Reorganization of the interchromosomal network during keratinocyte differentiation

Abstract

The well-established human epidermal keratinocyte (HEK) differentiation model was investigated to determine possible alterations in chromosome territory (CT) association during differentiation. The seven human chromosomes (1, 4, 11, 12, 16, 17, and 18) selected for this analysis are representative of the chromosome size and gene density range of the overall human genome as well as including a majority of genes involved in epidermal development and differentiation (CT1, 12, and 17). Induction with calcium chloride (Ca(2+)) resulted in morphological changes characteristic of keratinocyte differentiation. Combined multi-fluorescence in situ hybridization (FISH) and computational image analysis on the undifferentiated (0 h) and differentiated (24 h after Ca(2+) treatment) HEK revealed that (a) increases in CT volumes correspond to overall nuclear volume increases, (b) radial positioning is gene density-dependent at 0 h but neither gene density- nor size-dependent at 24 h, (c) the average number of interchromosomal associations for each CT is gene density-dependent and similar at both time points, and (d) there are striking differences in the single and multiple pairwise interchromosomal association profiles. Probabilistic network models of the overall interchromosomal associations demonstrate major reorganization of the network during differentiation. Only ~40 % of the CT pairwise connections in the networks are common to both 0 and 24 h HEK. We propose that there is a probabilistic chromosome positional code which can be significantly altered during cell differentiation in coordination with reprogramming of gene expression.

Chromosome territories and the global regulation of the genome

“…In addition, five of nine CT examined showed significant differences in radial position between normal breast epithelial cells and a corresponding malignant cell line . Even more striking, it was reported that the radial positioning of seven CT in undifferentiated human keratinocytes strongly correlated with gene density, while the positioning in the corresponding CT from late differentiating keratinocytes were highly altered with no relationship to gene density or chromosome size . These findings are consistent with the probabilistic radial positioning code proposed by Cremer and associates and support the view that the probabilistic positioning code is cell‐type‐specific and can undergo alterations during both malignant progression and cell differentiation where massive changes in the genomic expression programs generally occur.…”

“…Figure depicts typical CT multicolor labeled images from re‐FISH experiments with MCF10 normal human breast and malignant breast cancer cells (Figure A,B ), undifferentiated and differentiated human keratinocytes (Figure C,D), WI38 normal human fibroblasts (Figure E), and the NOR‐CT in WI38 cells (Figure F). An integrated computer imaging program was then applied to segment the individual CT and determine a large battery of measurements among the total population of labeled CT in the individual nuclei . These measurements included the 3‐D nearest neighbor distances between the borders of all labeled CT.…”

“…In studies involving nine CT pairs, a total of 153 pairwise CT interactions were determined . From this data, the percentages of pairwise associations (interactions) were calculated using a threshold distance of ≤4 pixels, which corresponded to ≤0.28 μm or the approximate resolution limitation of the fluorescence microscopy used for collecting the images …”

SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

“…FISH was performed as described previously (Sehgal et al 2016) on MCF-10A cells induced to express either shSCRAM or shSMARCA4 shRNAs. Probes from bacterial artificial chromosomes RP11-82D16 for Chr 1p, RP11-81J5 for Chr 1q, RP11-81L5 for Chr 4p; and RP11-196K19 for Chr 4q were used.…”

Super‐resolution structure of DNA significantly differs in buccal cells of controls and Alzheimer's patients

“…Chromosome positioning is not a constant feature of nuclei as positional changes were recorded during keratinocyte differentiation (Sehgal et al, ), during adipocyte differentiation (Charó, Rodríguez Ceschan, Galigniana, Toneatto, & Piwien‐Pilipuk, ; Kuroda et al, ), after PHA‐activation of lymphocytes (Ioannou, Kandukuri, Simpson, & Tempest, ), and after serum deprivation/stimulation (Mehta, Amira, Harvey, & Bridger, ). It thus appears that the nuclear organization of the genome is directly associated with its cellular function.…”