3D-Image analysis platform monitoring relocation of pluripotency genes during reprogramming

Abstract

Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization. We performed a comparative analysis of the relationship between spatial positioning of pluripotency genes with their genomic activity and determined the degree of similarity between fibroblasts, induced pluripotent stem cells and embryonic stem cells. Our analysis revealed a preferred positioning of actively transcribed Sox2, Oct4 and Nanog away from the nuclear periphery, but not from pericentric heterochromatin. Moreover, in the silent state, we found no common nuclear localization for any of the genes. Our results suggest that the surrounding gene density hinders relocation from an internal nuclear position. Altogether, our data do not support the hypothesis that the nuclear periphery acts as a general transcriptional silencer, rather suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts. Thus, our computational approach enables comparative analysis of topological relationships in spite of stark morphological variability typical of biological data sets.

Figure 1.

Correlation of subnuclear gene topology versus gene expression is hampered by drastic morphological variability. (a) 3D representation of a mouse mammalian nucleus (grey) with chromocenters highlighted in green (DNA staining with DAPI) and gene loci visualized by FISH in red. Nearest 3D distances are measured from the signal of interest (gene locus) to the chromocenter (1) and to the nuclear periphery (2). (b) Schematic representation of the pluripotency state of MEF, iPS and ES cells (ES). Confocal optical mid section of a DAPI stained representative MEF (lower), iPS (left) and ES (right) cell nucleus and corresponding 3D reconstructions highlighting their morphological variability. Scale bar 5 µm. (c) Bar histograms of nuclear volumes measured in MEFs, iPS and ES cells (n = 30). (d) The ratios between chromocenter versus nuclear volumes were compared using either BGT depicted in green, or fluorescent intensity threshold levels of 30 (blue) and 60 (red), respectively, and their relative frequency plotted. The largely diverging volumes demonstrate the dependency of these measurements from a given threshold setting (n = 133).

Figure 2.

Single cell-based normalization overcomes threshold dependent variability. (a) For data normalization, 10 000 random points were set throughout the 3D nuclear volume (Supplementary Movie 2) and all distances from these points (white) to the chosen nuclear landmarks measured. The target gene locus measurement (red) is set in relation to the random distribution obtained by the simulation. The fractions of random point measurements, which are smaller or equal to the gene locus distance measurements, are defined as quantile. (b) Gene loci distance measurements in MEF were normalized to a random distribution and the resulting quantiles plotted for each threshold setting (n = 133).

Figure 3.

Gene repositioning during gain/loss of pluripotency relative to chromosomal context and to distance from heterochromatin. Distances of pluripotency genes Sox2, Nanog and Oct4 to chromocenters and to the nuclear periphery in MEF, ES and iPS cells were normalized as shown in Figure 2A. (a) Quantile distribution relative to chromocenters (1) and to nuclear periphery (2) with histogram of the average quantiles underneath. A summary of expression and re-localization of the genes in the nucleus is added at the bottom. (b) Position of the genes within the corresponding mouse chromosome ideogram (Giemsa banding) with gene density plotted along the chromosome.