Differences of size and shape of active and inactive X‐chromosome domains in human amniotic fluid cell nuclei

Abstract

It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46,XX and 46,XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITCconjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi-and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISShybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area Axi for the inactive X-domain was smaller than the area A, , for the active domain (mean ratio Ax,/Ax, = 1.9 * 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P < .0001) difference both between A, , and Axi and between the ratios r(Xa) and r(Xi), calculated by dividing the maximum length L of each X-domain by its maximum width W. In most nuclei (26134) we found r(Xa)>r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 pm). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 t 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 k 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 t 2.6 S.D.)%. These findings indicate that differences in the 3D expansion of active and inactive X-chromosome domains are less pronounced than previously thought. A current model suggests that chromosome domains consist of a compact core surrounded by loosely coiled outer chromatin fiber loops. The latter fraction may be considerably larger in Xa-as compared to Xi-domains. We suggest that the interactive outlining procedure used in the 2D analyses included the loosely structured domain periphery more accurately, while the threshold algorithm applied to light optical sections delineated the more compact core of the domains, leading to smaller and more similar volume estimates of Xa and Xi. Present limitat...

Gene density and chromosome territory shape

“…Moreover, the gene rich CT 19 was reported to occupy a less compacted territory and have a more irregular shape than the gene poor CT 18 (Croft et al 1999). Similarly, the gene active CT Xa was found to have a more irregular and extended shape than its gene inactive CT Xi homolog (Bischoff et al 1993; Eils et al 1996; Walker et al 1991). Differences in CT shapes across the population, and a relative maintenance of these shapes for up to four hours in the cell cycle were further suggested from live cell analyses (Edelmann et al 2001; Muller et al 2010).…”

FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes

“…Therefore, it affords a larger S-phase window for following the replication status of a given locus. Moreover, since interphase chromosomes are folded and are each organized in a specific nuclear territory (especially those of the inactive X-chromosome), proximal and distal chromosomal regions of each chromosome become relatively close to one another (Dyer et al 1989;Cremer et al 1993;Bischoff et al 1993). Thus, Xsat may also be used as a suitable marker for loci located distal to the centromere, e.g., FMR1.…”

The “lnc” between 3D chromatin structure and X chromosome inactivation

“…DNA FISH probes that paint chromosomes have generally been employed to estimate chromosomal volume and through application of this technique, multiple studies using RNA/DNA FISH to identify the Xist RNA-coated Xi, have reported that the volume occupied by the Xi is smaller than that of the Xa. Using the total nuclear volume as a normalizer, a standard ratio of between 1.25 +/− 0.05 is consistently reported [55,94,95]. However, to decisively conclude that the nuclear space occupied by the Xi is indeed smaller than that of Xa, the difference between the Xa and Xi should be compared to, and significantly differ from, that between homologous autosomes.…”