Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei

Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to achieve this goal are discussed.