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

Abstract

BackgroundHeterochromatin 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.ResultsHere, 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.ConclusionIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0025-5) contains supplementary material, which is available to authorized users.

Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging (SNP-CLING)

“…Although individual alleles are stably positioned in space and time, different chromosomes have unique positions. These findings are consistent with results of fixed cells and the chromosome territory models 5 , 7 , 43 . We also found that allele positions are preserved and similar in live cells, irrespective of the genetic backgrounds (129S1/CAST) in 3D- and 4D-imaging.…”

“…Similar results were obtained by studying chromosomal territories, where each territory showed a unique pattern with minor differences between homologous chromosomes in fixed cells 29 . A broader analysis is required to dissect the characteristic chromosomal features of size, gene density 9 , transcriptional activity 44 , local chromatin states and protein recruitment 5 , 43 , and whether or not they directly influence allele positioning proportional to the spatial order of whole chromosomes.…”

“…The three-dimensional organization of DNA is critical in the establishment of cellular states and frequently dysregulated in disease 1 - 4 . This relies on regulatory aspects, such as non-random chromosomal positioning, chromosomal substructures and properties, and ultimately the position of a given locus in the nucleus 5 - 7 . Underscoring the importance of these processes, several recent studies have revealed mechanistic links between chromosomal topology and disease.…”

Replication timing and transcriptional control: beyond cause and effect — part III

“…Conversely, artificial anchoring of distinct genes to the nuclear lamina leads to their reversible repression [92–94], but this re-positioning occurs only after a passage through mitosis [93]. Tethering to the periphery is sufficient to suppress the expression of some but not all genes [92,95,96], implying the existence of different sensitivity classes of genes, as was found with the RT studies discussed above. Although RT was not measured in these studies, since the periphery is a late replicating compartment such analogies are reasonable to expect.…”

Reorganization of inter‐ chromosomal interactions in the 2q37‐deletion syndrome

“…Normally, the positioning of genes during interphase is constrained, and only mitosis-mediated changes or major gene expression changes can lead to gene repositioning within the nucleus (Chubb et al, 2002;Kumaran & Spector, 2008;Ferrai et al, 2010). Gene positions are dependent on the chromatin neighborhood and are not randomly located within the nucleus (Jost et al, 2015). Generally, transcriptionally active loci tend to be in the nuclear center, whereas silenced genes are located closer to the nuclear periphery, which is associated with heterochromatin (Peric-Hupkes et al, 2010).…”