title: Gene repositioning within the cell nucleus is not random and is determined by its genomic neighborhood.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
authors.forename: K Laurence
authors.initials: KL
authors.lastname: Jost
authors.affiliation: Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
authors.forename: Bianca
authors.initials: B
authors.lastname: Bertulat
authors.affiliation: Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Rapp
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Brero
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Hardt
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Domaing
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Gösele
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Schulz
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Hübner
authors.affiliation: Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Cardoso
keywords: 3D-FISH measurements; Chromocenters; Gene position; Gene silencing; Genomic context; Heterochromatin proximity; MeCP2; Myogenesis; Nuclear periphery; Transcriptional profiling