Philipp M. Diesinger

Localization Microscopy Reveals Expression-Dependent Parameters of Chromatin Nanostructure

A combined approach of 2D high-resolution localization light microscopy and statistical methods is presented to infer structural features and density fluctuations at the nuclear nanoscale. Hallmarks of nuclear nanostructure are found on the scale below 100 nm for both human fibroblast and HeLa cells. Mechanical measures were extracted as a quantitative tool from the histone density fluctuations inside the cell to obtain structural fluctuations on the scale of several micrometers. Results show that different mechanisms of expression of the same nuclear protein type lead to significantly different patterns on the nanoscale and to pronounced differences in the detected compressibility of chromatin. The observed fluctuations, including the experimental evidence for dynamic looping, are consistent with a recently proposed chromatin model.

Histone Depletion Facilitates Chromatin Loops on the Kilobasepair Scale

The packing of eukaryotic DNA in the nucleus is decisive for its function; for instance, contact between remote genome sites constitutes a basic feature of gene regulation. Interactions among regulatory proteins, DNA binding, and transcription activation are facilitated by looping of the intervening chromatin. Such long-range interactions depend on the bending flexibility of chromatin, i.e., the ring-closure probability is a directly measurable indicator of polymer flexibility. The applicability of a wormlike chain model to naked DNA has been widely accepted. However, whether this model also suffices to describe the flexibility of eukaryotic interphase chromatin is still a matter of discussion. Here we compare both 5C data from a gene desert and data from fluorescence in situ hybridization with the results of a Monte Carlo simulation of chromatin fibers with and without histone depletion. We then estimate the ring-closure probabilities of simulated fibers with estimates from analytical calculations and show that the wormlike chain model grossly underestimates chromatin flexibility for sharp bends. Most importantly, we find that only fibers with random depletion of linker histones or nucleosomes can explain the probability of random chromatin contacts on small length scales that play an important role in gene regulation. It is possible that missing linker histones and nucleosomes are not just simple, unavoidable, randomly occurring defects, but instead play a regulatory role in gene expression.

Depletion Effects Massively Change Chromatin Properties and Influence Genome Folding

We present a Monte Carlo model for genome folding at the 30-nm scale with focus on linker-histone and nucleosome depletion effects. We find that parameter distributions from experimental data do not lead to one specific chromatin fiber structure, but instead to a distribution of structures in the chromatin phase diagram. Depletion of linker histones and nucleosomes affects, massively, the flexibility and the extension of chromatin fibers. Increasing the amount of nucleosome skips (i.e., nucleosome depletion) can lead either to a collapse or to a swelling of chromatin fibers. These opposing effects are discussed and we show that depletion effects may even contribute to chromatin compaction. Furthermore, we find that predictions from experimental data for the average nucleosome skip rate lie exactly in the regime of maximum chromatin compaction. Finally, we determine the pair distribution function of chromatin. This function reflects the structure of the fiber, and its Fourier-transform can be measured experimentally. Our calculations show that even in the case of fibers with depletion effects, the main dominant peaks (characterizing the structure and the length scales) can still be identified.

The Influence of the Cylindrical Shape of the Nucleosomes and H1 Defects on Properties of Chromatin

We present a model improving the two-angle model for interphase chromatin (E2A model). This model takes into account the cylindrical shape of the histone octamers, the H1 histones in front of the nucleosomes, and the distance d between the in and outgoing DNA strands orthogonal to the axis of the corresponding nucleosome cylinder. Factoring these chromatin features in, one gets essential changes in the chromatin phase diagram: Not only the shape of the excluded-volume borderline changes but also the orthogonal distance d has a dramatic influence on the forbidden area. Furthermore, we examined the influence of H1 defects on the properties of the chromatin fiber. Thus, we present two possible strategies for chromatin compaction: The use of very dense states in the phase diagram in the gaps in the excluded-volume, borderline, or missing H1 histones can lead to very compact fibers. The chromatin fiber might use both of these mechanisms to compact itself at least locally. Line densities computed within the model coincident with the experimental values.

Two-angle model and phase diagram for chromatin

We have studied the phase diagram for chromatin within the framework of the two-angle model. Only a rough estimation of the forbidden surface of the phase diagram for chromatin was given in a previous work of Schiessel. We revealed the fine structure of this excluded-volume borderline numerically and analytically. Furthermore, we investigated the Coulomb repulsion of the DNA linkers to compare it with the previous results.

Monte Carlo Simulations indicate that Chromati: Nanostructure is accessible by Light Microscopy

A long controversy exists about the structure of chromatin. Theoretically, this structure could be resolved by scattering experiments if one determines the scattering function - or equivalently the pair distribution function - of the nucleosomes. Unfortunately, scattering experiments with live cells are very difficult and limited to only a couple of nucleosomes. Nevertheless, new techniques like the high-resolution light microscopy supply a new approach to this problem. In this work we determine the radial pair distribution function of chromatin described by our E2A model and find that the dominant peaks which characterize the chromatin structure are very robust in several ways: They can still be identified in the case of chromatin fibers with reasonable linker histone and nucleosome defect rates as well as in the 2D case after a projection like in most high-res light microscopy experiments. This might initiate new experimental approaches like optical microscopy to finally determine the nanostructure of chromatin. Furthermore, we examine the statistics of random chromatin collisions and compare it with 5C data of a gene desert. We find that only chromatin fibers with histone depletion show a significant amount of contacts on the kbp-scale which play a important role in gene regulation. Therefore, linker histone and nucleosome depletion might not only be chromatin defects but even be necessary to facilitate transcription. PACS codes: 82.35.Pq, 87.16.A-, 87.16.af

The Relation Between the Gene Network and the Physical Structure of Chromosomes

Human cells contain 46 chromosomes with a total length of about 5 cm beads-ona- string type of nucleosomal fibre, called chromatin. Packaging this into a nucleus of typically 5–20 μm diameter requires extensive compatification. This packaging cannot be random, as considerable evidence has been gathered that chromatin folding is closely related to local genome function. However, the different levels of compactification are ill understood and not easily accessible by experiments.

Structure and Dynamics of Epithelial Cell Cortical Actomyosin Networks

Cortical actomyosin networks consist of filamentous actin (F-actin) bundles crosslinked into highly dynamic networks that are linked physically to the extracellular matrix via integrins. These networks play an active role in tissue morphogenesis during development as well as metastasis in cancer, by enabling rapid remodelling of cell shape via F-actin polymerization and interaction with myosin-II motor proteins. Here we investigate a newly discovered isotropic actomyosin network on the dorsal surface of epithelial MDCK cells and characterize its structure and dynamics using computational analysis of timelapse fluorescence microscopy data provided by Lifeact, a highly specific marker for F-actin in living cells. At low cell density, F-actin bundles depolymerise and polymerize continuously, splitting and merging into large bundle clusters. Spatial Temporal Image Correlation Spectroscopy and Optical Flow are used to characterize the oscillatory dynamics of the bundled networks, and reveal characteristic length- and time-scales associated with their motions.