The active and inactive X (Xa;Xi) territory with its seemingly highly compacted Barr body in nuclei of female mammalian cells provide a key example for studies of structure/function relationships in homologous chromosomes with different functional properties. Here we used about 300 human X-specific large insert clones to generate probe sets, which target physically or functionally defined sub-chromosomal segments. We combined 3D multicolor FISH with quantitative 3D image analysis in order to compare the higher order organization in Xi-and Xa-territories in human diploid fibroblasts (HDFs) at various length scales ranging from about 50 Mb down to 1 Mb. Xi-territories were characterized by a rounder shape as compared to the flatter and more extended shape of Xa-territories. The overall compaction of the entire Xi-territory, including the Barr body, was only 1.2-fold higher than the Xa-territory. Significant differences, however, were noted between distinct subchromosomal segments: At 20 Mb length scales higher compaction in Xi-territories was restricted to specific segments, but higher compaction in these segments was not correlated with gene density, transcriptional activity, LINE content or histone markers locally enriched in Xi-territories. Notably, higher compaction in Xi-territories observed for 20 Mb segments was not reflected accordingly by inclosed segments of 1-4 Mb. We conclude that compaction differences result mainly from a regrouping of ~1 Mb chromatin domains rather than from an increased condensation of individual domains. In contrast to a previous report, genes subject to inactivation as well as escaping from inactivation were not excluded from the interior of the Barr body.
We present an intensity-based nonrigid registration approach for the normalization of 3-D multichannel microscopy images of cell nuclei. A main problem with cell nuclei images is that the intensity structure of different nuclei differs very much; thus, an intensity-based registration scheme cannot be used directly. Instead, we first perform a segmentation of the images from the cell nucleus channel, smooth the resulting images by a Gaussian filter, and then apply an intensity-based registration algorithm. The obtained transformation is applied to the images from the nucleus channel as well as to the images from the other channels. To improve the convergence rate of the algorithm, we propose an adaptive step length optimization scheme and also employ a multiresolution scheme. Our approach has been successfully applied using 2-D cell-like synthetic images, 3-D phantom images as well as 3-D multichannel microscopy images representing different chromosome territories and gene regions. We also describe an extension of our approach, which is applied for the registration of 3D + t (4-D) image series of moving cell nuclei.
(14) provided a first case-in-point. They performed FISH experiments in normal, phytohemagglutinin (PHA)-stimulated human lymphocytes with a probe covering a segment of the Angelman syndrome/Prader-Willi syndrome (AS/PWS) locus on HSA 15q11-13 and measured 3D interdistances between the two signals in samples of 50 nuclei at different interphase stages. Approximately 38% of nuclei at G 1 , Ϸ18% at early S phase, Ϸ58% at late S phase, and Ϸ18% at G 2 revealed distances Յ2 m, which were considered as a proximity criterion for a possible functional interaction between the two AS/PWS loci. Notably, the significant increase of associations at late S phase was not found in cells from PWS and AS patients. Furthermore, LaSalle and Lalande (14) mentioned that they obtained similar results for the Beckwith-Wiedemann syndrome (BWS) locus, a second imprinted locus on HSA 11p15.5. Based on these findings, LaSalle and Lalande formulated the kissing hypothesis, which states that a transient spatial association between the oppositely imprinted regions during late S phase is a necessary condition to maintain the imprinted status of cycling cells from one cell cycle to the next. This hypothesis was widely cited and further supported by a 2D FISH study from Riesselmann and Haaf (15), who measured 2D interhomolog distances between oppositely imprinted loci on MMU 7 as a fraction of the nuclear diameter in flattened mouse fibroblast nuclei obtained in cytospin preparations. Somatic pairing was assumed when hybridization signals apparently overlapped or touched each other. By this criterion, significantly higher values were found for BrdU-positive nuclei (13%; 27/208 nuclei) compared with BrdUnegative nuclei (9.2%; 21 of 228 nuclei). No increase of pairing frequencies during S phase was found for control regions not subject to imprinting. In contrast, Nogami et al. (16), who studied the frequency of associations between the two AS loci with probes for the imprinted SNRPN alleles in 3D preserved interphase nuclei of human myeloid leukemia HL60 cells, did not find evidence to further support the hypothesis.Importantly, a large variability of 3D positions was noted for pairs of HSA 11 and HSA 15 chromosome territories (CTs) both within nuclei and between nuclei of different cells. Given this variability, a transient kissing event during late S phase between loci widely separated at other stages of interphase requires specific, long-range, directed chromatin movements. The present study was initiated with the intent to clarify whether such movements lead to the transient juxtaposition of entire homologous CTs or, alternatively, whether homologous CTs stay in place, whereas giant loops harboring genes poised for a kissing event expand from territory surfaces and meet each other in between (1,6,8,17). We visualized the two AS/PWS regions in PHA-stimulated lymphocytes by 3D-FISH with a BAC contig covering most of the AS/PWS locus and measured the 3D distances between the two regions in G 1 , early, mid, and late S phase, G 2 , as well as in...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.