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Despite all the efforts the three-dimensional higher-order architecture and dynamics in the cell nucleus are still debated. The regulation of genes, their transcription, replication, as well as differentiation in Eukarya is, however, closely connected to this architecture and dynamics. Here, an evaluation and review framework is setup to investigate the folding of a 30 nm chromatin fibre into chromosome territories by comparing computer simulations of two different chromatin topologies to experiments: The Multi-Loop-Subcompartment (MLS) model, in which small loops form rosettes connected by chromatin linkers, and the Random-Walk/Giant-Loop (RW/GL) model, in which large loops are attached to a flexible non-protein backbone, were simulated for various loop, rosette, and linker sizes. The 30 nm chromatin fibre was modelled as a polymer chain with stretching, bending, and excluded volume interactions. A spherical boundary potential simulated the confinement by other chromosomes and the nuclear envelope. Monte Carlo and Brownian Dynamics methods were applied to generate chain configurations at thermodynamic equilibrium. Both the MLS and the RW/GL models form chromosome territories, with different morphologies: The MLS rosettes form distinct subchromosomal domains, compatible in size as those from light microscopic observations. In contrast, the big RW/GL loops lead to a more homogeneous chromatin distribution. Only the MLS model agrees with the low overlap of chromosomes, their arms, and subchromosomal domains found experimentally. A review of experimental spatial distance measurements between genomic markers labelled by FISH as a function of their genomic separation from different publications and comparison to simulated spatial distances also favours an MLS-like model with loops and linkers of 63 to 126 kbp. The chromatin folding topology also reduces the apparent persistence length of the chromatin fibre to a value significantly lower than the free solution persistence length, explaining the low persistence lengths found various experiments. The predicted large spaces between the chromatin fibres allow typically sized biological molecules to reach nearly every location in the nucleus by moderately obstructed diffusion and disagrees with the much simplified assumption that defined channels between territories for molecular transport as in the Interchromosomal Domain (ICD) hypothesis exist. All this is also in agreement with recent selective high-resolution chromosome interaction capture (T2C) experiments, the scaling behaviour of the DNA sequence, the dynamics of the chromatin fibre, the nuclear diffusion of molecules, as well as other experiments. In summary, this polymer simulation framework compared to experimental data clearly favours only a quasi-chromatin fibre forming a stable multi-loop aggregate/rosette like genome organization and dynamics whose local topology is tightly connected to the global morphology and dynamics of the cell nucleus., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

After now more than 170 years of research the dynamic three-dimensional chromatin architecture of genomes and the co-evolved interaction networks of regulatory elements which create genome function - i.e. the storage, expression, and finally replication of genetic information - involves ever more investigative efforts in respect to not only the pure understanding of living organisms, but also diagnosis, treatment, and even future genome engineering. To study genomic interactions, we developed a novel and superior high-quality selective high-resolution, high-throughput chromosome interaction capture method - T2C (targeted chromatin capture) - which allows to arbitrarily balance resolution, frequency range of interactions, and the investigated general genetic region or single interactions in a highly cost-effective manner in respect to the obtainable result and compared to other techniques. Beyond, T2C has such a high signal-to-noise ratio at high resolution that the "genomic" statistical mechanics level can be reached. With the guided T2C protocol described here, we were already able to finally determine the chromatin quasi-fiber conformation and its folding into stable multi-loop aggregates/rosettes connected by a linker. Actually, this guided T2C protocol provides the means for architectural genome sequencing from the level of the single base pair to the entire cell nucleus and thus to analyze genetic interactions in respect to genome function in a systems biological manner in general as well as in settings ranging from basic research, via diagnostics and treatment, to genome engineering. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

The human small glutamine-rich TPR-containing protein (hSGT) is essential for cell division since RNA-interference-mediated strong reduction of hSGT protein levels causes mitotic arrest (M. Winnefeld, J. Rommelaere, and C. Cziepluch, The human small glutamine-rich TPR-containing protein is required for progress through cell division, Exp. Cell Res. 293 (2004), 43-57). Analysis of HeLa cells expressing a histone 2A-YFP fusion protein revealed the continuous presence of few mislocalized chromosomes close to the spindle poles as possible cause for hSGT depletion-dependent prometaphase arrest. Cells unable to rescue these mislocalized chromosomes into the metaphase plate died at this stage through apoptosis. In order to address hSGT function at the molecular level, mass spectrometry analysis of proteins which co-immunoprecipitated with Flag-tagged hSGT was performed. Thereby, Hsp70 and Bag-6/Bat-3/Scythe were identified as novel hSGT interaction partners while interaction with Hsc70 was confirmed. Results obtained with truncated versions of the hSGT protein revealed that Bag-6/Bat-3/Scythe and Hsp70 or Hsc70 were independently able to form complexes with hSGT. Interaction of hSGT with Hsc70, Hsp70 or Bag-6/Bat-3/Scythe was demonstrated in prometaphase, thereby suggesting a possible role for complexes containing hSGT and distinct (co)-chaperones during mitosis. Finally, cells from populations with reduced levels of Bag-6/Bat-3/Scythe also displayed persistence of mislocalized chromosomes and mitotic arrest, which strongly indicated that hSGT-Bag-6/Bat-3/Scythe complexes could be directly or indirectly required for complete chromosome congression.