosome chain into a chromatin fiber is a central factor for controlling the DNA access of protein factors involved in transcription, DNA replication and repair. Force spectroscopy experiments with chromatin fibers are ideally suited to dissect the interactions that drive this process, and to probe the underlying fiber conformation. However, the interpretation of the experimental data is fraught with difficulties due to the complex interplay of the nucleosome geometry and the different energy terms involved. Here, we apply a Monte Carlo simulation approach to derive virtual chromatin fiber force spectroscopy curves. In the simulations, the effect of the nucleosome geometry, repeat length, nucleosome-nucleosome interaction potential, and the unwrapping of the DNA from the histone protein core on the shape of the force-extension curves was investigated. These simulations provide a framework for the evaluation of experimental data sets. We demonstrate how the relative contributions of DNA bending and twisting, nucleosome unstacking and unwrapping the nucleosomal DNA from the histone octamer can be dissected for a given fiber geometry.

Exploring the Conformational Space of Chromatin Fibers and Their Stability by Numerical Dynamic Phase Diagrams

The three-dimensional structure of chromatin affects DNA accessibility and is therefore a key regulator of gene expression. However, the path of the DNA between consecutive nucleosomes, and the resulting chromatin fiber organization remain controversial. The conformational space available for the folding of the nucleosome chain has been analytically described by phase diagrams with a two-angle model, which describes the chain trajectory by a DNA entry-exit angle at the nucleosome and a torsion angle between consecutive nucleosomes. Here, a novel type of numerical phase diagrams is introduced that relates the geometric phase space to the energy associated with a given chromatin conformation. The resulting phase diagrams revealed differences in the energy landscape that reflect the probability of a given conformation to form in thermal equilibrium. Furthermore, we investigated the effects of entropy and additional degrees of freedom in the dynamic phase diagrams by performing Monte Carlo simulations of the initial chain trajectories. Using our approach, we were able to demonstrate that conformations that initially were geometrically impossible could evolve into energetically favorable states in thermal equilibrium due to DNA bending and torsion. In addition, dynamic phase diagrams were applied to identify chromatin fibers that reflect certain experimentally determined features.

Affinity, stoichiometry and cooperativity of heterochromatin protein 1?(HP1) binding to nucleosomal arrays

Heterochromatin protein 1 (HP1) participates in establishing and maintaining heterochromatin via its histone-modification-dependent chromatin interactions. In recent papers HP1 binding to nucleosomal arrays was measured in vitro and interpreted in terms of nearest-neighbour cooperative binding. This mode of chromatin interaction could lead to the spreading of HP1 along the nucleosome chain. Here, we reanalysed previous data by representing the nucleosome chain as a 1D binding lattice and showed how the experimental HP1 binding isotherms can be explained by a simpler model without cooperative interactions between neighboring HP1 dimers. Based on these calculations and spatial models of dinucleosomes and nucleosome chains, we propose that binding stoichiometry depends on the nucleosome repeat length (NRL) rather than protein interactions between HP1 dimers. According to our calculations, more open nucleosome arrays with long DNA linkers are characterized by a larger number of binding sites in comparison to chains with a short NRL. Furthermore, we demonstrate by Monte Carlo simulations that the NRL dependent folding of the nucleosome chain can induce allosteric changes of HP1 binding sites. Thus, HP1 chromatin interactions can be modulated by the change of binding stoichiometry and the type of binding to condensed (methylated) and non-condensed (unmethylated) nucleosome arrays in the absence of direct interactions between HP1 dimers.

Visualization in Health Grid Environments: A Novel Service and Business Approach

Advanced visualization technologies are gaining major importance to allow presentation and manipulation of high dimensional data. Since new health technologies are constantly increasing in complexity, adequate information processing is required for diagnostics and treatment. Therefore, the German D-Grid initiative started to build visualization centers in 2008, which have recently been embedded into the existing compute and storage infrastructure. This paper describes an analysis of this infrastructure and the interplay with life science applications for 3D and 4D visualization and manipulation. Furthermore, the performance and business aspects regarding accounting, pricing and billing are investigated. The results show the viability and the opportunities for further optimization of this novel service approach and the possibilities for a sustainable business scenario.

Solutions for biomedical grid computing—Case studies from the D-Grid project Services@MediGRID

The project Services@MediGRID consortium established a tool set of grid-based biomedical services since 2008. The services are related to genetic analysis, genome data visualization, and pharmacokinetic modeling. Furthermore, business concepts for these services have been examined which are supported by an accounting and billing service. While the tools cover a whole service chain for biomedicine, the business concepts are rather heterogeneous. However, the overall addressed target market areas show promising potential. In addition, a structured coaching process reduces friction in the technology transfer from grid computing to biomedicine. This should be considered for similar future endeavors.