Gene repositioning within the cell nucleus is not random and is determined by its genomic neighborhood

Background: Heterochromatin has been reported to be a major silencing compartment during development and differentiation. Prominent heterochromatin compartments are located at the nuclear periphery and inside the nucleus (e.g., pericentric heterochromatin). Whether the position of a gene in relation to some or all heterochromatin compartments matters remains a matter of debate, which we have addressed in this study. Answering this question demanded solving the technical challenges of 3D measurements and the large-scale morphological changes accompanying cellular differentiation. Results: Here, we investigated the proximity effects of the nuclear periphery and pericentric heterochromatin on gene expression and additionally considered the effect of neighboring genomic features on a gene's nuclear position. Using a well-established myogenic in vitro differentiation system and a differentiation-independent heterochromatin remodeling system dependent on ectopic MeCP2 expression, we first identified genes with statistically significant expression changes by transcriptional profiling. We identified nuclear gene positions by 3D fluorescence in situ hybridization followed by 3D distance measurements toward constitutive and facultative heterochromatin domains. Single-cell-based normalization enabled us to acquire morphologically unbiased data and we finally correlated changes in gene positioning to changes in transcriptional profiles. We found no significant correlation of gene silencing and proximity to constitutive heterochromatin and a rather unexpected inverse correlation of gene activity and position relative to facultative heterochromatin at the nuclear periphery. Conclusion: In summary, our data question the hypothesis of heterochromatin as a general silencing compartment. Nonetheless, compared to a simulated random distribution, we found that genes are not randomly located within the nucleus. An analysis of neighboring genomic context revealed that gene location within the nucleus is rather dependent on CpG islands, GC content, gene density, and short and long interspersed nuclear elements, collectively known as RIDGE (regions of increased gene expression) properties. Although genes do not move away/to the heterochromatin upon up-/down-regulation, genomic regions with RIDGE properties are generally excluded from peripheral heterochromatin. Hence, we suggest that individual gene activity does not influence gene positioning, but rather chromosomal context matters for sub-nuclear location

Measuring leukocyte adhesion to (primary) endothelial cells after photon and charged particle exposure with a dedicated laminar flow chamber

The vascular endothelium interacts with all types of blood cells and is a key modulator of local and systemic inflammatory processes, for example, in the adhesion of blood leukocytes to endothelial cells (EC) and the following extravasation into the injured tissue. The endothelium is constantly exposed to mechanical forces caused by blood flow, and the resulting shear stress is essential for the maintenance of endothelial function. Changes in local hemodynamics are sensed by EC, leading to acute or persistent changes. Therefore, in vitro assessment of EC functionality should include shear stress as an essential parameter. Parallel-plate flow chambers with adjustable shear stress can be used to study EC properties. However, commercially available systems are not suitable for radiation experiments, especially with charged particles, which are increasingly used in radiotherapy of tumors. Therefore, research on charged-particle-induced vascular side effects is needed. In addition, a-particle emitters (e.g., radon) are used to treat inflammatory diseases at low doses. In the present study, we established a flow chamber system, applicable for the investigation of radiation induced changes in the adhesion of lymphocytes to EC as readout for the onset of an inflammatory reaction or the modification of a pre-existing inflammatory state. In this system, primary human EC are cultured under physiological laminar shear stress, subjected to a proinflammatory treatment and/or irradiation with X-rays or charged particles, followed by a coincubation with primary human lymphocytes (peripheral blood lymphocytes (PBL)). Analysis is performed by semiautomated quantification of fluorescent staining in microscopic pictures. First results obtained after irradiation with X-rays or helium ions indicate decreased adhesion of PBL to EC under laminar conditions for both radiation qualities, whereas adhesion of PBL under static conditions is not clearly affected by irradiation. Under static conditions, no radiation-induced changes in surface expression of adhesion molecules and activation of nuclear factor kappa B (NF-κB) signaling were observed after single cell-based high-throughput analysis. In subsequent studies, these investigations will be extended to laminar conditions

Heterochromatin and gene positioning: inside, outside, any side?

All cellular processes depend on the expression and repression of the right sets of genes at the right time. As each cell contains the same DNA, transcriptional and epigenetic factors have to maintain tight control over gene expression. Even a small divergence from the correct transcriptional program can lead to severe defects and even death. Having deciphered the complete linear genetic information, we need to clarify how this information is organized into the dynamic and highly heterogeneous three-dimensional space of the eukaryotic cell nucleus. Observations on the higher order organization of DNA into differentiated condensation levels date back to the early twentieth century, and potential implications of these structural features to gene expression were postulated shortly after. In particular, proximity of genes to condensed regions of heterochromatin was proposed to negatively influence their expression and, henceforward, the concept of heterochromatin as subnuclear silencing compartment emerged. Methodological advances fueled a flurry of recent studies, which only, in part, led support to this concept. In this review, we address how (hetero)chromatin structure and proximity might influence gene expression and discuss the challenges and means to unravel this fundamental biological question